RU2368384C2 - Biotransplant, treatment method of chronic liver diseases and treatment method of liver cirrhosis and portal hypertension - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and namely to hepatology and biotechnology, and deals with treatment of chronic diffusion liver diseases as well as treatment of liver cirrhosis and portal hypertension by means of the biotransplant (BT) obtained for that purpose. BT contains mixed cell pool in which 40% comprise stem cells, including stromal mesenchymal and hepatocyte bipotent stem cells, and the rest 60% - progenitor cells in the various phase of differentiation, including new immature hepatoblasts and progenitor cells of erythroid row, hemopoietic cells. Mixed BT cell pool is characterised with the expression of the following surface markers: CD13, CD29, CD44, CD90, CD117 (c-Kit) and absence of CD34 expression. For the purpose of treatment there injected is the above BT in the form of suspension in physiological solution in quantity of 2-4 mln cells per 1 kg of the patient's weight. At liver cirrhosis and portal hypertension, BT is injected in the form of suspension in physiological solution at total number of BT cells of 350 to 500 mln.

EFFECT: BT injection given above is an independent treatment method of liver diseases, which provides stable decrease of the process activity, decrease of degenerative dystrophic changes, and recurrence-free period of 12 months long.

3 cl, 3 ex, 9 tbl, 12 dwg

Description

The invention relates to medicine and biotechnology, and for the production of a biograft containing stem cells (mesenchymal stem cells, bipotent stem cells, progenitor cells of varying degrees of differentiation), and a method for the treatment of chronic diffuse liver diseases such as chronic hepatitis, liver cirrhosis, portal hypertension, using this biograft.

The main methods of treating patients with cirrhosis of the liver (CP) and portal hypertension (PG) at the present stage are still the supportive method, including the use of a specialized diet and various medications, as well as a surgical method that includes palliative interventions aimed at combating complications diseases (bleeding, ascites), and radical - orthotopic liver transplantation.

Known, for example, is the method of surgical treatment of hypertension by removing the spleen (Course of surgical surgery and topographic anatomy. G.E. Ostroverkhov et al. - M .: Medicine, 1964. - 839 p.).

There is another method for the treatment of portal hypertension by creating extraorgan vascular anastomoses between the portal and inferior vena cava systems. Surgical surgery with topographic anatomy of childhood (Yu.F. Isakov et al. - M.: "Medicine", 1977. - 639 p.).

From SU 1232229 (published May 23, 1986), a method for treating portal hypertension is known, which includes partial movement of the liver into the retroperitoneal space and that, in order to provide a persistent hypotensive effect in Budd-Chiari disease by increasing the number of extraperitoneal vascular connections into the retroperitoneal space the spleen is also moved, while the capsule is removed from both organs in the areas of movement and the dissected parietal peritoneum is hemmed to its edges.

However, the known methods are supplemented by a mandatory plelectomy, which in itself is a traumatic palliative intervention.

From RU 2135098 (published August 27, 1999), a method for treating portal hypertension and ascites in cirrhosis of the liver is known. According to this method, a video laparoscope is conducted through a trocar mounted in the fifth intercostal space along the anterior and posterior axillary lines. Fix the lower lobe of the right lung to the liver through the window in the diaphragm. Impose endo stitches with extracorporeal knotting. The method allows to reduce the morbidity and duration of the intervention.

For each of these areas there are certain disadvantages. Despite the wide range of proposed conservative measures and the large arsenal of drugs used, the effectiveness of maintenance therapy is significantly limited. It would be difficult to imagine that in this way it is possible to significantly improve the activity of hepatocytes that carry a huge amount of information and perform up to 500 different functions. In the short term, one can’t rely on the fact that organ transplantation can radically change the situation in the treatment of this severe suffering, due to the difficulties associated with the organization of the donor stage and a number of other, primarily economic, problems. The range of palliative surgical interventions is very wide, but the main goal of these interventions is to combat the clinical manifestations of GHG and its complications, which does not affect the most important clinical syndrome of the disease - chronic liver failure.

Chronic hepatocellular insufficiency (CHCH) is the most important syndrome that determines the clinical course and prognosis of the disease in patients with CP and PG. This applies to patients with clearly defined both therapeutic and surgical problems, including patients on the waiting list for organ transplantation. At the present stage, the main strategic line in the treatment of chronic hepatitis C can be the transition from the fight against "biochemical disorder in diseased cells" to the timely directed replenishment of the existing deficit of the information field in the liver. A special place in this is cell transplantation. Clinical experience in this area is extremely small, which does not allow us to properly assess the role and place of cell therapy in the complex of therapeutic measures in patients with CP and PG. There are many unresolved issues in this issue.

The only effective method for treating the final stages of hepatic cell failure is liver transplantation. However, a shortage of donor organs is a limiting factor. In this regard, technologies of cell replacement therapy, which are an alternative to organ transplantation, are of great interest. It is known that the high regenerative ability of the liver is due to the proliferation of hepatocytes. Therefore, one of the directions of cell replacement therapy is transplantation of mature hepatocytes. Isolated hepatocytes can be introduced into the subcutaneous tissue, spleen, and liver parenchyma (Hepatocyte transplantation: clinical and experimental application. Ohashi K., Park F., Kay MA // J. Mol. Med. - 2001. - Vol. 79. - P. 617-630).

At the same time, not only adult hepatocytes, but also fetal liver cells can be used as donor material (Hagihara M. et al: Effects of iso- and xeno-fetal liver fragments transplantation on acute and chronic liver failure in rats. Cell Transpant 3: 283-290, 1994,

Sukhikh G.T., Shtil A.A. Stem cell transplantation for treatment of liver diseases: From biological foundations to clinical experience (Reviev) // Inter. J. Mol. Med. - 2003. - Vol.11. - P.395-400).

So, a method is described according to which, in order to stop liver cell failure, autologous hepatocytes isolated from liver fragments were introduced into the spleen in patients with liver cirrhosis and chronic hepatitis (Mito M., Kusano M., Kawaraura Y. Hepatocyte transplantation in man // Transplant Proc. - 1992. - P.3052-3053).

Transplantation was not accompanied by the development of any complications, however, due to the use of autologous material and the receipt of a small number of hepatocytes, no noticeable clinical effect was noted. There is also known a method according to which allogeneic cells (in the amount of about a billion cells) obtained from the liver of 9-17-week-old human fetuses were administered to patients under a capsule of the rectus abdominis muscle. The introduction of such cells was not accompanied by the development of transplant reactions and in some patients was accompanied by a decrease in the manifestations of liver cell failure (V.I. Shumakov., E.I. Galperin, E.A. Neklyudova. Liver transplantation. - M., 1981).

The disadvantage of the above methods is that the introduction of hepatocytes can only temporarily improve liver function, without significantly affecting the repair processes of damaged hepatic parenchyma. The big problem is the isolation of donor hepatocytes and the maintenance of their functional activity during storage, the risk of infection through the use of allogeneic donor material and the possibility of developing transplant reactions. Subsequent studies have shown that, along with hepatocytes, an important role in liver regeneration, especially with chronic damage to liver cells, is played by liver stem cells that can differentiate into hepatocytes and cholangiocytes.

Currently, a new section is being formed in modern biomedicine - cell therapy, which allows using cell transplantation to make up for the insufficient functional activity of tissues and regenerate damaged organs. The function of updating and restoration of tissues in vivo is performed by stem cells, which are a pool of spare undifferentiated cell precursors of various types. In this regard, the use of stem cells is the most promising area of cell therapy, and the most relevant work on the isolation of stem cells from various human tissues.

Stem cells of the tissues of an adult organism serve as the cellular basis for maintaining homeostasis in it and underlie the reparative processes in the body. In this regard, the most relevant work on the isolation of stem cells from various human tissues. Currently, embryonic stem cells, as well as stem cells of adult tissues, are isolated - hematopoietic (blood cell precursors), neuronal (precursors of nerve tissue cells), mesenchymal (cells that can differentiate into tissue cells of mesenchymal origin, and are also able to transdifferentiate into tissue cells other germinal leaves), etc.

The relative simplicity of isolation and cultivation, the ability to proliferate over time in vitro and a wide range of differentiation make this type of cell attractive for basic research and open up the possibility for clinical use.

It is known that bone marrow stem cells can participate in liver regeneration. So, Goff and Peterson proposed a method for transplanting bone marrow cells to stimulate regeneration and restoration of the liver [Patent No. WO 0050048, published August 31, 2000]. According to this method, in order to stimulate regeneration, the bone marrow cells are introduced into the recipient in a dose sufficient to ensure the formation of new hepatocytes, cholangiocytes and / or oval liver cells. An enriched fraction of oval liver cells isolated using antibodies recognizing the Thy 1 antigen can also be used as a source of stem cells. Bone marrow cells or oval cells can be genetically modified to express a functional active protein, transplanted on a matrix, or treated with growth factors prior to transplantation. Before transplantation, bone marrow cells can be enriched by isolation of cells expressing stem cell markers (Thy 1, CD34, Flt-3 ligand, c-Kit), using magnetic bead separation methods, affinity chromatography, or flow cytometry. Moreover, the appearance in the liver of cells (hepatocytes, cholangiocytes, oval cells) of donor bone marrow origin was proved in experimental models of rats by the appearance of hepatocytes with markers of donor stem cells.

Also, from RU 2283113, a method of treating chronic diffuse liver diseases is known, including the use of hepatoprotectors and transplantation of bone marrow stem cells, which include using stem cells directly isolated from a patient’s bone marrow aspirate, the portion of which is calculated so that the number of CD34-positive cells in it ranged from 40 to 240x10 cells, which are divided into two halves, the first is administered intrahepatic, and the second intravenously.

So, as follows from the above sources, in modern medicine, a separate direction has clearly formed, using tissue and cell transplants for therapeutic purposes. One of the scientific and practical results of this direction is the recognition that fetal tissues and cells and their associates, as well as biologically active substances from them, have greater therapeutic efficacy than mature tissues and differentiated cells of an adult. This is due to the special properties of cells during embryogenesis.

The therapeutic effect of fetal transplants depends not only on the number of implanted fetal tissues, cells and their associates, but also on strict adherence to the conditions ensuring the content of cells in the transplant that possess all of the unique properties listed above.

From RU 2272638, published 03/27/2006, a biograft is known, a method for its production and a method for the treatment of chronic hepatitis and cirrhosis.

The well-known biograft is characterized in that it contains a culture of human fetal hepatocytes from the liver of human embryos of the 1-2 trimester of pregnancy, selectively propagated under cultivation conditions, and is a suspension of cell culture in physiological solution with a concentration of cellular elements of 1-15 million cells per 1 kg patient weight. A method of treating chronic hepatitis and cirrhosis of the liver is that the previously mentioned biografts are used, while a suspension of mesenchymal stem or fetal hepatocytes or a combination thereof is administered as a suspension in physiological solution with a concentration of cellular elements of 1-15 million cells per 1 kg of patient weight .

Known from RU 2272638 biograft and method according to the technical nature and the achieved result are the closest to the claimed group of inventions.

The objective of the claimed group of inventions is to study and determine the necessary and sufficient properties of a biograft for the most effective (complex) effect on the affected human liver and the development of the most effective method for the treatment of chronic hepatitis, liver cirrhosis and other chronic liver diseases, such as treatment of portal hypertension.

The task is achieved by the claimed group of inventions, which includes a biograft with its specific characteristics, as well as methods of treating chronic diffuse liver diseases, in particular, such as methods of treating liver cirrhosis and treatment of portal hypertension.

To identify the necessary characteristics of the biograft that solve the problem posed earlier, we studied biotransplants obtained from the fetal liver of a mammal.

It was found that the necessary properties are possessed by a biograft containing a mixed population of cells, in which 40% are stem cells, including stromal mesenchymal and hepatic bipotent stem cells, and the remaining 60% are progenitor cells at various stages of differentiation, including young immature hepatoblasts and cells erythroid precursors, hematopoietic cells, while a mixed population of biograft cells is characterized by the expression of the following surface markers: CD13, CD29, CD44, CD90, CD117 (c-Kit), lack of expression of CD34.

Such a biograft heterostructure can be obtained from various sources, not only from human fetal tissues, but also from other mammals, such as pigs.

The biograft according to the invention is used to treat liver diseases such as hepatitis, cirrhosis, acute liver failure, portal hypertension.

So, another invention of the claimed group is a method for the treatment of chronic diffuse liver diseases, characterized in that they use the developed biograft containing a mixed population of cells, including stromal mesenchymal stem cells, hepatic bipotent stem cells, as well as progenitor cells at different stages of differentiation, for why it is administered as a suspension in physiological saline with a concentration of cellular elements of 2-4 million cells per 1 kg of patient weight.

In particular, the method is intended for the treatment of hepatitis, cirrhosis of the liver, acute liver failure, chronic liver failure, portal hypertension and is characterized by the fact that the developed biograft is administered in the form of a suspension in physiological solution, while the total number of biograft cells is from 200 to 500 million.

The composition of the cell transplant was analyzed using flow cytometry and immunohistochemical analysis.

Upon receipt of the biograft, particular attention should be paid to the expression of specific markers of hepatocytes or hepatoblasts - AFP (alpha-fetoprotein) and albumin (Table 1).

Table 1. Antigenic characteristics of the cell preparation Antigens results CD13 Positive (+) CD29 Positive (+) Cd34 Negative (-) CD44 Positive (+) Cd90 Positive (+) CD117 (c-Kit) Positive (+) Albumin Positive (+) AFP Positive (+)

The above table 1 indicates that, using specific markers in the used mixed cell population, various stem cells, as well as progenitor cells capable of performing certain functions (synthesis of specific proteins) characteristic of hepatoblasts proper (alpha-fetoprotein), as well as more differentiated liver cells that are closer to hepatocytes (albumin).

So, the used mixed population of stem and progenitor cells immediately contains various stem cells, including: stromal mesenchymal stem cells, hepatic bipotent stem cells proper, as well as progenitor or progenitor cells.

The composition of the cell transplant was analyzed using flow cytometry and immunohistochemical analysis.

Using a wide range of designated monoclonal antibodies, specific antigens were identified, among which particular attention should be paid to the expression of specific hepatocyte markers - AFP and albumin (Table 1).

Direct introduction of the cell suspension (biotransplant) is carried out in normal saline (0.9% NaCl solution).

The course of treatment consists, in particular, in repeated intravenous injections of an allogeneic stem and progenitor cell biotransplant (ASPK). The multiplicity of intravenous cell transplants depends on the specific clinical situation, the severity of the disease and the degree of compensation of liver failure. Nevertheless, the optimal scheme includes 3 procedures with an interval of 1 and 3 months, and then additional sessions are possible after 6 months and a year after the first transplant. For one group of patients with a relatively compensated course of the disease and low disease activity, a single injection of cells may be sufficient. For other patients with a subcompensated or even decompensated course of the disease, repeated transplants may be required over a relatively long period of time (1-2 years), most often with an interval of 3-6 months. Although in some of the most difficult situations, forced transplant technologies may be required (3 transplants within 1.5 months).

During transplantation, up to 200-500 million mixed cell populations are introduced, of which 40% are bipotent and mesenchymal liver stem cells, and the rest are progenitor cells, including hepatoblasts (Fig. 1) and hematopoietic cells. Cell viability before transplantation is 90-95%. The number of viable cells was evaluated in a trypan blue test.

The antiviral effect of cell transplantation can be achieved with any method of their introduction: intraorgan (in the liver or spleen) or systemic, when the cell suspension is injected through any peripheral vein. Given the fact that the elimination of viral persistence is the most important task in patients with hepatitis, and in patients with cirrhosis, this problem is not leading, that is, it is impractical to complicate the technology of transplantation by using more invasive (intraorgan) methods of introducing cells.

Nevertheless, in patients with cirrhosis of the liver of viral etiology, the route of cell administration is regulated by other factors, while both more invasive methods and a simpler systemic transplantation route can be used.

In an experiment in rats with the systemic introduction of the developed cell biograft we developed, it was found that these cells are able to incorporate into the liver tissue and produce human albumin (Fig. 2). The data from this experiment suggest that even with systemic administration of cells, the applied cell population (biograft) has a tropism for the liver tissue, and having “navigation memory”, under the influence of certain signals, these cells rush to the recipient’s liver, take root and function in it .

So far, all the mechanisms by which the antiviral effect of allotransplantation of stem and progenitor liver cells is achieved are not known exactly. Studies have shed light on one possible and quite important mechanism.

The ability of stem and progenitor hepatic cells to take root and perform specific functions in the liver of the recipient determine the first and most important mechanism of the therapeutic effect of ASA. Another important mechanism that apparently provides morphologically confirmed, anti-inflammatory, anti-fibrotic, and regenerative effects of ASPA is the effect of cell transplant on the content and profile of cytokines and growth factors in the recipient's liver tissue. The cytokines studied in the liver tissue of the recipient were divided into 2 groups: pro-inflammatory and anti-inflammatory. They are presented in table 2.

table 2 Cytokines Pro-inflammatory Anti-inflammatory IL-1 If-gamma IL-4 IL-10 IL-6 Hgf TNF-α TGF-β PDGF

Table 3 presents the average values of the relative units of cytokines before and after ASPK.

Table 3 The average values of the relative units of cytokines before and after ASPK Cytokines The average value of cytokines To ASPK After ASPK IL-1 4.1 ± 1.7 4,5 ± 1,8 IL-4 6.1 ± 3.6 3.22 ± 2.9 IL-6 5.4 ± 1.7 6.2 ± 3.7 TNF-α 22.9 ± 10.4 6.22 ± 4.7 PDGF 8.25 ± 2.02 2.85 ± 1.85 TGF-β 14.6 ± 6.2 10.35 ± 8.3 IL-10 2.36 ± 0.96 8.54 ± 5.8 If-gamma 3.75 ± 1.1 7.75 ± 2.75 Hgf 11.9 ± 4.8 22.4 ± 10.08 IF-gamma / IL-4 0.61 ± 0.3 2.4 ± 0.94

As can be seen from table 3, when studying the spectrum of pro-inflammatory cytokines in the liver biopsy after transplantation, there is a tendency to increase the average values of IL-1 and IL-6. At the same time, we observed a significant decrease in the levels of TNF-α, PDGF and TGF-β. Inhibition of PDGF and IL-4, which, in turn, blocks the synthesis of TGF-P, plays a large role in the processes of “blocking” the factors that damage the liver, since the rate of progression of cirrhotic changes in the liver depends on the level of these cytokines.

By the control period, an increase in the average values of anti-inflammatory cytokines IL-10 from 2.36 ± 0.96 to 8.54 ± 5.8, IF-gamma from 3.75 ± 1.1 to 7.75 ± 2.75 and HGF was noted from 11.9 ± 4.8 to 22.4 ± 10.08. An increase in IF-gamma and a decrease in the content of IL-4 in the liver 7 days after transplantation provided an increase in the coefficient IF-gamma / IL-4 from 0.61 ± 0.3 to 2.4 ± 0.94, indicating a pronounced immunomodulating effect of ASPK .

In a control study, the following changes occurred in the system of pro-inflammatory cytokines: IL-1 expression increased in 4 patients, IL-6 in 4 patients, TGF-β in 3 patients, and TNF-α in 1 patient. A decrease in IL-1 production was observed in 4 patients, IL-6 in 4, TGF-β in 5 and TNF-α in 7 patients. All patients showed a significantly lower level of expression of IL-4 and PDGF compared to baseline. The results of the study of pro-inflammatory cytokines are presented in table 4.

Table 4 The dynamics of gene expression of pro-inflammatory cytokines Gene expression Number of patients IL-1 IL-4 IL-6 TGF-β PDGF TNF-α ↓ lowered n four 8 four 5 8 7 ↑ increased n four - four 3 - one

As can be seen from Table 4, along with an increase in some patients with some pro-inflammatory cytokines, such as IL-1, and IL-6 and TGF-β, in all patients there was a decrease in the expression of IL-4 and PDGF, and in most patients (in 7 patients) there was a decrease in the expression of the gene that controls the formation of TNF-α.

The results of the study of anti-inflammatory cytokines are presented in table.5. A control study showed an increase in the expression of HGF, IL-10, and IF-gamma in the spectrum of anti-inflammatory cytokines in all patients.

Table 5 The dynamics of gene expression of anti-inflammatory cytokines Gene expression Number of patients If-gamma IL-10 Hgf ↓ lowered n - - - ↑ increased n 8 8 8

The results of the study of cytokines in the liver tissue after transplantation suggest that the mechanisms of the positive effects of the developed biotransplant on the morphofunctional state are obviously mediated by the activation of powerful anti-inflammatory cytokines: IF-gamma, IL-10 and HGF. This group of cytokines realizes their action in many directions, including enhancing regenerative processes in the liver, immunomodulating effects, intensification of antiviral activity, etc. But this conclusion is quite simplified, since it does not fully take into account changes in the spectrum of pro-inflammatory cytokines. The data obtained do not allow us to fully appreciate the role of the complex mosaic of the relationships between the main components of these two groups of cytokines, especially in the temporal aspect, on which the result of transplantation largely depends. Nevertheless, the available data allow us to schematically represent the overall picture and mechanisms of cellular influences on the course of the pathological process in the liver (Fig. 3).

This scheme (Fig. 3) is rather arbitrary, it cannot characterize the temporal relationships characterizing the sequence of inclusion of certain cytokines. Nevertheless, it provides a rather informative hypothesis about the possible mechanism of the action of ASPK on the morphofunctional state of the liver, which is mediated by complex cascading interactions of various groups of liver cytokines.

In the clinical use of a biotransplant with the presented composition, there were no any adverse post-transplant complications due to the absence of class 2 histocompatibility complex antigens.

The following are illustrative examples.

Example 1. The patient history Voronin P.A. started from 10 years old when he was treated for hemorrhagic vasculitis in a children's hospital. The treatment was carried out by glucocorticoids for 8 months. At that moment, there was no talk of liver cirrhosis, although an increase in liver size (functional tests normal), changes in the kidneys (high protein, hematuria), immune tests were generally without pathology, but a tendency to their decrease was noted. In 1997, an examination for the military registration and enlistment office revealed a collapse of the esophagus of 2–3 mm, thrombocytopenia (in 1998, the difference in resistance was 10 mm). In 1998, the diagnosis was established: cryptogenic cirrhosis of the liver, hepatosplenomegaly. No significant changes were detected in the analyzes. Since 1998, there has been an increase in the level of transaminases (he was actively involved in sports - ice hockey). I felt satisfactory, and therefore for 5 years the doctors were not observed. For the first time in April 2003 - an increase in the liver, heaviness in the right hypochondrium, palpation and ultrasound enlargement of the liver, spleen, portal vein up to 14 mm and splenic up to 9 mm. With endoscopy - VRV 1.0-1.5 mm.

09/29/03 - first bleeding after a trip to Turkey.

10.27.03 - a gastrotomy was performed with flashing of the BPH of the esophagus and stomach. In the period of the 10th day, raspberry stool. Colonoscopy - angiopathy, vasculitis of the terminal section of the colon and small intestine.

On examination: the liver is moderately enlarged, compacted. Blood flow - 700 ml / min. PV -58%. With endoscopic hypertrophy of the stomach: VRVP and a stomach of 3 degrees. With scintigraphy: liver accumulation - 44%, spleen - 56%. TC-52.1, LC-3.4, AlAt-147, AcAt-180, PV-14.7, APTT-34, fibrinogen 3.0, albumin 53.42, gamma 22.45. Virus C was detected in 2003.

Subsequently, six months later, from February 24, 2004 to March 4, 2004, he was in the RSCH — stem and progenitor cells of the corresponding composition of the biotransplant were transplanted through the splenic artery into the spleen. March 4, 2004 - repeated intravenous administration of a biograft. During 2004, the patient monitored biochemical parameters and blood parameters. There was a tendency towards a decrease in transaminases (up to 2 norms), but an increase in bilirubin. Normal bilirubin levels were achieved by the end of 2004.

05/17/04 - virus C was detected, genotype - 1c, 5.08.04 - virus C was not detected.

Ultrasound: normal sized liver, blood flow - 1000 ml / min, umbilical vein recanalized. Liver scintigraphy from 8/10/04: liver accumulation - 52%, spleen - 48.

On an outpatient examination: 02/09/05 Ultrasound - a liver of normal size.

01/31/2005 virus C was not detected.

Hospitalization January 28, 2005 - February 11, 2005 in a planned manner.

Clinically achieved complete compensation for the disease.

The cell concentration during transplantation averages 2-4 million per kg of patient weight (recipient).

The data from studies on the clinical use of cell biograft in patients with cirrhosis of the liver suggest that using this method of treatment can not only achieve morphofunctional and clinical improvement during the disease, but, most importantly, affect the persistence of the virus in the patient’s body.

The antiviral effect of the allotransplantation of stem and progenitor liver cells of the developed biograft was observed in 20 of 27 patients with the viral origin of the disease, that is, in 74% of patients, which is significantly higher than the potential capabilities of existing antiviral therapy technologies, the effectiveness of which ranges from 20-40%.

It should be emphasized that the ideology of the revealed phenomenon of the antiviral effect of cell therapy is fundamentally different from existing methods of antiviral treatment in patients with hepatitis and cirrhosis.

Existing methods are nothing but chemotherapy methods, with many side effects in many areas of the functioning of the body. The proposed method mobilizes and uses the body's own reserves in the fight against the virus. Important mechanisms in this process are:

- an increase in the concentration of cytokines with a pronounced anti-inflammatory effect and a decrease in the concentration of cytokines with a predominantly pro-inflammatory effect;

- immunomodulating effect of cell transplantation, manifested in the form of an increase in the level of gamma interferon and the ratio between the level of interferon and interleukin-4;

- stimulation of the formation in the body of growth of liver factors that enhance regenerative processes in the liver.

Example 2. B-th B., 66 years old, with a severe decompensated course of liver cirrhosis, which manifested itself as diuretic-resistant ascites, high activity of the process in the liver and various manifestations of chronic liver failure, had, according to virological studies, the average level of hepatitis virus replication " C "(genotype 1b). After 4 sessions of biotransplant administration performed over 1.5 years, markers for hepatitis C virus were not detected, which, along with other effects of cell transplantation, provided significant clinical improvement with the achievement of disease compensation.

Example 3. Patient M., 54 years old, who was in the department with a diagnosis of liver cirrhosis, portal hypertension. A history of endoscopic ligation of varicose veins of the esophagus in connection with an episode of esophageal-gastric bleeding. Examination revealed a viral etiology of the disease: positive markers for hepatitis C virus (genotype 3a) with an average degree of virus replication (30 thousand copies per μl) were identified. 2 sessions of systemic administration of a biograft were performed (total 300 million cells) with an interval of 3 months. During the control virological study, 5 months after the first transplantation session, the virus replication intensity decreased sharply (1 thousand copies per μl), laboratory liver tests and the general condition of the patient improved significantly.

The following is a detailed description of the treatment (using the claimed biotransplant) of liver cirrhosis (CP) and portal hypertension (GP) related to chronic liver diseases.

The main group of patients was 32 patients who underwent transplantation. Of these, 21 (65.6%) patients were males, 11 (34.4%) were females. The age of patients ranged from 17 to 68 years (mean age 40.9 ± 12.47 years). The duration of the disease from the time of diagnosis ranged from 22 years to 1 year and averaged 5.7 ± 4.4 years.

According to the etiological attribute, CP was distributed as follows:

viral etiology was recorded in 19 (59%) patients (hepatitis C virus was detected in 10 patients, hepatitis B virus in 7 patients, of which three had superinfection "D", a combination of viruses "C" and "B" - in 2 patients), chronic alcohol intoxication was the cause of the disease in 5 (16%) patients, in 1 (3%) the patient was diagnosed with autoimmune cirrhosis, in 1 (3%) the patient was diagnosed with primary biliary cirrhosis (PBC), in the rest cases - in 6 (19%) patients it was not possible to establish the cause of the disease.

Characterization of research methods

Before the introduction of the developed biograft containing ASPA, as well as 3, 6, and 12 months after it, all patients underwent a complete clinical examination, including a physical examination and general instrumental and laboratory research methods. In addition, within the described periods, special research methods were performed that allowed a more detailed assessment of the functional state of the liver.

In addition to physical methods, the general clinical examination of patients included the following laboratory tests:

- clinical blood test

- blood chemistry

- blood serology for the presence of: HbsAg, anti-HCV, anti-HIV, Wasserman reaction

- general urine analysis

- chest x-ray

- electrocardiography

- ultrasound examination (ultrasound) of the abdominal cavity

- esophagogastroduodenoscopy (endoscopy)

Special examination methods:

- ultrasound dopplerography of the vessels of the portal system (USDG)

- electroencephalography (EEG)

- liver and spleen scintigraphy

- morphological examination of the liver

- study of the nutritional status of patients and blood aminograms

- angiographic studies

- study of the expression of liver cytokines.

During transplantation, 200 to 500 million mixed biotransplant cells were introduced as a suspension in physiological saline. Patients were given informed consent to perform all transplant related activities.

Technical features of the introduction of biotransplant

The main target organs during transplantation were the liver (20 observations) and the spleen (12 observations). Transplantation into the spleen was performed in the presence of signs of significant compromise of the morphofunctional state of the liver: a small liver, a decrease in blood flow along the hepatic artery and portal vein, a sharply depleted vascular bed in the liver, decompensation of the disease.

The elimination of the threat of gastrointestinal bleeding was one of the important conditions preceding transplantation. It was carried out by any method in accordance with the severity of the course of the disease: surgical (superimposed PKSh-7 patients, gastrotomy with stitching VRVPZh - 8 patients), endoscopic (ligation of VRVPZh - 4 patients, sclerosis of VRVPZh - 3 patients), or a combination thereof.

Transplantation was carried out in two ways - intraoperatively (11 patients) and X-ray endovascular method (20 patients). In one of the observations, the introduction of cells by percutaneous puncture of the spleen was used. However, due to the high risk of developing intra-abdominal bleeding, we later abandoned this method.

Evaluation of the effectiveness of biotransplantation and treatment results in patients with liver cirrhosis and portal hypertension

Assessment of the effectiveness of transplantation was carried out after 3, 6 and 12 months and was based on an analysis of the dynamics of the clinical status and the results of laboratory and instrumental research methods.

The main syndromes of the disease:

- asthenovegetative

- hepatic encephalopathy

- jaundice

- cytolytic

- protein-energy malnutrition (BEN)

- hemorrhagic

- ascitic

Additional research methods:

- separate static scintigraphy of the liver and spleen

- morphological examination of the liver

- a study of liver cytokines

An important criterion for assessing the effectiveness of transplantation was the change in the functional class of the studied patients in the post-transplant period.

There were 3 possible transplantation outcomes: good, satisfactory and unsatisfactory.

- A good transplant result was characterized by an improvement or complete disappearance of the main clinical syndromes of the disease, normalization of laboratory parameters and the achievement of compensation for the disease with the transition of the patient to a higher functional class according to Child-Pugh.

- A satisfactory result was characterized by a decrease in the main clinical manifestations of the disease, an improvement in laboratory parameters and stabilization of the condition of patients without transition to a higher functional class according to Child-Pugh.

- An unsatisfactory result was characterized by the absence of positive changes in the clinical course of the disease and the progression of the severity of the course of liver failure.

The effect of biotransplantation on the course of the main clinical syndromes of the disease in patients with liver cirrhosis and portal hypertension

In the course of the work, the dynamics of key syndromes accompanying the clinical course of CP and PG was monitored. The frequency of clinical syndromes in the studied patients before and after transplantation is presented in table.6.

Table 6 The frequency of clinical syndromes before and after transplantation Syndrome Before transplantation 1-3 months later After 3-6 months After 6-12 months abs. (%) abs. (%) abs. (%) abs. (%) asthenovegetative 26 81 fifteen 47 6 19 8 26 encephalopathy twenty 63 12 38 10 32 eleven 35 icteric 27 84 eighteen 56 eleven 35 13 41 cytolytic 25 78 17 53 four 13 8 26 protein-energy deficiency 26 81 twenty 63 8 26 8 26 hemorrhagic 27 84 21 66 12 39 eighteen 58 ascitic 27 84 21 67 eleven 35 eleven 35 Number of patients n = 32 n = 32 n = 31 n = 31

There is a clearly recorded decrease in the frequency of clinical manifestations of the main syndromes of the disease, the peak of which falls on the period from 3 to 6 months after transplantation and, to some extent, lasting up to 12 months of observation.

The effect of biotransplantation on asthenovegetative syndrome and hepatic encephalopathy

The results of a dynamic study of the clinical manifestations of hepatic encephalopathy are presented in table.7.

Table 7 Dynamics of hepatic encephalopathy Degree of encephalopathy Exodus 3 months 6 months 12 months (n = 32) (n = 32) (n = 31) (n = 31) Without encephalopathy 12 twenty 21 twenty latent 9 8 7 5 I degree 7 3 3 3 II degree four - - 2 III-IV degree - one - one

Improvement of the neurological picture and electroencephalography data was observed in 19 (61%) patients in the period from 3 to 6 months after transplantation. After 6 months, latent encephalopathy was determined in 7 (23%), I degree of encephalopathy - in 3 (10%) patients. Patients with a more severe degree of encephalopathy were not observed.

Dynamics of nutritional and metabolic status

6 months after transplantation, we noted a decrease in the deficiency of somatic protein parameters according to anthropometric measurements from -24.2 ± 8.6 to -2.1 ± 3.4 (p <0.05). After a year, the data of anthropometric measurements remained practically unchanged, and the somatic protein deficit was -2.3 ± 2.2.

In the visceral protein sector, the level of serum albumin before transplantation was 30.6 ± 0.8 g / L. By 3 months, the level of albumin in the blood increased to Z6.6 ± 3.1 g / l, reaching a value of 38.6 ± 2.2 g / l by 6 months.

The effect of biotransplantation on the activity of the process in the liver

After 6 months in the vast majority of patients, the activity of the inflammatory process in the liver decreased - only 4 (13%) patients had a cytolytic syndrome.

The effect of biotransplantation on hemorrhagic syndrome

By the 6th month, there was a significant positive dynamics in laboratory blood parameters characterizing the hemostatic system, which coincided with the clinical symptoms of this syndrome: in 14 (55%) patients, the manifestations of hemorrhagic syndrome completely disappeared.

The effect of biotransplantation on ascites syndrome

The dynamics of ascites syndrome is presented in figure 4.

Ascitic syndrome of varying severity was noted in 27 (84%) patients: in 5 (17%) patients, according to ultrasound, there was a slight ascites determined in the flat areas of the abdominal cavity, in 11 (34%) - moderate ascites that did not go beyond the lower floor of the abdominal cavity, and in 11 (34%) patients, pronounced diuretic-resistant ascites was observed, occupying more than two floors of the abdominal cavity. By the 6th month, severe ascites was observed in only 2 (6.4%) patients. In 3 patients with initially expressed ascites, moderate ascites was noted, in 3 - insignificant. In 3 patients, ascites completely disappeared. It should be noted that the regression of ascites syndrome in 2 patients occurred as a result of a combination of transplantation with the application of porto-caval vascular anastomoses.

In patients with moderate and mild ascites, regression of ascites syndrome was also noted. Thus, 6 months after transplantation, according to the severity of ascites syndrome, the patients were distributed as follows: with severe ascites, 2 (6.4%) patients remained, with moderate ascites, 4 (13%) and with insignificant, 5 (16%). In 15 (48%) patients, ascites completely disappeared.

Dynamics of biochemical parameters

Table 8 presents the results of biochemical studies that were carried out to assess the functional state of the liver before and at various periods after transplantation.

Table 8. Biochemical indicators of the functional state of the liver before and at various periods after transplantation Indicators before transplantation n = 32 after 3 months n = 32 after 6 months n = 31 after 12 months n = 31 Albumin (g / l) 30.7 ± 0.9 36.6 ± 3.1 * 38.8 ± 1.2 * 34.4 ± 2.1 Total bilirubin (μmol / L) 55, ± 14.3 33.1 ± 9.8 * 22.7 ± 7.4 * 32.3 ± 9.1 * AsAT (UNIT) 118.5 ± 14.6 54.3 ± 13.4 * 46.2 ± 6.4 * 48.4 ± 4.3 * AlAT (ED) 126.4 ± 15.3 52.3 ± 12.2 * 46.6 ± 10.2 * 54.8 ± 8.3 * PI (%) 63.4 ± 11.1 72.6 ± 6.4 80.6 ± 8.4 * 79.2 ± 6.4 * PV (sec) 16.4 ± 0.9 14.8 ± 0.4 13.2 ± 0.3 * 13.8 ± 0.6 * * - significance of differences compared with the initial data p <0.05

In relation to samples characterizing the functional state of the liver, a clear pattern was revealed, which consisted in the fact that after transplantation in the next six months there is a maximum improvement in basic biochemical parameters, which after a year of observation somewhat worsened, not reaching, as a rule, the initial level. At the same time, 50% of patients maintained a steady improvement in these indicators.

Dynamics of liver scintigraphy parameters

According to hepatoscintigraphy, by the sixth month, an increase in the accumulation of radiopharmaceuticals in the liver was observed in 23 patients out of 27 patients on average from 48.8 ± 11.1% to 56.9 ± 10.8% (p> 0.05). While maintaining pronounced uneven distribution, attention was drawn to increased intensity and expansion of the drug accumulation fields in the liver.

A decrease in the intensity of drug accumulation from 46.1 ± 6.1% to 42.8 ± 6.1% on average (p> 0.05) was observed in 4 patients. In 2 of them, these changes progressed, which was associated with the developed "stealing syndrome" of the liver due to the complete reduction of portal blood flow.

According to our observations, difficulties in interpreting the results of liver scintigraphy arise after performing shunt operations, as well as in patients with initial pronounced splenomegaly and a very high accumulation of the drug in the spleen.

Nevertheless, in a number of patients, changes in the parameters of liver scintigraphy were quite obvious and obvious, well correlated with clinical data. As an example, scan scintigrams of a patient S. 37 years old (Figs. 5 and 6) and a patient O. 51 years old (Figs. 7 and 8) performed before and 6 months after transplantation can be presented.

Morphological changes in the liver after transplant biotransplant

A morphological substrate of the positive results of transplantation in patients with CP was a decrease in the total activity of the cirrhotic process in the liver. The diagram (Fig. 7) shows the distribution of patients according to the degree of total activity of the process in the liver based on morphological data at the end and 6 months after transplantation.

As can be seen from the diagram (Fig. 7), low overall activity in the outcome was observed in 1 patient, moderate in 5 and pronounced in 5 patients. 6 months after transplantation, a low overall activity of the cirrhotic process was observed in 6, moderate in 3 and pronounced in 2 patients.

A histological examination 6 months after transplantation showed positive dynamics in 8 patients. It manifested itself in a significant decrease in lymphoid cell and inflammatory infiltration, in a decrease in the degree of fibrosis and hepatocyte dystrophy. On Fig and 9 presents microhistophotograms of the liver of patient O., 51 years old, performed before and 6 months after transplantation, while it was noted:

- marked activity of the cirrhotic process (lymphomacrophagous infiltration of the stroma and parenchyma, fatty and protein dystrophy of hepatocytes), a complete violation of the beam structure of the organ in the field of view;

- the activity of the cirrhotic process significantly decreased (weak lymphoid infiltration of the stroma). In the observed field of view, the beam structure is traced.

Simultaneously with a decrease in the activity of the process in the liver, a decrease in dystrophic changes in hepatocytes, positive changes occurred that characterize the functional state of hepatocytes. In particular, there was an increase in the accumulation of glycogen in the liver, which can be clearly seen on the microhistophotograms of patient Sh., 39 years old, performed during surgery (outcome) and 6 months after transplantation (Figs. 10 and 11):

- marked activity of the cirrhotic process (lymphomacrophagous infiltration of the stroma and parenchyma, extensive areas of hepatocyte necrosis), a complete violation of the beam structure of the organ in the field of view;

- low activity of the cirrhotic process. In the observed field of view, a beam structure is traced. The accumulation of glycogen uniformly increased.

Biograft and degree of liver failure compensation

Changes in clinical and laboratory parameters were accompanied by the transition of patients from one functional class to another, which was the most important criterion for the effectiveness of transplantation. The distribution of patients according to Child-Pugh at different periods after transplantation is presented in Fig. 12.

The data presented indicate that transplantation is an effective method of compensating for chronic heart failure and should be used as an important component of the comprehensive preoperative preparation of patients with CP and PG.

The role of biograft in the postoperative rehabilitation of patients with liver cirrhosis and portal hypertension

The effect of the developed biograft on the immediate postoperative period

The early postoperative period in all 15 patients undergoing ASPC proceeded smoothly, and we did not observe any complications associated with the operation. In the control group, 5 (31%) patients had various complications.

The characteristics of postoperative complications of the studied groups are presented in table.9.

Table 9 Characterization of postoperative complications Early p / o complications Patient groups Operation + ASPK control Bleeding from ARVI - one Acute Hepatic Cell Failure - one Large ascites loss - 2 Hypocoagulation - one Acute Hepatic Encephalopathy - one Suppuration p / o wounds - one Total amount - 7

On average, the duration of treatment since hospitalization in the group of operated patients with ASPK was 26 ± 6 days, in the control group - 32 ± 11 days (p> 0.05). At the same time, the terms of inpatient treatment in the postoperative period in the group of patients with ASA and the control group were 15 ± 2 and 20 ± 6 (p> 0.05) days, respectively.

The influence of the developed biograft on the course of the remote postoperative period

Within 3 years of observation in the group of operated patients with simultaneous cell transplantation, two cases of fatal outcomes were recorded. In the control group, 6 patients died during the same observation period.

Summarizing the data presented, the following conclusions should be made.

1. Transplantation of stem and progenitor liver cells of the developed biograft is a promising method of compensating for chronic hepatocellular insufficiency, contributing to the regression of the main syndromes of the disease, changing the functional class of patients with CP, and the effectiveness of its clinical effect depends on the initial reserve potential of the recipient's liver.

2. Transplantation of stem and progenitor cells of the developed biograft helps to improve the morphofunctional state of the liver by reducing the activity of the process, reducing the degree of degenerative-dystrophic changes in hepatocytes, restoring functional liver indices, and therefore can be recommended as an important component in the complex of therapeutic measures for preoperative preparation patients with cirrhosis and portal hypertension.

3. Transplantation of stem and progenitor cells of the developed biograft, helping to reduce the frequency of complications, improves the course of the early postoperative period and the immediate results of surgical treatment of patients with liver cirrhosis and portal hypertension.

4. Influencing the survival of patients, stem and progenitor cell transplantation of the developed biograft is an effective method of postoperative rehabilitation of patients with liver cirrhosis and portal hypertension, which improves the prognosis of the course of the disease. Cell therapy can be considered as an independent method of treating patients with CP and PG.

Claims (3)

1. A biograft for the treatment of chronic diffuse liver diseases, containing a mixed population of cells, in which 40% are stem cells, including stromal mesenchymal and hepatic bipotent stem cells, and the remaining 60% are progenitor cells at various stages of differentiation, including young immature hepatoblasts and cells erythroid precursors, hematopoietic cells, while a mixed population of biograft cells is characterized by the expression of the following surface markers: CD13, CD29, CD44, C D90, CD117 (c-Kit), lack of expression of CD34. 2. A method for the treatment of chronic diffuse liver diseases, characterized in that the biograft according to claim 1 is used, which is administered as a suspension in physiological saline, wherein the number of introduced cells is 2-4 million cells per 1 kg of patient weight. 3. A method for the treatment of liver cirrhosis and portal hypertension, characterized in that they use the biograft according to claim 1 in the form of a suspension in physiological solution, while the total number of biotransplant cells is from 350 to 500 million

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