WO2007091919A1 - Method for treating chronic illnesses (variants), method for producing a biograft (variants) and a biograft (variants) - Google Patents

Abstract

The invention relates to medicine and can be used for treating chronic illnesses. The aim of said invention is to increase the regeneration potential of autologous mononuclear cells, thereby increasing the efficiency of treatment of a large range of chronic illnesses. The inventive method for treating chronic illnesses consists in sampling a patient cell material for subsequently separating the autologous mononuclear cells therefrom, cultivating the mononuclear autologic cells for obtaining a biograf and subsequently transplanting said biograf to the patient, wherein the cultivation is carried out by adding a tissue-specific antigen which corresponds to the injured member or to the separated autologous mononuclear cells, prior to the cultivation, lymphoid and/or dendrite cells, which are pre-cultivated by addition of the tissue-specific antigen corresponding the injured member, are added. The methods for obtaining the biograft and the biograft obtained by said method are also disclosed.

Description

 A method for the treatment of chronic diseases (options), a method for producing a biograft (options), a biograft (options).

State of the art

The invention relates to medicine and can be used to treat chronic diseases.

Known methods for treating heart disease (Yu.L. Shevchenko, “Cellular technologies in cardiology)), Vestnik RAMS, N ° ll, 2003r, p. 6-10), based on the suspension of fetal cells from abortive donor material containing stem and progenitor cells, and their introduction into the myocardium. The disadvantages of this analogue are:

1. The illegitimacy of the use of fetal material, which cannot be used in a clinic in patients.

2. The risk of infection of the recipient with unrecognized infections contained in the donor material.

3. Fetal cells are allogeneic material, the effectiveness of which is gradually reduced due to immune rejection.

Another analogue is known (BeI A., Mess. E., Agbulut O., et al. Transplantation of autologous frès frope maropowt muosardium: A ward of cautiop. // Sirculiop. - 2003.-24. ), proposed for the treatment of heart disease by cell therapy. This method is based on obtaining an autologous freshly isolated bone marrow containing hematopoietic and stromal cells and returning them to the myocardium or coronary bed. The advantage of this method over the well-known one is the absence of legal and ethical problems; no risk of introducing infection into the recipient's body.

The disadvantages of this analogue, which reduce the effectiveness of restoration processes in a damaged organ with the help of cell therapy include:

1. The development of bone tissue in the area of cell introduction, due to the differentiation of stromal cells along the osteogenic pathway (BeI A., Mess. E., Agbulut O., et al. Transplantation of autologous frèsh fropom ipofartud muosardium: autopodium. // Sirculatiop. - 2003. - 108. - P. P-247)

2. The initially reduced regenerative potential of cell suspension, because the source of the cells is the patient himself, whose regenerative potential (including bone marrow cells) is reduced due to the disease.

3. The absence or a sharp decrease in tissue suspension of tissue-specific cell clones (lymphoid) that contribute to the regeneration of the corresponding chronically damaged organ, and this drawback, to a still greater extent reduces the regeneration potential of the used cell suspension, and thereby significantly reduces the effectiveness of the method.

The closest analogue of the claimed invention is the work carried out at the Institute of Transplantology (V.I.Shumakov et al., Abstracts at the 11th All-Russian Congress of Cardiovascular Surgeons, Moscow, October 23-26, 2005, p. 296; Abstract A. Bersenev’s PhD thesis, “Transplantation of embryonic liver cells and bone marrow stem cells for the correction of dyslipidemia and early stages of atherogenesis”, Moscow, 2003), in which for the treatment of chronic diseases of internal of organs, cultured autologous mononuclear cells containing stem and progenitor cells obtained from the patient’s bone marrow are used.

This method eliminates the possibility of bone tissue appearing in the cell injection zone due to cultivation. However, the use of bone marrow cells taken from patients with chronic diseases does not significantly increase the effectiveness of the method, because the suspension used consists of cells with a reduced regenerative potential and almost does not contain cells having a tissue-specific orientation of the regenerative effect on the corresponding damaged organ. The latter is due to the fact that in case of chronic organ damage in the blood and bone marrow, the content of tissue-specific cell clones is reduced due to the total decrease in it of viable functioning tissue capable of producing tissue-specific clones. This explains why the effectiveness of the method used does not exceed 60-70% (V.I. Shumakov et al. VestRAMN 2004, Ns9, p. 44-47).

The claimed inventions are aimed at eliminating the above disadvantages and achieving a technical result, which consists in increasing the regeneration potential of autologous mononuclear cells, which in turn provides an increase in the effectiveness of treatment of a wide range of chronic diseases.

The technical result is achieved due to the fact that in a method for the treatment of chronic diseases, including the collection of cellular material from a patient with subsequent isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells to obtain biograft and subsequent transplantation of the latter to the patient, cultivation is carried out with the addition of tissue-specific antigen corresponding to the damaged organ.

And also by the fact that in a method of treating chronic diseases, including collecting cellular material from a patient with subsequent isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells to produce a biotransplant and subsequent transplantation of the latter to the patient, add mononuclear autologous cells to the isolated cells before culturing lymphoid and / or dendritic cells, previously cultured with the addition of tissue-specific antigen, The appropriate organ damage.

And also due to the fact that in the method of treating chronic diseases previously, before the collection of cellular material, the patient has immunocorrection.

And also due to the fact that in the method of treating chronic diseases for immunocorrection, the stimulation index is determined as the ratio of the total production of reactive oxygen species by blood cells treated with a cAMP-phosphodiesterase inhibitor to the total production of reactive oxygen species by intact cells.

And also due to the fact that in the method for producing a biograft, which includes taking cellular material from a patient with subsequent isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells in a growth medium in a growth medium at a tissue-specific antigen corresponding to the damaged organ is added to the culture.

And also due to the fact that in the method for producing a biograft, including sampling cellular material from a patient with subsequent isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells in a growth medium, lymphoid and / or are added to the isolated mononuclear autologous cells before cultivation dendritic cells, previously cultured with the addition of tissue-specific antigen corresponding to the damaged organ.

And also due to the fact that the biograft, which includes isolated mononuclear autologous cells, contains stem cells that were previously cultured with a tissue-specific antigen corresponding to the damaged organ.

And also due to the fact that the biograft, including the isolated autologous mononuclear cells, contains lymphoid and / or dendritic cells, previously cultured with the addition of tissue-specific antigen corresponding to the damaged organ.

The claimed methods of treating chronic diseases eliminate the above disadvantages of analogues due to the fact that even before the collection of autologous mononuclear cells (AMK) in a patient, the immune status is examined and immunocorrection is carried out under the control of the “blood cell stimulation index”, which can significantly increase the regenerative potential of these cells. To ensure the formation of tissue of a specific orientation the regenerative potential of cells intended for cell therapy, at the stage of their cultivation, a tissue-specific antigen identical to the organ whose regeneration must be restored in the patient’s body is introduced into the growth medium.

As a result of using the claimed inventions, a quick and almost one hundred percent improvement in the functioning of damaged organs occurs during their chronic damage (for example, myocardium).

The invention is illustrated by the following illustrations. FIG. 1.- graph for calculating the stimulation index.

FIG. 2. - a graph for predicting a favorable result of the introduction of autologous cells to a patient with a stimulation index> 1.0. FIG. 3. - graph for predicting an unfavorable result of the introduction of autologous cells to the patient with a stimulation index <1.0.

FIG. 4. - graph for predicting an unfavorable result of the introduction of KKM of patient M with a stimulation index <1.0.

FIG. 5. - Schedule of individual selection of immunocorrectors by chemiluminescence.

FIG. 6. - graph for predicting a favorable outcome for the administration of AMK to patient M with a stimulation index> 1.0.

FIG. 7. - the dynamics of changes in the cellular composition of AMK depending on cultivation conditions (without and by the addition of TA).

FIG. 8. - quantitative assessment of regional changes in perfusion according to standard projections of static myocardial scintigraphy with Tc-99m in patient JI. before and after 1 month. after administration of AMK (cultivation with

TA). SUMMARY OF THE INVENTION

The proposed method includes: immunological examination of patients and immunocorrection under the control of “stimulation index)) of mononuclear cells from the blood of patients (normalization of which indicates the restoration of immunological homeostasis); autologous cell collection (bone marrow, blood); isolating from them fractions of nucleus-containing cells, culturing cells in a growth medium in the presence of a tissue-specific antigen (TA) identical to the damaged organ for 1-15 days and transplanting the prepared cell culture into a patient with a chronic disease of internal organs.

Evaluation of the immune status of patients was carried out by suspension of nucleated cells from the blood before and after immunocorrection.

Evaluation of the stimulation index of cells of the immune system includes the following steps:

To obtain nucleated cells, 2 ml of heparinized blood was mixed with 1 ml of a 3% gelatin solution heated to 37 ^° C and incubated in an incubator at 37 ^° C for 30 minutes. After erythrocyte sedimentation, the plasma layer enriched in leukocytes was taken into plastic tubes and washed with a 10-fold volume of phosphate-saline buffer (pH = 7.4). Then the leukocytes by centrifugation for 10 min at 1000 rpm were resuspended in a Hanks solution without Ca ²⁺ and Mg ²⁺ . After counting the number of nucleated cells, the concentration of the latter was brought to 10 cells / ml.

The determination of the stimulation index was carried out as follows.

In the 2nd cuvette, the control and experimental cells were introduced with 40 * 10 ³ cells. An interferon inducer containing a cAMP-phosphodiesterase inhibitor, a derivative of acridonoacetic acid (10-carboxymethyl-9-acridanone) and N-methylglucamine was additionally added to the experimental cuvette. Hanks solution was added to the control cuvette. The control and experimental cuvettes were incubated in a thermostat for 60 min at 37 ^° C. After cells were incubated, the chemiluminescent cell response curves were recorded in the control and experimental cuvettes using a Xelum-2001 01/01 chemiluminometer (Russia) in combination with an IBM computer -PC at the temperature of the counting chamber 37 ⁰ C ₅ for 15 minutes Luminol (5-amino-2,3-dihydro-l, 4-phthalazinedione, Sigma) at a final concentration of 10 ³ M was used as a luminescence enhancer, and PMA (forbol-12- myristate-13-acetate, Sigma) at a concentration of 10 ^" M.

Then, the total production of reactive oxygen species (ROS) was calculated as the area under the chemiluminescent reaction curve. The stimulation index (IS) of the drug used was calculated as the ratio of the area of the chemiluminescent reaction in the control and experiment (Fig. 1).

Stimulation index <1.0 predicts an unfavorable result of the introduction of autologous cells in this patient. A stimulation index> 1.0 predicts a favorable result of the introduction of autologous cells to a given patient (Fig. 2), a stimulation index <1.0 predicts an unfavorable result of the introduction of autologous cells to a given patient (Fig. 3).

Then the patient is immunocorrection with one of the following drugs used in therapeutic doses: Polyoxidonium Imunofan

Neovir Splenopid Reaferon Roferon Roncoleukin T-activin Timogen Cycloferon

The course of immunocorrection with a stimulation index below 1.0 is 14 days. To control the effectiveness of immunocorrection in patients with an initial stimulation index less than 1.0, a re-determination of the stimulation index is carried out. Cellular material is taken in patients only with a stimulation index greater than 1.0.

Obtaining autologous mononuclear cells (AMK) containing stem and progenitor cells from bone marrow (or peripheral blood) for transplantation to a patient is as follows.

For bone marrow collection, access from the posterior iliac crest is used. Bone marrow is taken under operating conditions, subject to the same aseptic rules, as with other surgical interventions. Pain sensations during bone marrow harvesting are mainly associated with a high sensitivity of the periosteum when piercing with a needle and require adequate pain relief. The choice between general, spinal, epidural or local anesthesia for analgesia depends on both the medical condition and the preferences of the donor and anesthetist. In 95% of cases, adequate analgesia by local anesthesia is possible. The donor is hospitalized in the morning on the eve of the fence and, in the absence of complications, can be discharged in the afternoon of the fence with the appointment of analgesics, recommendations for changing the dressing at the injection site.

After treating the skin with iodine-containing solutions in the area of the posterior crests, a puncture of the skin and subcutaneous fat is made, through which needles for aspiration are inserted. After that, the cortical plate of the iliac crest is pierced and the bone marrow is aspirated from the spongy bone. For the collection of 50-150 ml of bone marrow, it is necessary to make several punctures of the cortical plate of the bone, for this the skin and subcutaneous fat are moved with an aspiration needle. Classical technology requires aspirating the bone marrow from each injection in small portions (3-5) ml into a 20-ml syringe. After filling the syringe, the doctor disconnects it from the needle and passes it to the nurse, who transfers the contents of the syringe into a polymer container with an anticoagulant.

After the end of the fence, a bandage is applied in the area of skin puncture, and the CM is immediately sent to the laboratory for further processing in a special container with an anticoagulant at + 4C °. The volume of KM taken should be 150-200 ml.

An aspirate of bone marrow suspension of cells is placed in 50 ml tubes and centrifuged. Plasma and interphase with nucleated cells are collected and centrifuged again. Plasma is removed, and a lysing solution is added to the resulting pellet to eliminate red blood cells. The tube is closed and shaken vigorously, then centrifuged. The precipitate was resuspended in DMEM growth medium containing fetal serum, insulin, gentamicin and a tissue-specific antigen (for obtaining, see below). Cells are plated on Petri dishes and cultured at 37 ° C in an atmosphere containing 5% CO ₂ and at 95% humidity for 1-15 days. The biograft is the remaining mononuclear cells after cultivation and centrifugation at 1500 rpm for 5 minutes Hanks solution (5 ml) was added to the resulting pellet and transplanted to the recipient.

Obtaining another version of the biograft is as follows.

A leukocyte fraction of blood cells containing lymphocytes and monocytes is isolated from the patient’s blood, which is then cultured with a tissue-specific antigen. After cultivation, the leukocyte fraction containing lymphocytes and monocytes is washed from the tissue-specific antigen by centrifugation. The obtained leukocyte cells are added to the isolated autologous mononuclear cells containing stem cells and cultured at 37 ^° C. in an atmosphere containing 5% CO ₂ and at 95% humidity for 1-15 days. The remaining mononuclear cells after cultivation were centrifuged at 1500 rpm for 5 min, Hanks solution (5 ml) was added and transplanted to the recipient.

To obtain tissue-specific antigen, freshly isolated organ tissue identical to the damaged one was taken. Allogeneic or xenogenic tissue can be used as a source, and biopsy material can be used if possible.

The fabric is mechanically homogenized into fragments of not more than 0.5 mm ³ . The resulting homogenate is poured with a Hanks solution 1: 1 and placed in the freezer for 1 hour, then the fabric is thawed, this cycle is repeated 3 times. Then the homogenate is centrifuged at 2000 rpm for 10 minutes. The supernatant is taken and filtered through a filter with a pore diameter of 0.22 μm to ensure sterility of the solution. The protein concentration in the solution is determined by the Lowry method and should be in the range from 0.5 to 1 mg / ml.

The present invention is illustrated by a description of specific, but not exhaustive, examples of implementation.

The results of treatment of 80 patients who underwent transplantation of autologous mononuclear cells in the treatment of cardiovascular pathology were analyzed.

Recipients of autologous mononuclear cells were divided into 2 groups, depending on the initial functional activity of autologous mononuclear cells: a group of patients with IS> 1.0 (n = 40) and a group of patients with IS <1.0 (n = 15). The groups of patients did not differ from each other in medians of such parameters as age, duration of heart failure, and the number of myocardial infarctions. The parameters of intracardiac hemodynamics were determined in these patients before and after transplantation of autologous mononuclear cells: the final systolic volume of the left ventricle (KCO JDK), the final diastolic volume of the left ventricle (KDO JDK), the final systolic size of the left ventricle (KCP JDK), the final diastolic size of the left ventricle (JDK CDD), left ventricular expulsion fraction (PI JDK), and the growth of these indicators was calculated, which were designated as (Δ).

Statistically significant differences were found in indicators such as delta reduction in BWW JDK, KCO JDK, KCP JDK, KDR LV and FI after 1 month. (tab. 1) and 6 months. (tab. 2) after cell therapy according to echocardiography.

Table 1 Comparison of the parameters of intracardiac hemodynamics in patients with IC <1.0 and IO1.0 1 month after cell therapy (according to echocardiography).

"" -reliable with respect to the group with IC <1.0 (p <0.05)

Table 2 Comparison of the parameters of intracardiac hemodynamics in patients with IC <1.0 and IC> 1.0 6 months after cell therapy (according to echocardiography).

^ -reliable in relation to the group with IC <1.0 (p <0.05) As can be seen from table I ₅ in the group where IP> l, 0, after a month there is a significantly pronounced favorable decrease in BWW, KCO, KDR and KCP and an increase in FI compared with the group of recipients in which IP <l, 0. These data indicated a positive change in hemodynamics with a group with IC> 1.0 and the absence of a positive clinical effect when using cells with IC <1.0.

From table 2 it can be seen that after 6 months of observation, the noted positive trends when using cells with IC> 1.0 not only persist, but according to some indicators (KCO, KCP, FI) become more pronounced.

Thus, a comparative analysis showed that the clinical effect of cell transplantation directly depends on the initial value of the index of stimulation index.

Carrying out immunocorrection, before harvesting the bone marrow, it was possible to increase the effectiveness of the conducted cell therapy, because in all patients initially, before AMK, IP became> 1.0. The group without immunocorrection included patients with IP> l, 0 and IP <1.0. As can be seen from table 3 in the group of patients who underwent immunocorrection before taking AMK 6 months after transplantation, the clinical effect was significantly more pronounced compared with the group of patients without immunocorrection. Table 3. Comparison of the parameters of intracardiac hemodynamics in patients without and with preliminary immunocorrection 6 months after cell therapy (according to echocardiography).

* -reliable in relation to the group without immunocorrection (p <0.05)

Thus, preliminary immunocorrection increases the functional activity of AMK and significantly positively affects the processes of LV remodeling in patients with CH.

The need to add TA was studied in experiments with modeling dyslipidemic liver damage in guinea pigs and cryogenic damage to rat myocardium.

1. In guinea pigs with chronic dyslipidemia, fatty degeneration of the liver develops within 4 months and the morphometric characteristics of hepatocytes are sharply violated (table 4). To accelerate the recovery processes in the liver, a cell was performed using AMK cultured with and without the addition of TA. Tissue-specific antigen was obtained from the liver of healthy guinea pigs using the technology described above. TA was added to the bone marrow mononuclear cell culture and cultured on average for 7 days (1 to 15 days). Then the cells were washed with Hanks solution and transplanted into animals. As controls used animals that transplanted autologous bone marrow cultured without TA (control 2) and animals that were injected with saline (Control 1).

Table 4. The effect of transplantation of AMK and AMK cultured with TA on the regeneration of hepatocytes and damaged liver.

QпιяАuαif ШШМ ПМР.Т As can be seen from the data presented, transplantation of mononuclear stem cells increased the regenerative capacity of hepatocytes in case of fatty degeneration of the liver. However, this effect was not sufficiently pronounced. Co-cultivation of autologous mononuclear cells with TA dramatically increases the effect of cell transplantation and leads to a significant increase in the hepatocyte regeneration processes in the liver of experimental animals.

2. On the model of myocardial damage by rat cryodestruction (CD). On day 30, rounded scar tissue with a diameter of 6-7 mm was formed in the JDK damage zone. from the side of the epicardium and 3 mm. from the endocardium; LV wall thickness in the center of the scar did not exceed 1 mm. As can be seen from table 7, the decrease in the relative mass of the left ventricle (per unit mass) after cryodestruction was approximately 13%, and for the free wall of the left ventricle this indicator was 17%.

The area of necrosis after cryodestruction with respect to the total area of the left LV wall was 25.1 ± 3.6% on the epicardial side and 23.0 ± 4.2% on the endocardial side. The area of transmural necrosis after cryodestruction with respect to the total area of the free wall of the LV was 11.4 ± 0.7% from the side of the epicardium and 6.4 ± 2.5% from the side of the endocardium.

Thus, the morphometry of macro preparations of the damaged heart made it possible to establish a significant decrease in LV muscle mass, which would inevitably lead to a decrease in its contractile activity.

Tissue-specific antigen (TA) was obtained from the heart of intact rats according to the technology described above. TA was added to the culture mononuclear cells of the bone marrow and cultivation was carried out on average for 7 days (from 3 to 15 days). Then the cells were washed with Hanks solution and transplanted into animals. As a control, we used animals that were transplanted with autologous bone marrow cultured without TA, and animals that were injected with saline.

When autologous mononuclear cells cultured for 3-10 days were introduced into the myocardium in animals with CD, we observed an increase in myocardial mass, an improvement in the contractility of the heart, however, in none of the experiments we obtained a complete restoration of the heart muscle. In the group of animals with CD, which were injected with AMK, cultured with TA, we noted significantly more complete regeneration of the damaged organ. It should be noted that the mass of the left ventricle in rats with CD administered with AMA + TA did not significantly differ from the myocardial mass of intact animals (table 5).

Table 5. Comparison of the mass of intact hearts and hearts after CD without and with the introduction of AMK and AMK + TA.

Mass Free wall mass LV mass (mg) / animal mass, LV, (mg) / animal mass (g) g animal (g)

Intact animals

356.7 ± 5.8 1.70Sh.031 2.298 ± 0.062

Animals after cryodestruction and the introduction of physical. solution

366.7 ± 28.9 1.395 ± 0.125 * 2.004 ± 0.066 *

Animals <g cryodestruction after administration of AMK

360.0 ± 20.5 1.450 ± 0.O8 * 2.120 ± 0.02 *

Animals with v cryodestruction after administration of AMK cultivated with TA

352 .3 ± 12 .2 1.650 ± 0.02 + 2.188 ± 0 .08+

• * - significant in relation to the group of intact animals, p <0.05

• + - reliably in relation to the group with AMK ₅ p <0.05. Our experiments on animals demonstrated the feasibility of pre-culturing AMK with TA, which increased the efficiency of cell therapy.

An example implementation of the proposed method of treatment. A clinical example of the efficiency of transplantation of autologous mononuclear cells KM

Patient L., 67 years old, was admitted to the department of coronary surgery and heart transplantation with complaints of burning pains behind the sternum with minimal physical exertion, rarely at rest; shortness of breath.

From the anamnesis: anginal history of more than 3 years. Deterioration since July 2005, when an acute non-Q-forming MI was diagnosed in the area of PS JDK. More than 10 years suffering from arterial hypertension. Received for coronary angiography and determine further treatment tactics.

Concomitant diseases: GB 2 tbsp. Type 2 diabetes mellitus, moderate to severe. Condition after cholecystectomy (February 2005). ICD. Erosive gastritis.

Allergic history - not burdened.

At admission: general condition of moderate severity, increased nutrition, no edema. There is no noise over the common carotid and femoral arteries. In the lungs, harsh breathing is carried out in all departments, no wheezing, NPV-16 in 1 min. The borders of the heart are extended to the left, apical push spilled. Heart sounds are muffled, rhythm is correct, accent

2nd tone on JIA, heart rate - 68 in 1 min, blood pressure - 140/80 mm RT. Art. The abdomen on palpation is soft, b / w, the liver is not palpable. There is no dysuria, the shocking symptom is negative on both sides. No swelling. CNS - without gross pathology.

Examination in the department: data from general clinical blood and urine tests within normal limits. With R-graph organs gr. class pathology is not revealed.

ANALYSIS ON HIV, PB, HEPATITIS B and C are negative.

ECG: sinus rhythm, HCC = 69. Diffuse decrease in the amplitude of the tooth "T".

ECHOKG: Ao-3,4, LP-4,2, PZh-3,1, LV: KDP-4,6, KDO-95, KCP-3,1, KCO-36, UO-59, FI-62% . LV volumetric characteristics are not increased. Local contractility is not impaired. The valve apparatus is intact. DLA 12 mmHg Art.

KG: Type of coronary blood supply: right type. LEFT CORONARY ARTERY (LCA): Marked diffuse changes in the form of uneven contours. LCA trunk: grade 3 stenosis at the mouth with a clearance of about 2 mm. Anterior ventricular branch (LAD): extended stenosis of 2-3 degrees in the proximal third, subocclusion in the middle third, stenosis of 3 degrees on the border of the middle and distal third, two consecutive stenosis of 1-2 degrees in the distal third. Diagonal branch (DV): grade 3 stenosis at the mouth. Envelope branch (OB): long stenosis 2–3 degrees from the mouth. RIGHT CORONARY ARTERY (SC A): Two consecutive stenosis of 3 degrees in the middle third, stenosis of 1 degree in the distal third.

Based on the examination data, the patient was diagnosed with coronary heart disease: unstable (progressive) angina pectoris. PEAKS. HK 2a. GB 2 tbsp. Type 2 diabetes mellitus, moderate to severe. Chronic erosive gastritis without exacerbation.

Due to the severity of coronary artery disease and the clinic of unstable angina, the patient, according to vital signs, underwent surgery of aortocoronary bypass surgery of the anterior interventricular branch, diagonal branch and right coronary artery under conditions of cardiopulmonary bypass and pharmacological cold cardioplegia. During the operation, a biopsy of the ear of the right atrium was taken before the IR sectional method.

The early postoperative period was uneventful. Extubation in the operating room. The patient was transferred from the intensive care unit to the department the next day. In the department, paroxysms of MA were noted (December 3, 2005 and December 23, 2005), hemodynamically insignificant, stopped by potassium preparations and cordarone. Due to the low hemoglobin level (up to 7.8 g / dl) in the postoperative period, two eruptions were performed. masses (December 3, 2005 and December 5, 2005).

The patient was included in the scientific-clinical program for the transplantation of autologous stem cells of the bone marrow into the myocardium. 12/16/2005, bone marrow was collected with further separation of KKM. Autologous mononuclear cells were cultured for 5 days with tissue-specific antigen obtained from biopsy material during surgery. 12/22/2005 their intracoronary and systemic (descending aortic section) administration was performed. No complications.

During the control coronary-shuntography: in the place of subocclusion of the permanent pancreas, artery occlusion was recorded. We pass the shunt to the LAD, the distal and proximal sections of the LAD are well contrasted. The shunt in the Far East is well traversed, the entire LCA system is filled through the shunt, with the exception of the MAD, which is occluded in the proximal third. Shunt to PKA pass well, filling the entire PKA and a large branch of the sharp edge.

In the ward, the condition remained stable. On a series of control ECGs and echocardiography without negative dynamics. Postoperative wounds healed by primary intention, intradermal sutures.

The blood glucose level for the entire observation period is from 5.1 to 8.9 (12/26/2005: 9-7.5, 12-7.9, 15-8.1, 18-6.7, 21-7.5) . Total cholesterol is 7.1. GB -9.3. In the remaining analyzes without features.

In satisfactory condition, the patient is discharged under the supervision of a cardiologist at the place of residence.

The diagnosis is final: coronary heart disease: condition after AKHI permanent residence, DV and PKA from 30.11.2005. PEAKS. HK 1. GB 2 tbsp. condition after transplantation of autologous bone marrow stem cells from 12.22.2005. Type 2 diabetes mellitus, moderate to severe. Chronic erosive gastritis without exacerbation.

Before the fence was determined by the stimulation index, which was less than 1.0 (figure 4)

To increase the clinical effect of the patient, an individual selection of immunocorrectors was performed (Fig. 5). From the presented figure it can be seen that such a drug was POLYOXIDOID.

This drug was prescribed to the patient according to the scheme: 7 injections, 1st day 12 mg IM, then every other day 6 mg IM. At the end of the course, an analysis was repeated to determine the stimulation index (Fig.6), which showed the restoration of the IS to normal values. Then, the patient underwent bone marrow sampling, AMK were isolated, and their co-cultivation was performed with TA obtained from biopsy material. When culturing AMK with TA addition for 5 days, the phenotypic composition of cultured cells changes as compared with the outcome and those cells that were cultured without TA (Fig. 7). As can be seen from the presented figure, in the AMK culture, after the addition of antigen, there is an increase in the proportion of T-lymphocytes of ₅ especially T-helpers (CD4), which are served by literature (A.G. Babayeva, Separative processes and immunity)), Izvestia AN, Biological Series )) 1999, N ° 3, pp. 261-269), determine the effectiveness of regeneration processes in a damaged organ.

A follow-up examination 1 month after cell transplantation showed a pronounced increase in hemodynamics (ΔKDO -10%, ΔKSO -8.5%, ΔFI 12%) and myocardial perfusion (Fig. 8), which may be due to improved microcirculation due to neoangiogenesis . Where a. - myocardial perfusion before the introduction of AMK; b. - one month after the introduction of AMK; at. - initial myocardial perfusion data in% for segments of the left ventricle; G. - data on perfusion of the myocardium of the left ventricle by segments in% a month after intracoronary administration of AMK. Arrows indicate segments with an increase in myocardial perfusion of more than 5%

The claimed invention allows to treat any chronic diseases. This is ensured by the formation of tissue-specific orientation of the regenerative potential of cells intended for cell therapy. Clinical and experimental studies confirm the high efficiency of cell transplantation when a tissue-specific antigen identical to the organ whose regeneration must be restored in the patient's body is introduced at the stage of cultivation into the growth medium.

Claims

Claim 1. A method of treating chronic diseases, including sampling cellular material from a patient, followed by isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells to produce a biograft and subsequent transplantation of the latter to a patient, characterized in that the cultivation is carried out with the addition of tissue-specific antigen, corresponding to the damaged organ. 2. A method for the treatment of chronic diseases, including sampling cellular material from a patient, followed by isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells to produce a biograft and subsequent transplantation of the latter to a patient, characterized in that the isolated mononuclear autologous cells before cultivation add lymphoid and / or dendritic cells, previously cultured with the addition of tissue-specific anti ene corresponding to the damaged body. 3. A method for the treatment of chronic diseases according to claim 1, 2, characterized in that, prior to taking the cellular material, the patient has an immunocorrection. 4. The method according to PP. characterized in that for the implementation of immunocorrection, the stimulation index is determined as the ratio of the total production of reactive oxygen species by blood cells treated with a cAMP-phosphodiesterase inhibitor to the total production of reactive oxygen species by intact cells. 5. A method of producing a biograft, including sampling cellular material from a patient, followed by isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells in a growth medium, characterized in that a tissue-specific antigen corresponding to the damaged organ is added to the growth medium during cultivation. 6. A method for producing a biograft, including sampling cellular material from a patient, followed by isolation of mononuclear autologous cells containing stem cells from it, culturing mononuclear autologous cells in a growth medium, characterized in that lymphoid and / or dendritic cells are added to the isolated mononuclear autologous cells before cultivation cells pre-cultured with the addition of tissue-specific antigen corresponding to the damaged organ. 7. A biograft, including isolated autologous mononuclear cells, characterized in that it contains stem cells previously cultured with a tissue-specific antigen corresponding to a damaged organ. 8. A biotransplant comprising isolated autologous mononuclear cells, characterized in that it contains lymphoid and / or dendritic cells, previously cultured with the addition of tissue-specific antigen corresponding to the damaged organ.