TWI746799B - A use of a composition for preparing a medicine for treating collagen abnormal proliferation following peritoneal damage - Google Patents

Abstract

The present invention provides a use of a composition for preparing a medicine for treating symptoms or disorders of collagen abnormal proliferation following peritoneal and/or peritoneal cover organ damage in an individual in need thereof, wherein the composition comprises umbilical cord mesenchymal stem cells. The present invention also provides a composition for preparing a medicine for treating symptoms or disorders of collagen abnormal proliferation following peritoneal and/or peritoneal cover organ damage in an individual in need thereof, wherein the composition comprises an umbilical cord mesenchymal stem cell exsosome.

Description

Use of a composition for preparing medicine for treating abnormal collagen proliferation after peritoneal damage

The present invention relates to the use of a composition for the preparation of a medicament for treating the symptoms or disorders of abnormal collagen proliferation after damage to the peritoneum and/or peritoneal covering organs in an individual in need, wherein the composition contains umbilical cord mesenchymal stem cells or Umbilical cord mesenchymal stem cell exosomes (exsosome).

Peritoneum (peritoneum) is the serous membrane that covers the abdominal wall, the inner surface of the pelvic wall and the surfaces of the abdominal and pelvic organs. Among them, the peritoneum lining the abdominal wall, pelvic wall and the lower surface of the diaphragm is called parietal peritoneum, and The peritoneum covering the surface of the organ is called the visceral peritoneum. The peritoneum has the functions of support, fixation, protection, and defense. According to the degree to which the peritoneum covers the internal organs, the organs in the abdominal cavity and the pelvic cavity can be divided into two categories. The first category is intra-peritoneal organs, that is, the organs are completely or mostly covered by the peritoneum. Cover, such as stomach, upper duodenum, jejunum, ileum, cecum, appendix, transverse colon, sigmoid colon, spleen, ovary, fallopian tube, liver and gallbladder, elevating colon, upper rectum, bladder, uterus. The second major category is retro-peritoneal organs. Only a small part of these organs are covered by the peritoneum, such as the kidney, pancreas, ureter, and lower rectum.

When the mesothelial cells on the peritoneum are infected or injured After injury, the damaged mesothelial cells will become hypertrophic. Finally, the mesothelial cells on the peritoneum will fall off and migrate to the sub-mesothelial connective tissue below the mesothelial cells, which is called epithelial cell-mesenchymal transition ( epithelial-mesenchymal transition). The damaged peritoneum will release inflammatory factors, attract more white blood cells to infiltrate, and further, cause the deformed mesothelial cells to multiply and transform into activated fibroblasts (myo-fibroblast). At this time, a large number of activated fibroblasts in the connective tissue below the mesothelial cells will synthesize and release more collagen, resulting in an increase in the sub-mesothelial connective tissue (sub-mesothelial connective tissue), thus thickening of the peritoneum , Causing so-called peritoneal fibrosis (peritoneal fibrosis). Peritoneal fibrosis can cause the peritoneum to thicken, harden, and even calcify. The fibrotic peritoneum covers the internal organs, causing the internal organs in the abdominal cavity to stick to each other, the peristalsis of the organs becomes obstructed, and in severe cases, it can cause organ hypoxia and necrosis. Therefore, patients may experience loss of appetite, abdominal distension, abdominal pain, nausea, vomiting, constipation, and even cause organ necrosis and sepsis.

There are many causes of peritoneal fibrosis, such as genes, autoimmune diseases, drugs, tumors, trauma or surgery, radiation, hemorrhage of abdominal and pelvic organs, gastrointestinal inflammation, appendicitis, diverticulitis, various infections (such as tuberculosis, tissue cytoplasm) Mycosis, syphilis, etc.), peritoneal dialysis and other factors can cause peritoneal fibrosis, which is called secondary peritoneal fibrosis. In addition, a very large proportion of patients have unexplained peritoneal fibrosis, which is the so-called special Primary peritoneal fibrosis.

So far, there is no clinical medicine that can effectively cure the fibrotic peritoneum. It can only suppress the inflammation of the peritoneum by administering drugs such as steroids to the patient. Such drugs must be taken for a long time and accompanied by considerable side effects. Nevertheless, these drugs can only reduce the rate of deterioration of peritoneal fibrosis, and cannot treat the fibrotic peritoneum to restore it to its original state. Therefore, finding a treatment and reversing peritoneal fibrosis is a top priority.

The present invention relates to the transplantation of umbilical cord mesenchymal stem cells in Wharton's jelly in the umbilical cord of newborns into rats with peritoneal fibrosis to explore the feasibility and mechanism of treatment and reversal of peritoneal fibrosis.

High-glucose dialysis buffer (PD, containing 4.25% glucose) high-glucose dialysis buffer (PD, containing 4.25% glucose) containing 20 millimolar concentration (mM) glucose metabolite methylglyoxal (methylglyoxal, MGO) high-glucose dialysis buffer (PD) /MGO) was perfused into the abdominal cavity of rats for three consecutive weeks, after inducing peritoneal fibrosis in rats, the next day, low-dose (10 ⁶ ) and high-dose (10 ⁸ ) human umbilical cord mesenchymal stem cells were transplanted. One month later, observe the changes in peritoneal fibrosis. The results showed that in the rats in the PD/MGO treatment group for three weeks, the abdominal cavity showed the formation of abdominal cocoons, the parietal and visceral peritoneum were thickened, and the collagen in the peritoneum was also significantly increased. After PD/MGO treatment for three consecutive weeks, if no treatment is given, the rat’s peritoneum will become thicker and more collagen accumulation after one month. However, graft ¹⁰⁸ human umbilical mesenchymal stem cells into the peritoneal cavity of peritoneal fibrosis in rats, after one month, the thickness of rat peritoneum, and retroperitoneal fibrosis extent, are significantly reduced on. The mechanism of human umbilical cord mesenchymal stem cell therapy is to inhibit inflammation on the one hand and reduce the activation of fibroblasts to avoid the continuous production of new collagen. On the other hand, increase the production of matrix metalloproteinase-13 (matrix metalloproteinase-13, MMP-13, also known as collagenase-3, collagenase-3) to accelerate the decomposition of existing collagen. Through these two approaches, peritoneal fibrosis can be treated.

Furthermore, the conditioned medium of human umbilical cord mesenchymal stem cells cultured at normal oxygen concentration (20%) and low oxygen concentration (1%) was injected into the abdominal cavity of rats with peritoneal fibrosis, twice a week, continuously For four weeks, the results showed that the conditional medium for normal oxygen culture and hypoxic culture The conditioned medium has the effect of reducing peritoneal thickness and curing peritoneal fibrosis. Therefore, it is speculated that transplantation of human umbilical cord mesenchymal stem cells to treat and reverse peritoneal fibrosis is mainly based on stem cell releases containing a variety of biological hormones. Therefore, the conditioned medium also has the effect of treating peritoneal fibrosis.

The present invention relates to the use of a composition for preparing a medicament for treating the symptoms or disorders of abnormal collagen proliferation after damage to the peritoneum and/or peritoneal covering organs in an individual in need, wherein the composition contains umbilical cord mesenchymal stem cells.

In one embodiment, the umbilical cord mesenchymal stem cell line is derived from umbilical cord Watton's gel.

In another embodiment, the source species of the umbilical cord Watton's gel is the same or different from the individual species in need.

In one embodiment, the treatment of umbilical cord between the stromal cells of an effective dose of 10 ⁶ cells.

In one embodiment, the therapeutic effective dose of mesenchymal stem cells of umbilical cord ¹⁰⁸ between the cells.

In one embodiment, the composition further comprises umbilical cord mesenchymal stem cell exsosomes.

In one embodiment, the peritoneum refers to parietal peritoneum.

In one embodiment, the peritoneum refers to the visceral peritoneum.

In one embodiment, the peritoneum refers to the peritoneal reflections structure, including but not limited to omentum, mesenteric, and ligament.

In another embodiment, the omentum includes (but is not limited to) a greater omentum, a lesser omentum, and an omental bursa.

In another embodiment, the mesenteric system includes (but is not limited to) mesentery proper, transvese mesocolon, sigmoid mesocolon, and meso appendix.

In another embodiment, the ligament includes (but is not limited to) falciform ligament, ligamentum teres hepatis, coronary ligament, ligamentum venosus, phrenic ligament (phrenicocolic ligament), gastrosplenic or gastrolienal ligament, splenorenal or lienorenal ligament.

In another embodiment, the peritoneal covering organ refers to an intraperitoneal organ, including (but not limited to) stomach, upper duodenum, jejunum, ileum, cecum, appendix, transverse colon, sigmoid colon, spleen, ovary, and fallopian tube.

In another embodiment, the peritoneal covering organs refer to interperitoneal organs, including but not limited to liver and gallbladder, elevating colon, upper rectum, bladder, and uterus.

In another embodiment, the peritoneal covering organs refer to retroperitoneal organs, including (but not limited to) the descending and horizontal parts of the duodenum, adrenal glands, kidneys, pancreas, ureters, and lower rectum.

In one embodiment, the symptoms or symptoms of abnormal collagen proliferation include (but are not limited to) peritoneal thickening, peritoneal fibrosis, adhesion, and abdominal cocoon.

In another embodiment, the peritoneal fibrosis is caused by immunodeficiency, drugs, tumor, trauma, surgery, bleeding, urine extravasation, peritoneal dialysis, radiation, infection, or inflammation.

In another embodiment, the peritoneal fibrosis system includes idiopathic peritoneal fibrosis and secondary peritoneal fibrosis.

In another embodiment, the peritoneal fibrosis refers to cystic peritoneal sclerosis.

In one embodiment, the composition increases the expression of collagenase.

In another embodiment, the collagenase is matrix metalloproteinase.

In another embodiment, the matrix metalloproteinase is matrix metalloproteinase-13.

In one embodiment, the composition inhibits inflammation.

In one embodiment, the composition reduces the activation of fibroblasts.

In one embodiment, the composition reduces the number of macrophages.

The present invention also relates to the use of a composition for the preparation of a medicament for treating the symptoms or disorders of abnormal collagen proliferation after damage to the peritoneum and/or peritoneal covering organs in an individual in need, wherein the composition contains umbilical cord mesenchymal stem cells. Exsosome.

In one embodiment, the umbilical cord mesenchymal stem cell exocrine system is derived from the culture medium after culturing the umbilical cord mesenchymal stem cells in an environment with an oxygen concentration of not more than 20%.

In another embodiment, the umbilical cord mesenchymal stem cell exocrine system is derived from the culture medium after culturing the umbilical cord mesenchymal stem cells in an environment with an oxygen concentration of not more than 1%.

In one embodiment, the drug is administered to the individual in need at a frequency of twice a week.

In one embodiment, the drug is administered to the individual in need by intraperitoneal injection.

In one embodiment, the composition further comprises umbilical cord mesenchymal stem cells.

In one embodiment, the peritoneum refers to parietal peritoneum.

In one embodiment, the peritoneum refers to the visceral peritoneum.

In one embodiment, the peritoneum refers to the peritoneal reflections structure, including but not limited to omentum, mesenteric, and ligament.

In another embodiment, the omentum includes (but is not limited to) a greater omentum, a lesser omentum, and an omental bursa.

In another embodiment, the mesenteric system includes (but is not limited to) mesentery proper, transvese mesocolon, sigmoid mesocolon, and meso appendix.

In another embodiment, the ligament includes (but is not limited to) falciform ligament, ligamentum teres hepatis, coronary ligament, ligamentum venosus, phrenic ligament (phrenicocolic ligament), gastrosplenic or gastrolienal ligament, splenorenal or lienorenal ligament.

In another embodiment, the peritoneal covering organ refers to an intraperitoneal organ, including (but not limited to) stomach, upper duodenum, jejunum, ileum, cecum, appendix, transverse colon, sigmoid colon, spleen, ovary, and fallopian tube.

In another embodiment, the peritoneal covering organs refer to interperitoneal organs, including but not limited to liver and gallbladder, elevating colon, upper rectum, bladder, and uterus.

In another embodiment, the peritoneal covering organs refer to retroperitoneal organs, including (but not limited to) the descending and horizontal parts of the duodenum, adrenal glands, kidneys, pancreas, ureters, and lower rectum.

In one embodiment, the symptoms or symptoms of abnormal collagen proliferation include (but are not limited to) peritoneal thickening, peritoneal fibrosis, adhesion, and abdominal cocoon.

In another embodiment, the peritoneal fibrosis is caused by immunodeficiency, drugs, tumors, Caused by trauma, surgery, bleeding, urine extravasation, peritoneal dialysis, radiation, infection, or inflammation.

In another embodiment, the peritoneal fibrosis system includes idiopathic peritoneal fibrosis and secondary peritoneal fibrosis.

In another embodiment, the peritoneal fibrosis refers to cystic peritoneal sclerosis.

In one embodiment, the composition increases the expression of collagenase.

In another embodiment, the collagenase is matrix metalloproteinase.

In another embodiment, the matrix metalloproteinase is matrix metalloproteinase-13.

In one embodiment, the composition inhibits inflammation.

In one embodiment, the composition reduces the activation of fibroblasts.

In one embodiment, the composition reduces the number of macrophages.

Figure 1. The experimental flow chart of transplanting human umbilical cord mesenchymal stem cells into peritoneal fibrosis mice.

Figure 2. The macroscopic view of the abdominal cavity of a white mouse shows that the transplantation of human umbilical cord mesenchymal stem cells treats the peritoneal thickening, adhesion, and abdominal cocoon of the peritoneal fibrosis mouse. Before perfusion (A-E) and after fixation (a-e), the abdominal wall of the rats in each group was dissected, and photographs showed the macroscopic changes in the abdominal cavity of the rats in each group. Normal group (A, a), PD/MGO group (B, b), PD/MGO+solvent group (C, c), PD/MGO+HUMSCs (LD) group (D, d), PD/MGO+HUMSCs ( HD) group (E, e). The results showed that transplantation of human umbilical cord mesenchymal stem cells can treat the thickening, sticking, and abdominal cocoon of the peritoneum of rats with peritoneal fibrosis.

表示相較於PD/MGO+MSCs(LD)組p值小於0.05。 Figure 3. Transplantation of human umbilical cord mesenchymal stem cells reduces the thickness of the peritoneum in mice with peritoneal fibrosis. The parietal peritoneum (AE) on the rectus abdominis (AE) and the visceral peritoneum (GK) on the liver surface of each group of rats were stained by H&E. The images showed that the normal group (A, G), PD/MGO group (B, H) ), PD/MGO+solvent group (C, I), PD/MGO+HUMSCs (LD) group (D, J), PD/MGO+HUMSCs (HD) group (E, K). (F, L) Quantitative results showed that the thickness of the peritoneum was significantly increased after PD/MGO treatment for three weeks. The next day, transplantation of high-dose human umbilical cord mesenchymal stem cells can reduce the thickness of the peritoneum in rats with peritoneal fibrosis. Scale bar: 200 microns (μm). * Indicates that the p value is less than 0.05 compared to the normal group; # indicates that the p value is less than 0.05 compared to the PD/MGO group; ◆ indicates that the p value is less than 0.05 compared to the PD/MGO+solvent group;

It means that the p value is less than 0.05 compared to the PD/MGO+MSCs (LD) group.

表示相較於PD/MGO+MSCs(LD)組p值小於0.05。 Figure 4. Transplantation of human umbilical cord mesenchymal stem cells reduces collagen in the peritoneum of peritoneal fibrosis mice. The parietal peritoneum (AE) on the rectus abdominis muscle (AE) and the visceral peritoneum (GK) on the liver surface of rats in each group were stained with Sirius red, and collagen was identified. The image shows the normal group (A, G), PD/MGO group (B, H), PD/MGO+solvent group (C, I), PD/MGO+HUMSCs(LD) group (D, J), PD/MGO +HUMSCs (HD) group (E, K). (F, L) Quantitative results showed that after PD/MGO treatment for three weeks, the collagen content in the thickened peritoneum was significantly increased. The next day, transplantation of high-dose human umbilical cord mesenchymal stem cells can reduce the collagen in the peritoneum of rats with peritoneal fibrosis protein. Scale bar: 200 microns (μm). * Indicates that the p value is less than 0.05 compared to the normal group; # indicates that the p value is less than 0.05 compared to the PD/MGO group; ◆ indicates that the p value is less than 0.05 compared to the PD/MGO+solvent group;

It means that the p value is less than 0.05 compared to the PD/MGO+MSCs (LD) group.

Figure 5. Transplantation of human umbilical cord mesenchymal stem cells reduces the activation of fibroblasts in the peritoneum of rats with peritoneal fibrosis. In order to understand the inflammatory response in the peritoneum, the parietal peritoneum on the rectus abdominis of each group of rats was subjected to anti-αSMA tissue immunostaining (A-E, the lower row is the enlarged image of the box), The anti-αSMA antibody identifies activated fibroblasts. The images are the normal group (A), PD/MGO group (B), PD/MGO+solvent group (C), PD/MGO+HUMSCs(LD) group (D), PD/MGO+HUMSCs(HD) group ( E). The results showed that the number of activated fibroblasts in the thickened peritoneum increased significantly after PD/MGO treatment for three weeks. The next day, transplantation of high-dose human umbilical cord mesenchymal stem cells can reduce the activation of fibroblasts in the peritoneum of peritoneal fibrosis mice. number.

Figure 6. Transplantation of human umbilical cord mesenchymal stem cells reduces the number of macrophages in the peritoneum of rats with peritoneal fibrosis. In order to understand the inflammatory response in the peritoneum, the parietal peritoneum on the rectus abdominis of each group of rats was taken for tissue immunostaining with anti-ED1 (AE, the bottom row is the enlarged image of the box), and the anti-ED1 antibody identifies the macrophages . The images are the normal group (A), PD/MGO group (B), PD/MGO+solvent group (C), PD/MGO+HUMSCs(LD) group (D), PD/MGO+HUMSCs(HD) group ( E). The results showed that after PD/MGO treatment for three weeks, the number of macrophages marked in the thickened peritoneum increased significantly. The next day, transplantation of high-dose human umbilical cord mesenchymal stem cells can reduce the number of macrophages in the peritoneum of peritoneal fibrosis mice number.

Figure 7. Transplantation of human umbilical cord mesenchymal stem cells improves the ability of peritoneal fibrosis rats to decompose intraperitoneal collagen. In order to understand the breakdown of collagen in the peritoneum, fresh peritoneums of rats in each group were taken and analyzed by anti-MMP13 by Western blot method. MMP-13 can break down collagen. The results showed that the content of MMP-13 in the peritoneum was significantly reduced after PD/MGO treatment for three weeks. The next day, transplantation of high-dose human umbilical cord mesenchymal stem cells can increase the production of MMP-13 in the peritoneum of rats with peritoneal fibrosis.

Figure 8. The experimental flow chart of conditioned medium of human umbilical cord mesenchymal stem cells cultured in mice with peritoneal fibrosis.

Figure 9. The macroscopic view of the abdominal cavity of a white mouse shows that the human umbilical cord mesenchymal stem has been cultured The conditioned medium of cells can treat peritoneal thickening, sticking, and abdominal cocoons in rats with peritoneal fibrosis. After the rats in each group were perfused and fixed (A-E), the abdominal wall was dissected, and photographs showed the macroscopic changes in the abdominal cavity of the rats in each group. Normal group (A), PD/MGO group (B), PD/MGO+DMEM group (C), PD/MGO+Normaxia CM group (D), PD/MGO+Hypoxia CM group (E). The results show that the conditioned medium that has been cultured with human umbilical cord mesenchymal stem cells can treat the thickening, sticking, and cocoon of the peritoneum of peritoneal fibrosis mice.

表示相較於PD/MGO+Normaxia CM組p值小於0.05。 Figure 10. The conditioned medium of human umbilical cord mesenchymal stem cells was given to reduce the thickness of the peritoneum in rats with peritoneal fibrosis. The parietal peritoneum (AE) on the rectus abdominis (AE) and the visceral peritoneum (GK) on the liver surface of each group of rats were stained by H&E. The images showed that the normal group (A, G), PD/MGO group (B, H) ), PD/MGO+DMEM group (C, I), PD/MGO+Normaxia CM group (D, J), PD/MGO+Hypoxia CM group (E, K). (F, L) Quantitative results showed that the peritoneal thickness was significantly increased after PD/MGO treatment for three weeks. The conditioned medium of human umbilical cord mesenchymal stem cells can reduce the peritoneal thickness of rats with peritoneal fibrosis. Scale bar: 200 microns (μm). * Indicates that the p value is less than 0.05 compared to the normal group; # indicates that the p value is less than 0.05 compared to the PD/MGO group; ◆ indicates that the p value is less than 0.05 compared to the PD/MGO+DMEM group;

It means that the p value is less than 0.05 compared to the PD/MGO+Normaxia CM group.

表示相較於PD/MGO+Normaxia CM組p值小於0.05。 Figure 11. The conditioned medium of human umbilical cord mesenchymal stem cells was given to reduce the collagen in the peritoneum of peritoneal fibrosis rats. The parietal peritoneum (AE) on the rectus abdominis muscle (AE) and the visceral peritoneum (GK) on the liver surface of rats in each group were stained with Sirius red, and collagen was identified. The image shows the normal group (A, G), PD/MGO group (B, H), PD/MGO+DMEM group (C, I), PD/MGO+Normaxia CM group (D, J), PD/MGO+ Hypoxia CM group (E, K). (F, L) Quantitative results showed that after PD/MGO treatment for three weeks, the collagen content in the thickened peritoneum was significantly increased. The conditioned medium of human umbilical cord mesenchymal stem cells can reduce the intraperitoneal in peritoneal fibrosis mice. Collagen. Scale bar: 200 microns (μm). * Indicates that the p value is less than 0.05 compared to the normal group; # indicates that the p value is less than 0.05 compared to the PD/MGO group; ◆ indicates that the p value is less than 0.05 compared to the PD/MGO+DMEM group;

It means that the p value is less than 0.05 compared to the PD/MGO+Normaxia CM group.

The following examples are not for limiting purposes, but only to present various aspects of this invention.

Materials and Methods

Establishment of animal model of peritoneal fibrosis in rats

A 4.25% glucose dialysis mixture (4.25% glucose dialysis solution purchased from Baxter) containing 20 millimolar (mM) methylglyoxal (MGO) was injected into the rat through a PE160 buried tube Into the abdominal cavity, the total volume of injection is based on the ratio of 100 milliliters (ml) of dialysis mixture per kilogram of the weight of the rat, once a day for three consecutive weeks.

Isolation and culture of human umbilical cord mesenchymal stem cells

The umbilical cord after delivery was collected aseptically and stored in a buffer solution of Hanks' Balanced Salt Solution (HBSS, Hanks' Balanced Salt Solution) at 4°C. The umbilical cord interstitial cells were separated and cultured within 24 hours. The experimental equipment was all After being used after high temperature and high pressure sterilization, the whole experiment was disinfected with 75% alcohol, and it was overheated before use. Take out the umbilical cord in a sterile operating table, and after soaking in 75% alcohol for disinfection, place it in HBSS buffer solution. Next, the umbilical cord interstitial tissue (Wharton’s jelly, Wharton’s jelly) was separated and minced with a sterilized instrument at a rate of 4000 per minute. Centrifuge at rpm for 5 minutes. Remove the supernatant, add collagenase and trypsin to the interstitial tissue of the umbilical cord, and then add FBS (Gibco 10437-028) to stop the trypsin reaction. At this time, the umbilical cord mesenchymal tissue has been processed into human umbilical mesenchymal stem cells (Human Umbilical Mesenchymal Stem Cell; referred to as HUMSCs). Finally, the umbilical cord mesenchymal stem cells were added to an appropriate amount of 10% FBS DMEM to break up the cells, count the number of cells, and proceed to cell culture.

Implantation of human umbilical cord mesenchymal stem cells

After three weeks rats treated PD / MGO dialysate peritoneal fibrosis establishment mode, ^106, or ¹⁰⁸ human umbilical mesenchymal stem cells were dissolved in physiological saline, injected intraperitoneally. Please refer to Figure 1 for the experimental flow chart.

Grouping of experimental animals transplanted with stem cells

The first group is the normal group. The rats were given normal saline (100 ml per kilogram of body weight) for three consecutive weeks, and every other day, only 10 ml of saline was injected into the abdominal cavity, and sacrificed for observation one month later; The group is the PD/MGO group. The rats are continuously injected with a 4.25% glucose dialysis mixture containing 20 millimolar MGO (100 ml per kilogram of body weight) into the abdominal cavity for three consecutive weeks, immediately sacrificed for observation; the third group is PD/ In the MGO+solvent group, the rats were continuously injected with a 4.25% glucose dialysis mixture containing 20 millimolar MGO (100 ml per kilogram of body weight) into the abdominal cavity for three consecutive weeks. The next day, 10 ml of normal saline was injected into the abdominal cavity of the rats, and sacrificed for observation one month later; the fourth group was the PD/MGO+HUMSCs (LD) group, and the rats were given a dialysis mixture of 4.25% glucose containing 20 millimolar MGO. Liquid (100 ml per kilogram of body weight) for three consecutive weeks. The next day, the ¹⁰⁶ human umbilical mesenchymal stem cells in 10 ml of physiological saline, was injected into the abdominal cavity of rats, observed after one month sacrifice; fifth group of PD / MGO + HUMSCs (HD) group, comprising 20 rats given 4.25% glucose dialysis mixture with millimolar concentration of MGO (100 ml per kilogram of body weight) for three consecutive weeks. The next day, the umbilical mesenchymal stem cells was dissolved in 10 ml 10 ⁸ human physiological saline, was injected into the abdominal cavity of rats, observed after one month sacrifice.

Hematoxylin & Eosin Stain (H&E stain for short)

After the rats were anesthetized, they were perfused and fixed, the abdominal muscles and liver were taken out, and tissue sections and HE stain were performed to observe the changes in the parietal peritoneum, the thickness of the visceral peritoneum, and the pathology.

Tissue collagen staining

After the rats were anesthetized, they were fixed by perfusion, the abdominal muscles and liver were taken out, and the tissues were sectioned and stained with 0.1% sirius red dissolved in picric acid for 6 minutes to observe the parietal peritoneum. peritoneum), and changes in collagen in the visceral peritoneum. Collagen appears red after staining, and the area occupied by red collagen is quantified.

Tissue immunostaining or Western blotting analysis of anti-α-smooth muscle actin antibody and anti-ED1 antibody

The anti-α-smooth muscle actin antibody (anti-αSMA) is used to identify activated fibroblasts, while the anti-ED1 antibody (ED1 antibody) is used to identify activated lymphocytes and monocytes, with the purpose of detecting Measure the change of inflammation in the peritoneum.

The tissue sections of the abdominal wall containing the parietal peritoneum and the liver tissue of the visceral peritoneum were sectioned to perform tissue immunostaining with anti-α-smooth muscle actin antibody and anti-ED1 antibody.

Western blot analysis of anti-MMP-13 antibody

Matrix metallopeptidase-13 (Matrix metallopeptidase-13, MMP-13) has been published in papers, which can help the decomposition of collagen. The parietal peritoneum or the visceral peritoneum were taken and analyzed with the anti-MMP-13 antibody (MMP-13 antibody) by Western blot method to quantify the synthesis of MM-P13 protein.

Conditioned Medium Transplantation of Human Umbilical Cord Mesenchymal Stem Cells

After the rats were treated with PD/MGO dialysate for three weeks to establish a peritoneal fibrosis model, the culture medium of human umbilical cord mesenchymal stem cells, called stem cell conditioned medium, was injected into the abdominal cavity of the rats through a PE tube.

Experimental animal grouping of stem cell conditioned medium

This experiment is divided into five groups, and the experiment flow chart is shown in Figure 7: The first group is the normal group, that is, the rats are given normal concentration of normal saline (100 ml per kilogram of body weight) for three consecutive weeks. Then, twice a week, 10 ml of normal saline was injected into the abdominal cavity of the rats for four consecutive weeks, and finally sacrificed for observation; the second group is the PD/MGO group, that is, the rats are continuously injected with 4.25% glucose dialysis mixture containing 20mM MGO (100 ml per kilogram of body weight) in the abdominal cavity for three consecutive weeks, immediately sacrificed for observation; the third group is the PD/MGO+DMEM group, that is, rats are continuously injected with 4.25% glucose dialysis mixture containing 20mM MGO (per kilogram of body weight) 100ml) in the abdominal cavity for three consecutive weeks, and then twice a week, each 10ml DMEM was injected into the abdominal cavity of the rat for four consecutive weeks, and finally sacrificed for observation; the fourth group is the PD/MGO+Normoxia-CM group, namely The rats were given PD/MGO dialysate for three weeks, and then the conditioned medium (Normoxia-conditioned medium, Normoxia-CM) of human umbilical cord mesenchymal stem cells cultured in a normoxic environment, twice a week, 10 times each time Milliliter of Normoxia-CM was injected into the abdominal cavity of the rats for four consecutive weeks, and finally sacrificed for observation; the fifth group was PD/MGO+Hypoxia-CM, that is, the rats were given PD/MGO dialysis for three weeks Then, the conditioned medium (Hypoxia-conditioned medium, Hypoxia-CM) of human umbilical cord mesenchymal stem cells cultured in a hypoxic environment was injected twice a week, and 10 ml of Hypoxia-CM each time was injected into the abdominal cavity of the rat In, for four consecutive weeks, and finally sacrificed for observation.

Example 1. Macro view shows that transplantation of human umbilical cord mesenchymal stem cells can treat peritoneal adhesions and abdominal cocoons in mice with peritoneal fibrosis

Take each group of rats that have not been fixed by perfusion (Figure 2A-E) and after perfusion (Figure 2a-e), cut their abdominal wall and observe their appearance. The results showed that the peritoneum in the abdominal cavity of the normal group was clear and closely attached to the organs. Therefore, the surface of the liver and small intestine appeared smooth (Figure 2A and a). In rats in the PD/MGO group, treated with PD/MGO for three weeks, the peritoneum on the visceral surface was clearly visible, the liver became thick and swollen, and the small intestine became hypertrophy due to food accumulation (Figure 2B and b). The rats in the PD/MGO+solvent group were treated with PD/MGO for three weeks, and only saline was given the next day. One month later, it was found that the visceral peritoneum and parietal peritoneum were both thickened and adhered to each other, and the peritoneum covered the entire In the abdominal cavity, an abdominal cocoon is formed (Figure 2C and c). The rats in the PD/MGO+HUMSCs (LD) group were treated with PD/MGO for three weeks and transplanted with low-dose stem cells once every other day. One month later, the thickened peritoneum of the rats did not improve significantly (Figure 2D and d) ). The rats in the PD/MGO+HUMSCs (HD) group were treated with PD/MGO for three weeks and transplanted with a high dose of stem cells every other day. One month later, no abdominal calluses were found in the abdominal cavity of the rats, and no thickened peritoneum was seen. , The surface of the organs is smoother, and the small intestine returns to its normal shape (Figure 2E and e).

Example 2. Transplantation of human umbilical cord mesenchymal stem cells can reduce the thickening of parietal and visceral peritoneum in rats with peritoneal fibrosis

Take the parietal peritoneum (Figure 3A-E) and the visceral peritoneum (Figure 3G-K) of the rats after perfusion and fixation, and perform H&E staining to observe the changes in peritoneal thickness. The results show that it is normal Whether it is the parietal peritoneum on the surface of the rectus abdominis muscle or the visceral peritoneum on the surface of the liver, it is only a thin layer (Figure 3A and G). In the PD/MGO group, after three weeks of PD/MGO treatment, the thickness of the peritoneum was significantly increased (Figure 3B and H). The thickness of the visceral peritoneum and parietal peritoneum in the PD/MGO+solvent group was significantly higher than that in the PD/MGO group, and they also adhered to each other (Figure 3C and I).

In the PD/MGO+HUMSCs (LD) group, the peritoneal thickness of the rats did not significantly improve (Figure 3 D and J). In the PD/MGO+HUMSCs (HD) group, the thickness of the parietal peritoneum or the visceral peritoneum of the rats was significantly reduced, which was equivalent to the thickness of the normal group, and there was no statistical difference (Figure 3E and K). Statistics show that transplantation of high-dose human umbilical cord mesenchymal stem cells can improve the peritoneal thickness of rats with peritoneal fibrosis (Figure 3F and L).

Example 3. Transplantation of human umbilical cord mesenchymal stem cells can reduce the collagen in the parietal and visceral peritoneum of rats with peritoneal fibrosis

The parietal peritoneum (Figure 4A-E) and the visceral peritoneum (Figure 4G-K) of each group after perfusion and fixation were taken and stained with Sirius Red. The collagen was marked to observe the changes of collagen in the peritoneum. The results showed that there was a small amount of collagen in the parietal or visceral peritoneum of the normal group (Figure 4A and G). In the PD/MGO group, after three weeks of PD/MGO treatment, the thickened peritoneum, a large amount of collagen accumulated (Figure 4B and H). The parietal peritoneum and visceral peritoneum in the PD/MGO+solvent group not only became thicker, but also the collagen content in the thickened peritoneum and the collagen between muscle tissues were significantly increased (Figure 4C and I). In the PD/MGO+HUMSCs (LD) group, the collagen in the rectus abdominis of rats was slightly improved, but the collagen in the visceral peritoneum did not change significantly (Figure 4D and J). In the PD/MGO+HUMSCs (HD) group, the thickness of the parietal peritoneum or the visceral peritoneum of the rats was significantly reduced, and the collagen content in the peritoneum was also significantly reduced, which was comparable to that of the normal group. Statistical differences (Figure 4E and K). Statistics show that transplantation of high-dose human umbilical cord Carrying mesenchymal stem cells can improve the collagen content in the peritoneum of rats with peritoneal fibrosis (Figure 4F and L).

Example 4. Transplantation of human umbilical cord mesenchymal stem cells can reduce the inflammation in the peritoneum of peritoneal fibrosis mice

The parietal peritoneum of each group of rats after perfusion and fixation was taken, and anti-αSMA (Figure 5A-E) tissue immunostaining was performed. The anti-αSMA antibody identifies activated fibroblasts, which synthesize and release collagen. Therefore, activated fibroblasts represent an inflammatory phenomenon. The results showed that no activated fibroblasts were found in the peritoneum of the normal group (Figure 5A). In the PD/MGO group, after treating PD/MGO for three weeks, there was a large number of activated fibroblasts in the thickened peritoneum (Figure 5B). In the PD/MGO+solvent group, the number of activated fibroblasts in the thickened peritoneum was still large (Figure 5C). In the PD/MGO+HUMSCs (LD) group, in the thickened peritoneum of rats, the activated fibroblasts became slender (Figure 5D).

In the PD/MGO+HUMSCs (HD) group, in the parietal peritoneum of rats, activated fibroblasts showed a significant decrease (Figure 5E). The results show that transplantation of high-dose human umbilical cord mesenchymal stem cells can inhibit fibroblasts activated in the peritoneum of peritoneal fibrosis mice.

In addition, the parietal peritoneum of each group of rats after perfusion and fixation was taken for anti-ED1 (Figure 6A-E) tissue immunostaining. Anti-ED1 antibody identifies macrophages, which represent inflammation. Almost no macrophages were found in the peritoneum of the normal group (Figure 6A). In the PD/MGO group, after treating PD/MGO for three weeks, a large number of macrophages appeared in the thickened peritoneum (Figure 6B). There are still many thickened intraperitoneal macrophages in the PD/MGO+solvent group (Figure 6C). In the PD/MGO+HUMSCs (LD) group, there were fewer thickened intraperitoneal macrophages in rats (Figure 6D). In the PD/MGO+HUMSCs(HD) group, intraperitoneal macrophages with thickened parietal peritoneum of rats showed Significant reduction (Figure 6E). The results show that transplantation of high-dose human umbilical cord mesenchymal stem cells can inhibit macrophages in the peritoneum of peritoneal fibrosis mice, that is, inhibit the inflammatory response.

Example 5. Transplantation of human umbilical cord mesenchymal stem cells improves the ability of peritoneal fibrosis mice to decompose intraperitoneal collagen

Take the fresh peritoneum of each group of rats and perform MMP-13 western blotting method to quantify the concentration of MMP-13. MMP-13 is also called collagenase-3 (collagenase-3), which can break down collagen.

MMP-13 exists in the peritoneum of the normal group. The intraperitoneal MMP-13 in the PD/MGO group and PD/MGO+solvent group was almost unidentified. In the PD/MGO+HUMSCs(HD) group, the MMP-13 content in the peritoneum of the rats reappeared (Figure 7). The results show that transplantation of high-dose human umbilical cord mesenchymal stem cells can promote the expression of MMP-13 in the peritoneum of peritoneal fibrosis mice, and it is speculated that the ability to decompose intraperitoneal collagen is also improved.

Example 6. The conditioned medium for processing human umbilical cord mesenchymal stem cells can treat peritoneal adhesions and abdominal cocoons in rats with peritoneal fibrosis

Take the rats of each group after perfusion fixation, cut their abdominal wall and observe the appearance. The results showed that the peritoneum in the abdominal cavity of the normal group was tightly attached to the organs, so the liver, and the small intestine surface appeared smooth (Figure 9A). In rats in the PD/MGO group, after treating PD/MGO for three weeks, the peritoneum on the visceral surface was clearly visible, and the small intestine became hypertrophy (Figure 9B). The rats in the PD/MGO+DMEM group were treated with PD/MGO for three weeks, and then only given DMEM. One month later, it was found that the visceral peritoneum and parietal peritoneum became significantly thicker and also stick to each other, forming severe abdominal cocoons (Figure 9) C). The rats in the PD/MGO+Normaxia-CM group were treated with PD/MGO for three weeks, and then the conditioned medium (Normaxia-CM) of human umbilical cord mesenchymal stem cells cultured in a normal oxygen environment, twice a week, 10 times each time Milliliters Normaxia-CM was injected into the abdominal cavity of rats for four consecutive weeks, and the thickened peritoneum of the rats was significantly improved (Figure 9D). The rats in the PD/MGO+Hypoxia-CM group were treated with PD/MGO for three weeks, and then the conditioned medium (Hypoxia-CM) of human umbilical cord mesenchymal stem cells cultured in a hypoxic environment, twice a week, 10 times each time One milliliter of Hypoxia-CM was injected into the rat's abdominal cavity for four consecutive weeks, and the rat's abdominal cavity had no cocoon or thickened peritoneum, the surface of the organs was smoother, and the small intestine returned to its normal shape (Figure 9E).

Example 7. Treatment of conditioned medium of human umbilical cord mesenchymal stem cells can reduce the thickening of parietal and visceral peritoneum in rats with peritoneal fibrosis

Take the parietal peritoneum (Figure 10A-E) and the visceral peritoneum (Figure 10G-K) of the rats after perfusion and fixation, and perform H&E staining to observe the changes in peritoneal thickness. The results showed that in the normal group, whether it was parietal peritoneum or visceral peritoneum, there was only a thin layer (Figure 10A and G). In the PD/MGO group, after three weeks of PD/MGO treatment, the thickness of the peritoneum was significantly increased (Figure 10B and H). The thickness of parietal peritoneum and visceral peritoneum of PD/MGO+DMEM group was significantly increased compared with PD/MGO group (Figure 10C and I). In the PD/MGO+Normaxia-CM group, the peritoneal thickness of the rats was significantly improved compared with the PD/MGO group (Figure 10 D and J). In the PD/MGO+Hypoxia-CM group, the thickness of the parietal peritoneum or the visceral peritoneum of the rats was significantly reduced, which was equivalent to the thickness of the normal group, and there was no statistical difference (Figure 10E and K). The statistical results show that the conditioned medium given to human umbilical cord mesenchymal stem cells can improve the peritoneal thickness of rats with peritoneal fibrosis (Figure 10F and L).

Example 8. The conditioned medium for treating human umbilical cord mesenchymal stem cells can reduce the collagen in the parietal and visceral peritoneum of peritoneal fibrosis rats

The parietal peritoneum (Figure 11A-E) and the visceral peritoneum (Figure 11G-K) of each group after perfusion and fixation were taken and stained with Sirius red. The collagen was marked with Then observe the changes in collagen in the peritoneum. The results showed that there were trace amounts of collagen in the parietal or visceral peritoneum of the normal group (Figure 11A and G). In the PD/MGO group, after three weeks of PD/MGO treatment, the thickened peritoneum, a large amount of collagen accumulated (Figure 11B and H). The parietal and visceral peritoneum in the PD/MGO+DMEM group not only became thicker, but also the collagen content in the thickened peritoneum and the collagen between the muscle tissues were significantly increased (Figure 11 C and I). In the PD/MGO+Normaxia-CM group, the collagen in the parietal and visceral peritoneum of rats showed a significant decrease (Figure 11 D and J). In the PD/MGO+Hypoxia-CM group, the thickness of the parietal peritoneum or the visceral peritoneum of the rats was significantly reduced, and the collagen content in the peritoneum was also significantly reduced (Figure 11 E and K). The statistical results show that the conditioned medium given to human umbilical cord mesenchymal stem cells can improve the collagen content in the peritoneum of peritoneal fibrosis rats (Figure 11F and L).

In order for those with ordinary knowledge in the technical field of this invention to understand the method of making and using this technique, this invention has been described and exemplified in sufficient detail. However, various variations, modifications or improvements should be considered It is tantamount to the spirit and scope of this invention.

Those with ordinary knowledge in the technical field to which the present invention pertains can easily understand and achieve the objectives of the present invention, and obtain the results and advantages mentioned earlier. The cells, animals, and the processes and methods for producing them used in the present invention represent the best embodiments, are exemplary in nature, and are not intended to limit the scope of the present invention. Those skilled in the art and the modifications or other uses that will be produced when making or using this technique are all within the spirit of the present invention and are defined by the scope of rights.

Claims (14)

A composition is used to prepare a medicine for reversing the symptoms or disorders of abnormal collagen proliferation after damage to the peritoneum and/or peritoneal covering organs in an individual in need, wherein the composition contains umbilical cord mesenchymal stem cells. Such as the application of the first item in the scope of patent application, wherein the umbilical cord mesenchymal stem cell line is derived from umbilical cord Wharton’s jelly. Such as the use of the second item in the scope of patent application, wherein the source species of the umbilical cord Watton's gel is the same or different from the individual species in need. The patent application 242,800 of the first range, wherein the Treatment of umbilical mesenchymal stem cells effective dose of 10 ⁶ cells. The patent application 242,800 of the first range, wherein the Treatment of umbilical mesenchymal stem cells effective dose of 10 ⁸ cells. Such as the use of item 1 in the scope of the patent application, wherein the composition further comprises a conditioned medium of umbilical cord mesenchymal stem cells. A composition is used to prepare a medicine for treating the symptoms or disorders of abnormal collagen proliferation after damage to the peritoneum and/or peritoneal covering organs in an individual in need, wherein the composition is a conditioned medium containing umbilical cord mesenchymal stem cells. For example, the application of item 7 in the scope of patent application, wherein the conditioned medium of the umbilical cord mesenchymal stem cells is the culture medium after culturing the umbilical cord mesenchymal stem cells in a normal oxygen environment. For example, the application of item 7 of the scope of patent application, wherein the conditioned medium of umbilical cord mesenchymal stem cells is derived from the culture medium after culturing the umbilical cord mesenchymal stem cells in an environment with an oxygen concentration of not more than 1%. Such as the use of item 7 of the scope of patent application, wherein the drug is administered to the individual in need at a frequency of twice a week. Such as the application of item 7 of the scope of patent application, wherein the drug is administered to the individual in need by intraperitoneal injection. Such as the use of item 7 of the scope of patent application, wherein the composition further comprises umbilical cord mesenchymal stem cells. Such as the application of item 1 or 7 in the scope of patent application, wherein the composition can promote the expression of matrix metalloproteinase. Such as the use of item 13 in the scope of patent application, wherein the matrix metalloproteinase is matrix metalloproteinase-13 (matrix metalloproteinase-13).

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