Medicine, medicine composition and application of medicine and medicine composition in treatment of liver fibrosis

Abstract

The invention relates to a medicine, a medicine composition and application of the medicine and the medicine composition in treatment of liver fibrosis. The medicine and the medicine composition comprise an umbilical cord mesenchymal stem cell membrane; the umbilical cord mesenchymal stem cell membrane is prepared by the following steps: coating the surface of a temperature-sensitive culture dish with a matrix; adding the cell suspension of the umbilical cord mesenchymal stem cells into a temperature-sensitive culture dish for culturing; the temperature is reduced, the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by the umbilical cord mesenchymal stem cells are separated in a lamellar mode, and the umbilical cord mesenchymal stem cell membrane is obtained. The medicine and the medicine composition provided by the invention can effectively relieve liver fibrosis.

Description

Technical Field

[0001] The present disclosure relates to the fields of regenerative medicine and cell biology, and in particular to a drug, a pharmaceutical composition, and applications thereof in treating liver fibrosis. Background Art

[0002] Liver fibrosis is a common response of the liver to chronic damage, such as viral infections, alcohol, drugs, metabolic diseases, and autoimmune attacks on liver cells. Although many achievements have been made in the treatment of liver fibrosis in recent years, such as liver transplantation and the use of artificial livers, the number of patients suffering from liver disease continues to rise. The number of patients who are suitable for liver transplantation is relatively small, and the cost is high. Therefore, patients with liver fibrosis are in urgent need of effective alternatives to liver transplantation. Summary of the Invention

[0003] The present disclosure provides a drug, a pharmaceutical composition, and applications thereof in treating liver fibrosis to address deficiencies in related technologies.

[0004] According to a first aspect of the embodiments of the present disclosure, a medicine is provided, comprising an umbilical cord mesenchymal stem cell sheet, wherein the umbilical cord mesenchymal stem cell sheet is prepared by the following steps:

[0005] Coating the matrix on the surface of the thermosensitive culture dish;

[0006] The cell suspension of umbilical cord mesenchymal stem cells is added into a thermosensitive culture dish for culture;

[0007] The temperature is lowered, and the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by them are separated into sheets, thereby obtaining an umbilical cord mesenchymal stem cell membrane sheet.

[0008] In one aspect of the present disclosure, the extracellular matrix in the umbilical cord mesenchymal stem cell sheet comprises at least fibronectin and integrin family.

[0009] In one aspect of the present disclosure, the umbilical cord mesenchymal stem cell sheet is capable of secreting the following compounds: interleukin-6, transforming growth factor-β, prostaglandin E2, hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolic enzymes and nitric oxide synthase.

[0010] In one aspect of the present disclosure, the coating matrix comprises at least one of the following: collagen, gelatin, fibronectin, fibronectin, and vitronectin.

[0011] In one aspect of the present disclosure, the coating temperature is selected from 36°C to 39°C.

[0012] In one aspect of the present disclosure, coating is performed under saturated humidity and a concentration of 3% to 6% CO2.

[0013] In one aspect of the present disclosure, the coating time is selected from 0.5 to 48 hours.

[0014] In one aspect of the present disclosure, the culture process comprises: taking 1-4 mL of a total cell content of 1.8×10 5 ~1.8×10 7 The cell suspension of umbilical cord mesenchymal stem cells is added to the temperature-sensitive culture dish and cultured for 2 to 48 hours.

[0015] In one aspect of the present disclosure, the culture temperature is selected from 36°C to 39°C.

[0016] In one aspect of the present disclosure, the culturing is performed under saturated humidity and a CO2 concentration of 3% to 6%.

[0017] In one aspect of the present disclosure, after the culture is completed, the temperature-sensitive culture dish is moved to an environment of 4° C. to 32° C., and the umbilical cord mesenchymal stem cell sheet is peeled off from the temperature-sensitive culture dish.

[0018] In one aspect of the present disclosure, the umbilical cord mesenchymal stem cell sheet is circular or approximately circular with a diameter of 3 to 30 mm.

[0019] In one aspect of the present disclosure, the thickness of the umbilical cord mesenchymal stem cell sheet is selected from 50 to 1000 μm.

[0020] In one aspect of the present disclosure, the cell density in the umbilical cord mesenchymal stem cell sheet is selected from 1×10 6 ~1×10 8 .

[0021] In one aspect of the present disclosure, the aforementioned medicament is used to treat liver fibrosis.

[0022] According to a second aspect of the embodiments of the present disclosure, a pharmaceutical composition is provided, comprising the aforementioned drug and excipients.

[0023] In one aspect of the present disclosure, the aforementioned pharmaceutical composition is used to treat liver fibrosis.

[0024] According to a third aspect of the embodiments of the present disclosure, there is provided use of the aforementioned drugs and pharmaceutical compositions in the treatment of liver fibrosis.

[0025] In one aspect of the present disclosure, the administration method of the umbilical cord mesenchymal stem cell sheet satisfies at least one of the following conditions:

[0026] i) attaching the umbilical cord mesenchymal stem cell sheet to the surface of the liver and between the liver lobes;

[0027] ii) attaching the umbilical cord mesenchymal stem cell sheet between the liver lobes;

[0028] iii) attaching the umbilical cord mesenchymal stem cell membrane to other parts of the abdominal cavity.

[0029] The technical solutions provided by the embodiments of the present disclosure may have the following beneficial effects:

[0030] It can be seen from the above embodiments that the present disclosure provides a medicine and a pharmaceutical composition comprising an umbilical cord mesenchymal stem cell sheet for treating liver fibrosis.

[0031] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0033] Figure 1 is a pathological section image of the liver after treatment according to an exemplary embodiment.

[0034] Figure 2 FIG. 4 is a scoring image of liver glycogen according to an exemplary embodiment.

[0035] Figure 3 FIG. 1 is a liver glycogen pathological staining image according to an exemplary embodiment.

[0036] Figure 4 FIG. 1 is an image of cell apoptosis according to an exemplary embodiment.

[0037] Figure 5 FIG. 4 is a cytokine secretion image according to an exemplary embodiment. DETAILED DESCRIPTION

[0038] The exemplary embodiments are described in detail herein. In the following description, when referring to the accompanying drawings, identical numerals in different drawings represent identical or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments are not intended to represent all possible embodiments consistent with the present disclosure. Rather, they are merely examples of apparatuses and methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

[0039] The terms used in this disclosure are for the purpose of describing specific embodiments only and are not intended to limit the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," "the," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0040] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell sheet provided by the present disclosure retains an intact extracellular matrix and has adhesion properties, and does not require suturing during transplantation; moreover, the umbilical cord mesenchymal stem cell sheet provided by the present disclosure can release a variety of cytokines at the attachment site, and these multiple cytokines can repair liver tissue, eliminate or reduce inflammatory responses, and regulate the immune microenvironment, thereby improving liver function.

[0041] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell sheet is prepared by the following steps:

[0042] Coating the matrix on the surface of the thermosensitive culture dish;

[0043] The cell suspension of umbilical cord mesenchymal stem cells is added into a thermosensitive culture dish for culture;

[0044] The temperature is lowered, and the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by them are separated into sheets, thereby obtaining an umbilical cord mesenchymal stem cell membrane sheet.

[0045] In some embodiments of the present disclosure, the process of coating the surface of a temperature-sensitive culture dish with a matrix includes:

[0046] The coating matrix is ​​diluted with a physiological buffer, wherein the coating matrix comprises at least one of the following: collagen, gelatin, fibronectin, adhesion protein, vitronectin, laminin, poly-ornithine and poly-lysine, but is not limited thereto; the physiological buffer is selected from physiological saline or PBS buffer, but is not limited thereto;

[0047] The diluted solution is added to a thermosensitive culture dish, the lid of the thermosensitive culture dish is closed, and then the thermosensitive culture dish is placed in an environment with a coating temperature of 36°C to 39°C, saturated humidity, and a CO2 concentration of 3% to 6% for coating; specifically, the thermosensitive culture dish can be placed in an environment with a coating temperature of 37°C, saturated humidity, and a CO2 concentration of 5% for coating; the coating time is selected from 0.5 to 48 hours.

[0048] In some embodiments of the present disclosure, the coating temperature may be selected from 36°C to 37°C, 37°C to 38°C, or 38°C to 39°C, preferably 37°C.

[0049] In some embodiments of the present disclosure, the concentration of CO2 during coating can be selected from 3% to 4%, 4% to 5%, or 5% to 6%, preferably 5%.

[0050] In some embodiments of the present disclosure, the coating time is selected from 0.5-1 h, 1-2 h, 2-3 h, 3-4 h, 4-5 h, 5-6 h, 6-7 h, 7-8 h, 8-9 h, 9-10 h, 10-15 h, 15-20 h, 20-25 h, 25-30 h, 30-35 h, 35-40 h, 40-45 h or 45-48 h.

[0051] In some embodiments of the present disclosure, the culturing process comprises:

[0052] Remove the aforementioned coating matrix from the thermosensitive culture dish and take 1-4 mL of the culture medium with a total cell content of 1.8×10 5 ~1.8×10 7 The cell suspension and culture medium of umbilical cord mesenchymal stem cells are added into a temperature-sensitive culture dish;

[0053] The thermosensitive culture dish is placed in an environment with a culture temperature of 36° C. to 39° C., saturated humidity, and a concentration of 3% to 6% CO2 for culturing; specifically, the thermosensitive culture dish is placed in an environment with a culture temperature of 37° C., saturated humidity, and a concentration of 5% CO2 for culturing, and the culturing time is selected from 2 to 48 hours;

[0054] After the culture is completed, the temperature-sensitive culture dish is moved to a low-temperature environment of 4° C. to 32° C., and the umbilical cord mesenchymal stem cell membrane is peeled off from the temperature-sensitive culture dish in a sheet-like manner.

[0055] In some embodiments of the present disclosure, the culture medium is a serum-free medium; the serum-free medium may be a combination of a basal medium and nutrient additives, or a commercial serum-free medium.

[0056] In some embodiments of the present disclosure, the basal culture medium can be selected from any one of the following: 1640 cell culture medium, DMEM cell culture medium, α-MEM cell culture medium, DMEM / F12 cell culture medium, and F12 cell culture medium, but are not limited thereto; the nutrient additives can be selected from any one of the following: sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin, lutein, 1,4-butanediamine, biotin, sodium pyruvate, ethanolamine, vitamin Bt, amino acids, vitamin C, glutathione, linoleic acid, and linolenic acid, but are not limited thereto. In some embodiments of the present disclosure, commercial serum-free culture medium can also be used, wherein the commercial serum-free culture medium can be selected from CTSTM StemPro™ MSC SFM Kit, MesenCult™-ACF Medium, MesenCult™-ACF Plus Medium, and Mesen Cult™-XF Medium, but are not limited thereto.

[0057] In some embodiments of the present disclosure, during the culture process, the ratio of the culture medium in the temperature-sensitive culture dish is 0.1 to 0.5 mL / cm 2 (e.g. 0.1-0.2 mL / cm 2 , 0.2~0.3mL / cm 2 , 0.3~0.4mL / cm 2 or 0.4–0.5 mL / cm 2 , preferably 0.3 mL / cm 2 ).

[0058] In some embodiments of the present disclosure, the culture temperature may be selected from 36°C to 37°C, 37°C to 38°C, or 38°C to 39°C, preferably 37°C.

[0059] In some embodiments of the present disclosure, the concentration of CO2 during culture can be selected from 3% to 4%, 4% to 5%, or 5% to 6%, preferably 5%.

[0060] In some embodiments of the present disclosure, the culture time is selected from 0.5-1 h, 1-2 h, 2-3 h, 3-4 h, 4-5 h, 5-6 h, 6-7 h, 7-8 h, 8-9 h, 9-10 h, 10-15 h, 15-20 h, 20-25 h, 25-30 h, 30-35 h, 35-40 h, 40-45 h or 45-48 h.

[0061] In some embodiments of the present disclosure, the temperature of the low temperature environment of 4°C to 32°C may be 4°C to 5°C, 5°C to 6°C, 6°C to 7°C, 7°C to 8°C, 8°C to 9°C, 9°C to 10°C, 10°C to 12°C, 12°C to 14°C, 14°C to 16°C, 16°C to 18°C, 18°C ​​to 20°C, 20°C to 22°C, 22°C to 24°C, 24°C to 26°C, 26°C to 28°C, 28°C to 30°C or 30°C to 32°C.

[0062] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell membrane obtained by the present disclosure contains multiple layers of cells, which can be at least 2 layers, at least 3 layers, at least 4 layers, at least 5 layers, at least 6 layers, at least 7 layers, at least 8 layers, at least 9 layers, at least 10 layers, at least 12 layers, at least 14 layers, at least 16 layers, at least 18 layers, at least 20 layers or more layers.

[0063] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell membrane obtained in the present disclosure is circular with a diameter of 3 to 30 mm, which can be 3 to 4 mm, 4 to 5 mm, 5 to 6 mm, 6 to 7 mm, 7 to 8 mm, 8 to 9 mm, 9 to 10 mm, 10 to 12 mm, 12 to 14 mm, 14 to 16 mm, 16 to 18 mm, 18 to 20 mm, 20 to 22 mm, 22 to 24 mm, 24 to 26 mm, 26 to 28 mm or 28 to 30 mm.

[0064] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell membrane obtained in the present disclosure can also be a circular shape with a diameter of 3 to 30 mm, which can be 3 to 4 mm, 4 to 5 mm, 5 to 6 mm, 6 to 7 mm, 7 to 8 mm, 8 to 9 mm, 9 to 10 mm, 10 to 12 mm, 12 to 14 mm, 14 to 16 mm, 16 to 18 mm, 18 to 20 mm, 20 to 22 mm, 22 to 24 mm, 24 to 26 mm, 26 to 28 mm or 28 to 30 mm.

[0065] In some embodiments of the present disclosure, the thickness of the umbilical cord mesenchymal stem cell membrane obtained in the present disclosure is selected from 50 to 1000 μm, which can be 50 to 60 μm, 60 to 70 μm, 70 to 80 μm, 80 to 90 μm, 90 to 100 μm, 100 to 120 μm, 120 to 140 μm, 140 to 160 μm, 160 to 180 μm, 180 to 200 μm, 200 to 250 μm, 250~300μm, 300~350μm, 350~400μm, 400~450μm, 450~500μm, 500~550μm, 550~600μm, 600~6 50μm, 650~700μm, 700~750μm, 750~800μm, 800~850μm, 850~900μm, 900~950μm or 950~1000μm.

[0066] In some embodiments of the present disclosure, the cell density in the umbilical cord mesenchymal stem cell sheet obtained in the present disclosure is selected from 1×10 6 ~1×10 8 ; it can be 1×10 6 ~2×10 6 / cm 2 , 2×10 6 ~3×10 6 / cm 2 , 3×10 6 ~4×10 6 / cm 2 , 4×10 6 ~5×10 6 / cm 2 , 5×10 6 ~6×10 6 / cm 2 , 6×10 6 ~7×10 6 / cm 2 , 7×10 6 ~8×10 6 / cm 2 , 8×10 6 ~9×10 6 / cm 2 , 9×10 6 ~1×10 7 / cm 2 , 1×10 7 ~2×10 7 / cm 2 , 3×10 7 ~4×10 7 / cm 2 , 4×10 7 ~5×107 / cm 2 , 5×10 7 ~6×10 7 / cm 2 , 6×10 7 ~7×10 7 / cm 2 , 7×10 7 ~8×10 7 / cm 2 , 8×10 7 ~9×10 7 / cm 2 , 9×10 7 ~1×10 8 / cm 2 ; but not limited to this.

[0067] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell membrane obtained in the present disclosure is off-white, has a dense structure, and a smooth surface.

[0068] In some embodiments of the present disclosure, no additional reagents or materials are added when the umbilical cord mesenchymal stem cell membrane sheet and the extracellular matrix secreted therefrom are detached in sheets.

[0069] In some embodiments of the present disclosure, the medicine or pharmaceutical composition provided by the present disclosure may also include umbilical cord mesenchymal stem cell membrane fragments and / or split umbilical cord mesenchymal stem cell membrane sheets, wherein the umbilical cord mesenchymal stem cell membrane fragments and / or split umbilical cord mesenchymal stem cell membrane sheets are obtained by crushing or splitting the umbilical cord mesenchymal stem cell membrane sheets obtained by the present disclosure.

[0070] In some embodiments of the present disclosure, the diameter of the cell membrane fragments is about 0.1-3 mm, for example, about 0.1-0.2 mm, 0.2-0.3 mm, 0.3-0.4 mm, 0.4-0.5 mm, 0.5-0.6 mm, 0.6-0.7 mm, 0.7-0.8 mm, 0.8-0.9 mm, 0.9-1 mm, 1-1.1 mm, 1.1-1.2 mm, 1.2-1.3 mm, 1.3- 1.4mm, 1.4-1.5mm, 1.5-1.6mm, 1.6-1.7mm, 1.7-1.8mm, 1.8-1.9mm, 1.9-2mm, 2-2.1mm, 2.1-2.2mm, 2.2-2.3mm, 2.3-2.4mm, 2.4-2.5mm, 2.5-2.6mm, 2.6-2.7mm, 2.7-2.8mm, 2.8-2.9mm or 2.9-3mm. It should be noted that the diameter of the cell fragment refers to the longest line segment connecting two non-adjacent vertices in each cell fragment.

[0071] In some embodiments of the present disclosure, the umbilical cord mesenchymal stem cell membrane fragments are obtained by injecting the umbilical cord mesenchymal stem cell membrane fragments obtained in the present disclosure through a syringe needle, but are not limited thereto and can also be obtained by other means.

[0072] In some embodiments of the present disclosure, the cell membrane fragments are obtained by the following method:

[0073] 1) Load the cell membrane sheet into the syringe needle;

[0074] 2) After normal saline is drawn into the syringe barrel, the syringe needle loaded with the cell membrane sheet is installed onto the syringe barrel;

[0075] 3) Pushing the syringe piston to inject the cell membrane to obtain cell membrane fragments;

[0076] Optionally, in step 1), the cell membrane sheet is pre-cut (to accommodate different administration doses).

[0077] In some embodiments of the present disclosure, the needle gauge is 22G, 23G, 25G or 27G. The needle gauges in the present disclosure adopt international standards.

[0078] In some embodiments of the present disclosure, the needle is of gauge 22G or 27G.

[0079] In some embodiments of the present disclosure, the cell membrane fragments are no less than 2 pieces, for example, no less than 3 pieces, no less than 4 pieces, no less than 5 pieces, no less than 6 pieces, no less than 7 pieces, no less than 8 pieces, no less than 9 pieces, no less than 10 pieces, no less than 12 pieces, no less than 14 pieces, no less than 16 pieces, no less than 18 pieces, no less than 20 pieces, no less than 22 pieces, no less than 24 pieces, no less than 26 pieces, no less than 28 pieces, no less than 30 pieces or more pieces.

[0080] The present disclosure also provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which includes primary isolation and culture and subculture.

[0081] In some embodiments of the present disclosure, the primary isolation culture comprises the following steps: isolating Wharton's jelly from umbilical cord tissue; mincing the Wharton's jelly to obtain tissue blocks; plating the tissue blocks in a culture container for culturing; adding an appropriate amount of complete culture medium to cover the tissue blocks and continuing to culture; when cells (in a fibrous form) attached to the culture container appear around the tissue blocks and the cells grow to 70-100% confluence, removing the tissue blocks and performing a cell passaging operation.

[0082] In some embodiments, the subculture process includes the following steps: separating the cells from the culture vessel and evenly dispersing the cells in culture medium, followed by inoculation into the culture vessel; adding an appropriate amount of culture medium, replacing the culture medium with an appropriate amount of fresh medium every 1-5 days depending on the cell growth status; and repeating the subculture process when the cells reach 70-100% confluence. Each time the cells are subcultured, the number of generations increases by one. Umbilical cord mesenchymal stem cells adhere to the culture vessel and exhibit a uniform, fibroblast-like morphology.

[0083] In the present disclosure, the pharmaceutical composition provided further includes excipients, which can be any pharmaceutically acceptable excipients without specific limitation and can be selected according to actual needs.

[0084] The present invention will be further described below with reference to specific embodiments. However, these specific embodiments are not to be construed as limiting the scope of protection of the present invention. Those skilled in the art may make various changes or modifications to these specific embodiments without departing from the scope of the technical solution of the present invention, and such changes and modifications will still fall within the scope of protection of the present invention.

[0085] Hereinafter, the present invention will be described through specific embodiments.

[0086] The coating process and culture process provided by the present disclosure include a serum-free culture medium and a coating matrix matched with the culture medium, wherein:

[0087] 1. The composition of serum-free medium can be basal medium + nutrient additives, or commercial serum-free medium, as follows:

[0088] (1) Basic culture medium + nutrient additives:

[0089] The basal culture medium can be selected from 1640 cell culture medium, DMEM cell culture medium, α-MEM cell culture medium, DMEM / F12 cell culture medium and F12 cell culture medium;

[0090] The nutritional additives include one or more of sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin, progesterone, 1,4-butanediamine, biotin, sodium pyruvate, ethanolamine, vitamin Bt, amino acids, vitamin C, glutathione, linoleic acid and linolenic acid.

[0091] (2) Commercial serum-free culture medium:

[0092] Including but not limited to: CTSTMStemProTMMSC SFM Kit, MesenCultTM-ACF Medium, MesenCultTM-ACF Plus Medium, MesenCultTM-XF Medium, etc.

[0093] 2. Coating matrix matched with culture medium:

[0094] Including but not limited to collagen, gelatin, fibronectin, fibrinogen, adhesion protein, vitronectin, laminin, polyornithine, polylysine and the like.

[0095] Example 1: Isolation, culture and passage of umbilical cord mesenchymal stem cells

[0096] The collected umbilical cord of a newborn (sourced from Beijing Mingde Hospital and donated by pregnant women with signed informed consent, which has been approved by the Ethics Committee of Mingde Hospital) is washed with a physiological solution with an osmotic pressure equal to that of human body fluids, after which the arteries, veins, and adventitia are removed, Wharton's jelly is separated, and the cord is minced into small tissue blocks of 0.1 to 2 mm. The tissue blocks are evenly spread in a culture container coated with a matrix, with a spacing of 2 to 30 mm between the tissue blocks. The culture container is placed in a cell culture incubator and cultured for 2 to 7 days. An appropriate amount of complete culture medium is then added to cover the tissue blocks. The umbilical cord mesenchymal stem cells crawl out on approximately 8 to 21 days. The crawled-out cells grow adherently to the wall and have a uniform fibrous morphology. When the migrated cells grow to 70% to 100% confluence, the tissue blocks are removed and the cells are passaged.

[0097] In the subculture operation, the cells are separated from the culture container and then dispersed in the culture medium by methods including but not limited to stirring or vortexing, and the cells are plated at a rate of 500 to 100,000 cells / cm 2 The cells are seeded into a culture vessel at a density of 100 μg / mL. Then, an appropriate amount of culture medium is added. Depending on the cell growth status, an appropriate amount of fresh culture medium is replaced every 1 to 5 days. When the cells grow to 70% to 100% confluence, the subculturing operation is repeated.

[0098] Example 2: Identification of umbilical cord mesenchymal stem cells

[0099] 2.1 Umbilical cord mesenchymal stem cell growth curve detection:

[0100] Methods for determining the growth curve of umbilical cord mesenchymal stem cells include, but are not limited to, the MTT method, the WST method, the DNA content detection method, and the ATP detection method. The WST method is taken as an example for further explanation.

[0101] The umbilical cord mesenchymal stem cells obtained in Example 1 are dispersed, with reference to commercially available reagent instructions, inoculated into culture well plates with a certain density, and carried out liquid exchange operation according to the culture conditions commonly used in this area. At a fixed time every day for a period of time, cell activity is detected according to the instructions, and the data of cell activity or quantity are obtained. Within the range of 7 days, WST reagent is utilized to detect the activity of cells every day. The method is that WST reagent is added to the cell well plates being cultivated according to the instruction recommended ratio, and the absorbance value of the well plate liquid at a wavelength of 450nm is detected with a microplate reader or an ultraviolet spectrophotometer after incubation for a fixed time in a cell culture box. This value is positively correlated with the number of cells. From its result, it can be seen that with the extension of incubation time, the activity of cells in the chamber increases, and it can be inferred that the number of cells increases with the extension of incubation time.

[0102] 2.2 Identification of surface markers of umbilical cord mesenchymal stem cells:

[0103] The umbilical cord mesenchymal stem cells obtained in Example 1 were dispersed in a culture medium and centrifuged. Cell surface marker proteins, including but not limited to CD73, CD90, CD105, CD34, CD11B, CD19, CD45, and HLA-DR, were stained using serum or a physiological solution with a serum protein content of 1% to 20% and an osmotic pressure equivalent to that of human body fluids according to the instructions of commercially available reagents. The phenotypes for CD73, CD90, and CD105 were positive, with a ratio of not less than 95%, and the phenotypes for CD34, CD11B, CD19, CD45, and HLA-DR were negative, with a ratio of not more than 2%.

[0104] 2.3 Three-way differentiation of umbilical cord mesenchymal stem cells:

[0105] Umbilical cord mesenchymal stem cells have the ability to induce differentiation into bone, cartilage, and fat. The umbilical cord mesenchymal stem cells obtained in Example 1 were inoculated into a suitable culture vessel according to the proportions in the instructions for the three-way induction differentiation reagent. When the cells for osteogenic induction test grew to 50% to 90% confluence and the cells for adipogenic induction test grew to more than 90% confluence, osteogenic and adipogenic induction medium were added respectively. During chondrogenic induction, a certain number of cells were centrifuged to the bottom of the centrifuge tube, and then chondrogenic induction medium was added. After the cells were pelleted, the cell pellets were removed from the bottom of the tube to ensure complete contact with the induction medium. The cells were tested after induced culture for more than 7 days. Osteogenic induction can be stained with, but not limited to, Alizarin Red and anti-Osteocalcin. Adipogenic induction can be stained with, but not limited to, Oil Red O and anti-FABP4. Chondrogenic induction can be stained with, but not limited to, Alcian Blue, Safranin O, and anti-Aggrecan.

[0106] Example 3: Preparation of umbilical cord mesenchymal stem cell membrane

[0107] 3.1 Preparation process of umbilical cord mesenchymal stem cell membrane:

[0108] (1) Thermosensitive smart culture dish coating

[0109] First, a layer of matrix that facilitates the attachment of mesenchymal stem cells is coated on the surface of the temperature-sensitive smart culture dish, and the coated matrix is ​​diluted with a physiological buffer, wherein the coating matrix includes at least one of the following: collagen, gelatin, fibronectin, adhesion protein, vitronectin, laminin, poly-ornithine, and poly-lysine; and the physiological buffer is selected from physiological saline or PBS buffer;

[0110] The diluted solution is added to a thermosensitive culture dish, the lid of the thermosensitive culture dish is closed, and the thermosensitive culture dish is placed in an environment with a coating temperature of 37°C, saturated humidity, and 5% CO2 concentration for coating; the coating time is selected from 0.5 to 48 hours;

[0111] Remove the aforementioned coating matrix from the thermosensitive culture dish and take 1-4 mL of the culture medium with a total cell content of 1.8×10 5 ~1.8×10 7 The cell suspension of umbilical cord mesenchymal stem cells was added into a temperature-sensitive culture dish;

[0112] The thermosensitive culture dish is placed in an environment of 37°C, saturated humidity, and 5% CO2 for culturing for a period of 2 to 48 hours;

[0113] After the culture is completed, the temperature-sensitive culture dish is moved to an environment of 4°C to 32°C, and the umbilical cord mesenchymal stem cell membrane is peeled off from the temperature-sensitive culture dish in a sheet-like manner. The umbilical cord mesenchymal stem cell membrane is a circle or a similar circle with a diameter of 3 to 30 mm and a thickness of 50 to 1000 μm; the cell density in the umbilical cord mesenchymal stem cell membrane is selected from 1×10 6 ~1×10 8 The umbilical cord mesenchymal stem cell membrane obtained in Example 3 was off-white, had a dense structure, and a smooth surface.

[0114] Example 4: Functional testing of umbilical cord mesenchymal stem cell membranes

[0115] 4.1 Detection of secreted factors of umbilical cord mesenchymal stem cell membranes by enzyme-linked immunosorbent assay

[0116] The supernatant obtained in the preparation process of the umbilical cord mesenchymal stem cell membrane sheet obtained in Example 3 was tested according to the instructions of the enzyme-linked immunosorbent assay kit to determine the content of various factors secreted by the umbilical cord mesenchymal stem cell membrane sheet. The factors referred to here include but are not limited to interleukin-6 (IL-6), transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor (VEGF), insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolic enzyme (IDO) and nitric oxide synthase (iNOS), etc. Example 4 selected interleukin-6, hepatocyte growth factor, and vascular endothelial growth factor as examples to detect the secretion factors of the umbilical cord mesenchymal stem cell membrane sheet; the experiment was repeated three times (using umbilical cords from different sources); blank culture medium was used as a control, and the secretion of relevant cytokines was detected by ELISA method, such as Figure 5 As shown in the results, human umbilical cord mesenchymal stem cell membrane can secrete a large amount of hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and interleukin-8 (IL-8). The above content shows that this cell membrane product has the potential to promote cell growth, promote angiogenesis, eliminate inflammation, and regulate the microenvironment.

[0117] 4.2 Tissue sections of fibronectin in umbilical cord mesenchymal stem cell membranes

[0118] The umbilical cord mesenchymal stem cell membrane obtained in Example 3 is fixed with paraformaldehyde or formalin fixative, and then the cell membrane is made into a tissue section with a thickness of 4 to 10 μm by paraffin sectioning or frozen sectioning to be stained, and the protein content of the extracellular matrix in the cell membrane is observed, wherein the protein includes but is not limited to fibronectin, integrin, vitronectin, etc., and the cell nucleus is usually stained with fluorescent dyes such as DAPI or Hoechst33258 staining at the same time as the staining to assist in positioning. In addition to the immunofluorescence method, staining observation can also be performed using methods including but not limited to immunohistochemistry. Taking fibronectin and integrin-β1 as examples, fibronectin and integrin-β1 are both stained with fluorescently labeled antibody dyes, and the cell nucleus is stained with DAPI, so it can be seen that the prepared cell membrane contains a large amount of fibronectin and integrin-β1.

[0119] 4.3 Microstructure of umbilical cord mesenchymal stem cell membrane obtained by scanning electron microscopy

[0120] The umbilical cord mesenchymal stem cell membrane obtained in Example 3 was fixed with 2.5% glutaraldehyde, then dehydrated through a gradient of alcohol and air-dried to obtain a dried cell membrane for scanning electron microscopy analysis. The dried cell membrane was affixed to the surface of a sample stage using conductive double-sided tape and then gold-sputtered using vacuum magnetron sputtering to make the surface conductive. The sample stage was then placed in a scanning electron microscope (Hitachi S-4800) for observation.

[0121] Example 5: Activity test of umbilical cord mesenchymal stem cell membrane

[0122] The umbilical cord mesenchymal stem cell membrane obtained in Example 3 was placed in a preservation solution and temporarily stored at 4°C.

[0123] 5.1 Animal Modeling

[0124] (1) C57BL / 6J male mice were selected and injected intraperitoneally with CCl4 (0.7 μl / g) twice a week for 8 consecutive weeks.

[0125] (2) Blood biochemistry (ALT, AST) was measured after 4 weeks of use to preliminarily determine changes in liver function;

[0126] (3) At 7 weeks, two C57BL / 6J male mice were randomly selected for dissection to observe whether there were white particles on the liver surface and whether the liver texture had become hard.

[0127] 5.2 Patches:

[0128] (1) C57BL / 6J male mice were anesthetized by intraperitoneal injection of sodium pentobarbital. Care was taken in the operation to avoid damaging the abdominal organs. After anesthesia, the rats were placed supine on the operating table and their limbs were fixed with a fixator. The hair on the lower chest and abdomen was shaved and disinfected with iodine.

[0129] (2) Use scissors to make a semicircular incision in the middle of the rat's abdomen, turn the skin back, and expose the liver so that the surgical field is fully exposed.

[0130] (3) Then, use a cotton swab to gently push the liver out of the abdominal cavity, remove the umbilical cord mesenchymal stem cell membrane, transplant the membrane onto the surface of the liver, and after confirming that it is firmly attached, gently return the liver to the abdominal cavity. The membrane transplantation method is as follows: anesthesia, open the abdomen under the xiphoid process, squeeze out the liver, turn over the middle lobe of the liver, repeatedly wipe the surface of the left lobe of the liver with a cotton swab, place 1 / 4 of the membrane on the surface of the left lobe of the liver, cover the middle lobe of the liver, return the liver to the abdominal cavity and suture.

[0131] (4) Finally, suture the wound.

[0132] 5.3 Effectiveness test:

[0133] Multiple experimental groups with different doses were set up, G1 was the control group, that is, olive oil was used instead of CCl4 for intraperitoneal injection; G2 was the sham operation control group, G3 was the MSC suspension group, G4 was the MSC membrane group, and G5 was the modeling group.

[0134] Administration results: After 6-8 weeks of CCl4 induction in C57BL / 6J mice, gross pathological observations showed that, except for the normal control group, the livers of animals in the CCL4 model group were rough in texture, yellow-brown in color, slightly adhered to surrounding tissues, with little ascites, and picrosirius red staining of the liver was consistent with the characteristics of liver fibrosis.

[0135] After MSC transplantation, weight increased in the G1 and G5 control groups. Weight in the G2 to G4 groups initially decreased and then increased, consistent with the effects of surgery or tail vein injection on mice. Compared to the sham-operated group, the MSC suspension and membrane groups showed a trend toward improved liver damage. The number of mice in each group increased compared to the sham-operated group, and total bilirubin levels increased, indicating enhanced secretion and excretion.

[0136] In vivo imaging of MSCs transplanted into ICR mice revealed that the fluorescence signal decayed with the date of transplantation. The fluorescence signal in NPG mice decayed more slowly, but also dropped to a relatively low level after 7-10 days. In immunodeficient NPG mice, the fluorescence signal was essentially undetectable by 28 days.

[0137] At 14 days after MSC transplantation, there was no significant difference in fibrosis scores between the G3 and G2 groups (P = 0.0786); there was no significant difference in fibrosis scores between the G4 and G2 groups (P = 0.0786). At 31 days after MSC transplantation, there was a significant difference in fibrosis scores between the G3 and G2 groups (P = 0.0049); there was no significant difference in fibrosis scores between the G4 and G2 groups (P = 0.8937). When all samples at 14 and 31 days were analyzed together, there was a highly significant difference in fibrosis scores between the G3 (MSC suspension group) and the G2 (sham control group) (P = 0.0004); and there was a highly significant difference in fibrosis scores between the G4 (MSC sheet group) and the G2 (sham control group) (P = 0.0476).

[0138] The pathological sections of the liver after treatment in the control group, MSC suspension group and membrane group were as follows: Figure 1 As shown, it can be seen that both the MSC suspension group and the membrane sheet group can alleviate the ballooning degeneration and fibrosis of the liver, and the membrane sheet group has a more obvious effect in alleviating the ballooning degeneration and fibrosis of the liver.

[0139] The liver glycogen scores of the control group, sham operation group, MSC suspension group and membrane sheet group were as follows: Figure 2 As shown in Figure 2, the pathological staining of hepatic glycogen in the control group, sham operation group, MSC suspension group and membrane sheet group (day 14) was as follows: Figure 3 As shown in Figure 2, the membrane group promoted the synthesis of hepatic glycogen better. Figure 4 As shown, it can be seen that the number of apoptotic cells in the liver of the membrane group was significantly reduced.

[0140] In summary, both the MSC suspension and membrane groups can alleviate fibrosis, and the MSC membrane group is more effective in restoring liver function. In vivo imaging results of MSC transplanted into ICR and NPG mice showed that the cell fluorescence signal decayed with the date of transplantation, decreasing to a relatively low level after 7-10 days. The fluorescence signal of MSC transplanted into NPG mice decayed relatively slowly.

[0141] Other embodiments of the present disclosure will readily occur to those skilled in the art after considering the specification and practicing the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of the present disclosure that follow the general principles of the present disclosure and include common knowledge or customary techniques in the art not disclosed herein. The description and examples are to be considered as exemplary only, with the true scope and spirit of the present disclosure being indicated by the following claims.

[0142] It should be understood that the present disclosure is not limited to the exact structures that have been described above and shown in the drawings, and that various modifications and changes can be made without departing from the scope thereof. The scope of the present disclosure is limited only by the appended claims.

Claims

1. A drug, characterized in that The drug comprises an umbilical cord mesenchymal stem cell sheet; wherein the umbilical cord mesenchymal stem cell sheet is prepared by the following steps: Coating the matrix on the surface of the thermosensitive culture dish; The cell suspension of umbilical cord mesenchymal stem cells is added into a thermosensitive culture dish for culture; The temperature is lowered, and the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by them are separated into sheets, thereby obtaining an umbilical cord mesenchymal stem cell membrane sheet.

2. The drug according to claim 1, characterized in that The extracellular matrix in the umbilical cord mesenchymal stem cell membrane at least contains fibronectin and integrin family.

3. The drug according to claim 1, characterized in that The umbilical cord mesenchymal stem cell membrane can secrete one or more of the following cytokines: interleukin-6, transforming growth factor-β, prostaglandin E2, hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolic enzymes and nitric oxide synthase.

4. The drug according to claim 1, characterized in that The coating matrix includes at least one of the following: collagen, gelatin, fibronectin, fibronectin, vitronectin, laminin, polyornithine and polylysine.

5. The drug according to claim 1, characterized in that The coating temperature is selected from 36°C to 39°C.

6. The drug according to claim 5, characterized in that The coating was carried out in saturated humidity and 3% to 6% CO2 concentration.

7. The drug according to claim 1, characterized in that The coating time is selected from 0.5 to 48 hours.

8. The drug according to claim 1, characterized in that The culturing process comprises: Take 1-4 mL, the total cell content is 1.8×10 5 ~1.8×10 7 The cell suspension of umbilical cord mesenchymal stem cells is added to the temperature-sensitive culture dish and cultured for 2 to 48 hours.

9. The drug according to claim 8, characterized in that The culture temperature is selected from 36°C to 39°C.

10. The drug according to claim 9, characterized in that The culture was carried out under saturated humidity and 3% to 6% CO2 concentration.

11. The drug according to claim 1, characterized in that After the culture is completed, the temperature-sensitive culture dish is moved to an environment of 4° C. to 32° C., and the umbilical cord mesenchymal stem cell sheet is peeled off from the temperature-sensitive culture dish.

12. The drug according to any one of claims 8 to 11, characterized in that The umbilical cord mesenchymal stem cell sheet satisfies at least one of the following conditions: i) the umbilical cord mesenchymal stem cell membrane sheet is circular or approximately circular with a diameter of 3 to 30 mm; ii) the thickness of the umbilical cord mesenchymal stem cell membrane is selected from 50 to 1000 μm; iii) The cell density in the umbilical cord mesenchymal stem cell membrane is selected from 1×10 6 ~1×10 8 / cm 2 .

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the drug according to any one of claims 1 to 12, and excipients.

14. Use of the medicament according to any one of claims 1 to 12 or the pharmaceutical composition according to claim 13 in the treatment of liver fibrosis.

15. The use according to claim 14, characterized in that The administration method of the umbilical cord mesenchymal stem cell sheet satisfies at least one of the following conditions: i) attaching the umbilical cord mesenchymal stem cell sheet to the surface of the liver and between the liver lobes; ii) attaching the umbilical cord mesenchymal stem cell sheet between the liver lobes; iii) attaching the umbilical cord mesenchymal stem cell membrane to other parts of the abdominal cavity.

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