WO2024022079A9 - 人源间充质干细胞膜片在子宫瘢痕治疗中的用途 - Google Patents

人源间充质干细胞膜片在子宫瘢痕治疗中的用途 Download PDF

Info

Publication number
WO2024022079A9
WO2024022079A9 PCT/CN2023/106157 CN2023106157W WO2024022079A9 WO 2024022079 A9 WO2024022079 A9 WO 2024022079A9 CN 2023106157 W CN2023106157 W CN 2023106157W WO 2024022079 A9 WO2024022079 A9 WO 2024022079A9
Authority
WO
WIPO (PCT)
Prior art keywords
mesenchymal stem
stem cell
human mesenchymal
cell sheet
cells
Prior art date
Application number
PCT/CN2023/106157
Other languages
English (en)
French (fr)
Other versions
WO2024022079A1 (zh
Inventor
常德华
高爽
赵玉菲
刘帅
Original Assignee
京东方科技集团股份有限公司
京东方再生医学科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 京东方科技集团股份有限公司, 京东方再生医学科技有限公司 filed Critical 京东方科技集团股份有限公司
Publication of WO2024022079A1 publication Critical patent/WO2024022079A1/zh
Publication of WO2024022079A9 publication Critical patent/WO2024022079A9/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives

Definitions

  • the present disclosure relates to the technical field of stem cell application, and in particular to the use of a human mesenchymal stem cell membrane in the treatment of uterine scars.
  • the endometrium is a highly regenerative reproductive organ. Women will experience more than 400 shedding and regeneration of the endometrium in their lifetime. The thickness of the endometrium reflects the functional status of the endometrium. Scars are easily formed after the endometrium is damaged due to artificial abortion and cesarean section, which seriously affects the implantation of fertilized eggs and embryos and pregnancy. Although drugs and techniques are used clinically to repair damaged endometrium, the treatment effect is poor. In recent years, the in-depth development of stem cell research has provided possibilities and prospects for the repair of endometrial scars, and also brought hope to patients with uterine scars.
  • mesenchymal stem cells from various tissue sources can play a role in repairing, regenerating and treating endometrial scars.
  • Mesenchymal stem cells release angiogenic factors through paracrine effects, which can reduce local inflammatory reactions in the endometrium, improve the ability of angiogenesis in the affected area, reduce the formation of fibrotic scars, and play a repair and regeneration effect on the endometrium.
  • stem cell suspension is usually injected locally or intravenously. However, this method will cause a large amount of stem cell loss during the treatment process. The stem cells cannot be concentrated and gathered at the lesion to take effect. Due to the low cell utilization rate, the treatment effect is poor.
  • Another method is to use absorbable biomaterials as scaffolds for stem cell transplantation.
  • biological scaffolds and cells solves the problem of cell colonization, the final treatment effect is affected by the incomplete absorption of the biological scaffolds and the inflammatory response induced by the residual local scaffolds in the affected area.
  • the cell membrane technology uses a temperature-sensitive smart culture dish to culture cells and their extracellular matrix into a membrane as a sheet-type preparation.
  • the mesenchymal stem cell membrane prepared by this technology not only retains the secretion of mesenchymal stem cells intact, but also The extracellular matrix is an important basic substance for mesenchymal stem cells to maintain their physiological functions.
  • the cell membrane with extracellular matrix is not only conducive to the adhesion between the cell membrane and the local tissue, but also helps the secretion of cytokines and the transmission of physiological signals between cells, so that the cell membrane can play a greater therapeutic role.
  • the cell membrane solves the problem of local cell colonization and enhancing the efficacy of stem cells after mesenchymal stem cell transplantation.
  • the disclosed embodiments provide use of a human mesenchymal stem cell sheet in treating uterine scar in a subject, wherein the human mesenchymal stem cell sheet is topically applied to the uterus of the subject.
  • the disclosed embodiments provide use of a human mesenchymal stem cell sheet in preparing a composition for treating uterine scar in a subject, wherein the human mesenchymal stem cell sheet is topically applied to the uterus of the subject.
  • the disclosed embodiment provides a method for treating uterine scar in a subject, wherein the method comprises locally applying the human mesenchymal stem cell sheet to the uterus of the subject.
  • the diameter of the human mesenchymal stem cell sheet may be about 3-55 mm, semicircular or in a suitable shape with a similar area.
  • the human mesenchymal stem cell sheet can be attached to the endometrial suture incision site.
  • the human mesenchymal stem cell membrane sheet may be circular with a diameter of 13-20 mm, 22-28 mm, or 35-55 mm, or semicircular with a diameter of 13-20 mm, 22-28 mm, or 35-55 mm.
  • the human mesenchymal stem cell membrane sheet can be appropriately cut or divided to have other thicknesses or shapes suitable for applications.
  • the mesenchymal stem cells of the human mesenchymal stem cell cell sheet may be derived from umbilical cord, placenta, amnion, endometrium, fat, bone marrow, or dental pulp.
  • the human-derived mesenchymal stem cells may be umbilical cord mesenchymal stem cells.
  • the human mesenchymal stem cell membrane may be prepared using mesenchymal stem cells at passage numbers P0-P20, such as P2-P10.
  • the thickness of the human mesenchymal stem cell membrane may be 10-300 microns, such as 50-300 microns.
  • the cell density in the human mesenchymal stem cell sheet may be 1 ⁇ 10 5 to 1 ⁇ 10 7 /cm 2 , for example 3 ⁇ 10 5 to 5 ⁇ 10 6 /cm 2 .
  • the human mesenchymal stem cell sheet can be left to stand for 15-60 minutes after attachment to allow the human mesenchymal stem cell sheet to be completely attached to the endometrial tissue.
  • the human mesenchymal stem cell cell sheet can be left to stand for 15-30 minutes after being attached, and a moist sterile gauze can be used to keep the sheet attachment area moist during the static observation period.
  • the human mesenchymal stem cell sheets may be connected to each other through the extracellular matrix secreted by the human mesenchymal stem cell sheets, and the extracellular matrix is rich in fibronectin and integrin- ⁇ 1.
  • the human mesenchymal stem cell sheet is capable of secreting multiple cytokines, and the cytokines include one or more of HGF, IL-6, IL-8 and VEGF.
  • the human mesenchymal stem cell membrane contains the extracellular matrix secreted by human mesenchymal stem cells, and no additional suturing and gluing are required during the transplantation process, which is conducive to the surgical process.
  • the human mesenchymal stem cell membrane contains the extracellular matrix secreted by human mesenchymal stem cells. Compared with the treatment method using human mesenchymal stem cell suspension, it can achieve the targeted transplantation of mesenchymal stem cells. The cells are constrained by the extracellular matrix and will not be lost, which is conducive to the targeted release of cytokines by cells at the scar site and the performance of their functions.
  • human mesenchymal stem cell membrane Compared with human mesenchymal stem cell suspension, human mesenchymal stem cell membrane has better factor secretion function.
  • FIG1 shows a photograph of a cell membrane sheet attached to the model (the black arrow points to the cell membrane sheet).
  • FIG. 2 shows an umbilical cord mesenchymal stem cell sheet.
  • FIG3 shows the cell sheet attached to the uterine incision scar on day 0 after surgery (the dotted area is the cell sheet).
  • FIG4 shows the results of hematoxylin-eosin staining of uterine tissues in the experimental group and the control group after surgery (the black double arrows indicate the thickness of the myometrium).
  • FIG5 shows the Masson staining results of uterine tissues in the experimental group and the control group after surgery (the blue area indicated by the black arrow is the fibrotic area in the uterine myometrium).
  • FIG6 shows the statistical results of uterine myometrial thickness and fibrosis in the experimental group and the control group (*p ⁇ 0.05, ***p ⁇ 0.001).
  • Figure 7 shows a comparison of the amount of factors secreted by human umbilical cord mesenchymal stem cell membranes and human umbilical cord mesenchymal stem cell suspensions. It can be seen that the samples provided by three different donors all show that human umbilical cord mesenchymal stem cell membranes can secrete more influencing factors.
  • the term "about” in reference to a reference value may also include a range of values that are plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value.
  • the term “about” may mean within an order of magnitude of a value.
  • MSCs Mesenchymal stem cells
  • Extracellular matrix biological macromolecules secreted by cells into the extracellular matrix.
  • ECM constitutes a complex protein grid structure that supports, connects and regulates physiological activities and signal transduction between cells.
  • the present invention provides methods for making an endometrial scar animal model; selecting rabbits (or rats) as experimental animals; establishing a mesenchymal stem cell membrane pharmacodynamic evaluation model; and developing a uterine incision scar model that is repeatable, stable, and easy to promote.
  • the human mesenchymal stem cell membrane of the present invention promotes the repair of the scar uterus model (as reflected by the increase in muscle layer thickness after healing and the reduction in fibrosis area)
  • the culture system includes a serum-free culture medium and a coating matrix matching the culture medium, wherein
  • the serum-free culture medium may include (1) basal culture medium + nutrient additives:
  • the basic culture medium can be 1640, DMEM, ⁇ -MEM, DMEM/F12, F12, etc.
  • Nutritional additives include vitamin C, sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin (plant expression), progesterone, putrescine, biotin, sodium pyruvate, ethanolamine, carnitine, amino acids, vitamins, glutathione, linoleic acid, linolenic acid, etc.
  • the coating matrix compatible with the culture medium includes, but is not limited to, collagen, gelatin, fibronectin, adhesion protein, vitronectin, etc.
  • This culture system is suitable for the culture, preparation and detection of mesenchymal stem cells from various tissues, including but not limited to umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, Cells, bone marrow mesenchymal stem cells.
  • Umbilical cord mesenchymal stem cells were used in this example.
  • Umbilical cord mesenchymal stem cells are first isolated and cultured in primary culture and then cultured in subculture to expand the cell number. The procedure is as follows:
  • the obtained newborn umbilical cord (sourced from Beijing Mingde Hospital, donated by pregnant women who had given birth and signed an informed consent form, which has been approved by the Ethics Committee of Mingde Hospital) was washed with a physiological solution with the same osmotic pressure as human body fluids, and then the arteries, veins, and outer membranes were removed, and Wharton's jelly was separated and cut into small tissue blocks of 0.1-2 mm.
  • the tissue blocks were evenly spread in a culture container coated with a matrix, and the spacing between the tissue blocks was 2-30 mm.
  • the culture container was placed in a cell culture incubator, and after 2-7 days, an appropriate amount of complete culture medium was added to cover the tissue blocks.
  • the umbilical cord mesenchymal stem cells crawled out in 8-21 days.
  • the crawled-out cells grew attached to the wall and were fibrous with uniform morphology. When the crawled-out cells grew to 70-100% confluence, the tissue blocks were removed and the cells were passaged.
  • the cells are passaged: the cells are separated from the culture container, including but not limited to digestion with trypsin and similar substances, cell scraping, etc.
  • the cells are dispersed in the culture medium by stirring, vortexing and other methods (including but not limited to), and the cells are inoculated in the culture container at a density of 500-100000/ cm2 .
  • Add an appropriate amount of culture medium replace an appropriate amount of fresh culture medium every 1-5 days according to the growth status of the cells, and repeat the passage operation when the cells grow to 70-100% confluence.
  • the cultured umbilical cord mesenchymal stem cells grow adherently, in a fibrous shape, and have a uniform morphology.
  • the obtained umbilical cord mesenchymal stem cells were detected and identified as follows:
  • the method for determining the growth curve includes but is not limited to the MTT method, WST method, DNA content detection method, ATP detection method, etc.
  • the WST method is taken as an example here for further explanation. Disperse the umbilical cord mesenchymal stem cells, inoculate them into the culture well plate at a certain density according to the reagent instructions, and perform the liquid change operation according to normal culture conditions. At a fixed time every day for a period of time, the cell activity is detected according to the instructions to obtain data on cell activity or quantity.
  • the WST reagent is used to detect the activity of the cells every day within 7 days.
  • the method is to add the WST reagent to the cell well plate being cultured according to the recommended ratio in the instructions, and incubate in the cell culture incubator for a fixed time and then detect with an enzyme marker or ultraviolet spectrophotometer.
  • the absorbance value of the liquid in the well plate chamber at a wavelength of 450nm is positively correlated with the number of cells. From the results, it can be seen that as the culture time increases, the cell activity in the chamber increases, which can be inferred that the cell number increases as the culture time increases.
  • the identification method of umbilical cord mesenchymal stem cells includes but is not limited to flow cytometry detection of cell surface marker proteins, three-way differentiation of umbilical cord mesenchymal stem cells, PCR detection of cell expression genes, etc. Flow cytometry and three-way differentiation are used as examples for description.
  • Umbilical cord mesenchymal stem cells are dispersed in a culture medium and centrifuged, and the cell surface marker proteins are stained according to the purchased reagent instructions in a physiological solution with a serum or serum protein content of 1-20% and an osmotic pressure equal to that of human body fluids, including but not limited to CD73, CD90, CD105, CD34, CD11B, CD19, CD45, and HLA-DR.
  • the phenotypes of CD73, CD90, and CD105 are positive, and the ratio should be no less than 95%
  • the phenotypes of CD34, CD11B, CD19, CD45, and HLA-DR are negative, and the ratio should be no more than 2%.
  • Umbilical cord mesenchymal stem cells have the ability to induce differentiation into bone, cartilage, and fat.
  • the specific operation of osteogenic and adipogenic differentiation detection is to inoculate the cells in a suitable culture vessel according to the proportions in the instructions of the three-way induction differentiation reagent.
  • the cells for osteogenic induction detection grow to 50-90% confluence and the cells for adipogenic induction detection grow to more than 90% confluence, add osteogenic and adipogenic induction medium respectively.
  • chondrogenic induction a certain number of cells are centrifuged to the bottom of the centrifuge tube, and then chondrogenic induction medium is added. After the cells are clustered into small balls, the cell balls are allowed to leave the bottom of the tube to ensure complete contact with the induction medium.
  • Osteogenic induction can be stained with, but not limited to, Alizarin Red and anti-hOsteocalcin
  • adipogenic induction can be stained with, but not limited to, Oil Red O and anti-mFABP4
  • chondrogenic induction can be stained with, but not limited to, Alcian Blue, Safranin O and anti-hAggrecan.
  • the procedure for preparing the umbilical cord mesenchymal stem cell membrane is as follows
  • a layer of matrix that is conducive to the attachment of mesenchymal stem cells should be coated on the surface of the temperature-sensitive smart culture dish in advance.
  • the matrix includes but is not limited to collagen, gelatin, fibronectin, vitronectin, laminin, poly-ornithine, poly-lysine, etc.
  • the coating matrix is diluted with physiological buffers such as physiological saline and PBS, and then added to the temperature-sensitive smart culture dish and covered with the culture dish cover. Then the temperature-sensitive smart culture dish is placed in a 37°C, saturated humidity, 5% CO2 incubator for coating for 0.5-48h.
  • thermosensitive smart culture dish After coating, the coating matrix is removed from the smart culture dish, and the mesenchymal stem cell suspension is added to the thermosensitive smart culture dish.
  • a thermosensitive smart culture dish with a diameter of 35 mm is used, the amount of cells added is 1.8 ⁇ 10 5 -1.8 ⁇ 10 7 (most preferably 1 ⁇ 10 7 ), and the amount of culture fluid added is 1-4 ml (most preferably 3 ml). Then the thermosensitive smart culture dish is placed in a 37°C, saturated humidity, 5% CO 2 incubator for 2-48h.
  • thermosensitive smart culture dish After the culture is completed, the thermosensitive smart culture dish is moved to a 4-32°C environment, and the cells will detach from the bottom of the thermosensitive smart culture dish in sheets without adding any additional reagents or materials to form a cell membrane.
  • the cell membrane is off-white, compact, and has a smooth surface.
  • the size is a circle with a diameter of 3-30 mm and a thickness of 50-1000 ⁇ m. Tissue section observation shows that the cell membrane contains a multilayer cell structure and contains extracellular matrix components secreted by the cells.
  • the functional assays of the formed cell membrane sheets are as follows:
  • the supernatant in the process of preparing the cell membrane sheet was detected to determine the content of various factors secreted by the cell membrane sheet.
  • the factors referred to herein include but are not limited to interleukin-6 (IL-6), transforming growth factor- ⁇ (TGF- ⁇ ), 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 (indoleamine 2,3-dioxygenase, IDO) and nitric oxide synthase (inducible nitric oxide synthase, iNOS), etc.
  • interleukin-6 (IL-6), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) are used as examples to detect the secretion factors of
  • the cell membrane obtained above is fixed with paraformaldehyde or formalin fixative, and then the cell membrane is made into a tissue section with a thickness of 4-10 ⁇ m by paraffin sectioning or frozen sectioning, and then stained to observe the protein content of the extracellular matrix in the cell membrane, wherein the protein includes but is not limited to fibronectin, integrin family, vitronectin, etc., and the cell nucleus is usually stained with fluorescent dyes such as DAPI or Hoechst33258 staining at the same time to assist in positioning.
  • staining observation can also be performed using methods including but not limited to immunohistochemistry.
  • fibronectin and integrin- ⁇ 1 are used as examples for illustration.
  • Both fibronectin and integrin- ⁇ 1 are stained with antibody dyes labeled with fluorescein, and the cell nucleus is stained with DAPI. It can be seen that the prepared cell membrane contains a large amount of fibronectin and integrin- ⁇ 1.
  • the cell membrane obtained above was fixed with 2.5% glutaraldehyde fixative, and then dehydrated with gradient alcohol. After natural drying, the dried cell membrane was obtained for scanning electron microscope analysis.
  • the dried cell membrane was pasted on the surface of the sample stage with conductive double-sided tape, and then the surface was gold-sprayed by vacuum magnetron sputtering to make the surface of the observed sample conductive.
  • the sample stage was placed in a scanning electron microscope (Hitachi S-4800) for observation.
  • the prepared membrane is temporarily stored at 4°C.
  • the steps for making an animal model of uterine incision scar are as follows: (1) inhalation or intravenous injection to induce animal anesthesia; (2) shaving hair, preparing skin, disinfecting, and then making an abdominal incision to expose the uterus; (3) making a uterine penetrating wound to construct an injury model; (4) intermittently or continuously suturing the uterus; (5) returning the uterus to the abdominal cavity; and (6) suturing and closing the abdominal cavity after disinfection.
  • anesthesia can be performed by gas anesthesia (isoflurane, Lunan Beite Pharmaceutical Co., Ltd.) or intravenous injection (sodium pentobarbital, 30 mg/kg dose);
  • a sharp scalpel is used to cut through the uterine serosa, myometrium and endometrium to form an incision; the direction of the incision is consistent with the extension direction of the uterine horn, and the length is 1-20 mm.
  • Step (4) Suturing the uterus.
  • the endometrial layer, myometrium, and serosa should be aligned with the corresponding layers and sutured layer by layer.
  • Step (6) Closure of the abdominal cavity.
  • the abdominal muscles should be sutured layer by layer in order to close the abdominal cavity.
  • the animal model was used to verify the effectiveness of the umbilical cord mesenchymal stem cell membrane sheet.
  • the operation is as described in step (4) above.
  • the mesenchymal stem cell membrane sheet is transplanted and attached to the affected part of the uterine serosa (i.e., the sutured part) and maintained for 30-60 minutes.
  • the above step (5) is continued and the uterus is returned to the abdominal cavity (6) and the abdominal cavity is sutured and closed.
  • the "mesenchymal stem cell membrane” in the present invention is a temperature-sensitive culture dish used to culture mesenchymal stem cells.
  • the surface of the culture dish is a layer of temperature-sensitive polymer material.
  • the temperature decreases, the surface tension of the culture dish changes, and the bonding force between the culture dish and the mesenchymal stem cells decreases, causing the cells to naturally fall off from the surface of the culture dish in a film layer and then the cell membrane is harvested.
  • Umbilical cord mesenchymal stem cells are digested into single cells, then planted in a thermosensitive culture dish with a polyacrylamide thermosensitive polymer material connected to the surface of the culture dish, and cultured in an incubator (Thermo Fisher, model 371) at 37°C, 5% CO 2 , and 95% humidity. After the cells proliferate and fully fuse, they are moved to a 20°C environment, and the cells will quickly separate from the bottom of the culture dish in sheets, and the umbilical cord mesenchymal stem cell membrane can be harvested (as shown in Figure 2).
  • mesenchymal stem cells may be derived from umbilical cord, placenta, amnion, endometrium, fat, bone marrow, dental pulp, etc.
  • mesenchymal stem cells in treating uterine incision scars is as follows: after transplantation of mesenchymal stem cell sheets, they secrete a variety of growth factors and cytokines at the wound site, such as HGF, VEGF, IL-8, etc. These factors can regulate the microenvironment of the uterine scar site, reduce inflammatory response, promote local angiogenesis, reduce the progression of fibrosis, and improve the repair of the endometrial myometrium.
  • the length should be 8-15mm
  • the joints should be sutured neatly;
  • the abdominal muscles are sutured layer by layer with non-absorbable sutures to close the abdominal cavity.
  • the animal is sent to the feeding area and waited for it to wake up after anesthesia. Only drinking water is provided within 1 hour after surgery. After stabilization, regular feed can be provided and warmth is provided if necessary. Penicillin-streptomycin is taken orally for three consecutive days after surgery to prevent postoperative infection.
  • the effectiveness of the umbilical cord-derived mesenchymal stem cell membrane sheet is verified by using an animal model.
  • the operation is to cut the umbilical cord-derived mesenchymal stem cell membrane sheet into a semicircular shape with a diameter of about 3-30 mm or a suitable shape with a similar area after the above step (4), and attach it to the affected part of the endometrial suture incision.
  • the umbilical cord mesenchymal stem cell membrane sheet is preferably a semicircular shape with a diameter of 13-20 mm. This shape and size is conducive to the attachment of the membrane sheet and is not easy to fall off.
  • the surface of the membrane sheet is flat after attachment (as shown in Figure 1).
  • the thickness of the mesenchymal stem cell membrane sheet is 10-300 microns, such as 50-300 microns.
  • the cell ratio of mesenchymal stem cells in the mesenchymal stem cell membrane sheet is at least 90%, such as at least 95%.
  • the cell density in the mesenchymal stem cell membrane sheet is 1 ⁇ 10 5 to 1 ⁇ 10 7 /cm 2 , such as 3 ⁇ 10 5 to 5 ⁇ 10 6 /cm 2.
  • the mesenchymal stem cell membrane sheet is prepared using mesenchymal stem cells with a passage number of P0-P20, such as P2-P10.
  • the umbilical cord mesenchymal stem cell membrane After the umbilical cord mesenchymal stem cell membrane is attached, it is left to stand for 30-60 minutes to allow the umbilical cord mesenchymal stem cell membrane to be completely attached to the endometrial tissue. The best time is 15-30 minutes. During the static observation process, moist sterile gauze can be used to keep the membrane attachment area moist.
  • the animal model was used to verify the effectiveness of the umbilical cord mesenchymal stem cell membrane.
  • the uterine samples of the experimental group and the control group were fixed and stained with hematoxylin-eosin and Masson's staining.
  • the endometrial tissues of the two groups of animals were observed under a microscope.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Cell Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Reproductive Health (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

人源间充质干细胞膜片制备治疗子宫瘢痕药物中的用途，以及人源间充质干细胞膜片在制备用于治疗受试者中子宫瘢痕的组合物中的用途。其中，所述人源间充质干细胞膜片局部应用于受试者的子宫。所述人源间充质干细胞膜片可以实现间充质干细胞的定点移植，细胞受细胞外基质约束，不会发生流失，有利于细胞在瘢痕部位定向释放细胞因子，发挥功能。

Description

人源间充质干细胞膜片在子宫瘢痕治疗中的用途 技术领域
本公开涉及干细胞应用技术领域，尤其涉及人源间充质干细胞膜片在子宫瘢痕治疗中的用途。
背景技术
子宫内膜是高度再生的生殖器官，女性一生将经历400多次子宫内膜的脱落和再生，子宫内膜的厚度反应了内膜功能状态。人工流产和剖宫产等子宫内膜受损后易形成瘢痕，严重影响了受精卵胚胎着床和妊娠。虽然临床上应用药物，手法等修复受损的子宫内膜，但治疗效果欠佳。近年，干细胞研究的深入发展为子宫内膜瘢痕修复提供了可能性和前景，也为子宫瘢痕患者带来希冀。近年，国内外大量的动物试验与临床研究表明，多种组织来源的间充质干细胞对子宫内膜瘢痕均可起到修复再生和治疗作用。间充质干细胞通过旁分泌作用释放血管生成因子等，可以减少子宫内膜局部炎性反应、提高患处血管新生能力、减少纤维化瘢痕的形成，对子宫内膜起到修复和再生效果。干细胞治疗过程中多采用局部或静脉注射干细胞悬液，但该方法在治疗过程中将伴有大量干细胞流失，干细胞无法集中和汇集于病灶处起效，由于细胞利用率低，治疗效果欠佳。另外一种方法是利用可吸收性生物材料作为支架进行干细胞移植。生物支架和细胞的组合虽然解决了细胞的定植问题，但又因生物支架吸收不完全、残留在患处局部诱发炎性反应而影响最终治疗效果。
发明内容
为了解决以上干细胞移植方法等问题，申请人选用无支架细胞膜片技术提高干细胞移植率和子宫内膜瘢痕修复治疗效果。细胞膜片技术利用温度敏感智能培养皿，将细胞及其细胞外基质培养成一张膜片作为片层型制剂。利用该技术制备的间充质干细胞膜片，不仅完好地保留了间充质干细胞所分泌 的细胞外基质，而且膜片中细胞与细胞之间紧密相连接。细胞外基质是间充质干细胞维持其生理功能的重要基础物质，保留有细胞外基质的细胞膜片，不仅有利于细胞膜片与局部组织之间的贴附，还有助于细胞因子的分泌和细胞间生理信号传递，从而使细胞膜发挥更大的治疗作用。细胞膜片解决了间充质干细胞移植后细胞局部定植和增强干细胞疗效的难题。
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本公开实施例提供了人源间充质干细胞膜片在治疗受试者中子宫瘢痕中的用途，其中，所述人源间充质干细胞膜片局部应用于所述受试者的子宫。
本公开实施例提供了人源间充质干细胞膜片在制备用于治疗受试者中子宫瘢痕的组合物中的用途，其中，所述人源间充质干细胞膜片局部应用于所述受试者的子宫。
本公开实施例提供了一种用于治疗受试者中子宫瘢痕的方法，其中，所述方法包括将所述人源间充质干细胞膜片局部应用于所述受试者的子宫。
在一些示例性实施方式中，所述人源间充质干细胞细胞膜片的直径可以为约3-55mm半圆形或近似面积的适当形状。
在一些示例性实施方式中，所述人源间充质干细胞细胞膜片可以贴附在子宫内膜缝合切口患处。
在一些示例性实施方式中，所述人源间充质干细胞膜片的直径可以为13-20mm、22-28mm或35-55mm的圆形，或者直径为13-20mm、22-28mm或35-55mm的半圆形。
在一些示例性实施方式中，所述人源间充质干细胞膜片可以被适当裁剪、分割，使其具有适于应用的其他厚度或形状。
在一些示例性实施方式中，所述人源间充质干细胞细胞膜片的间充质干细胞可以来源于脐带、胎盘、羊膜、子宫内膜、脂肪、骨髓、牙髓。
在一些示例性实施方式中，所述人源间充质干细胞可以是脐带间充质干细胞。
在一些示例性实施方式中，所述人源间充质干细胞膜片可以是使用传代数为P0-P20，例如P2-P10的间充质干细胞制备的。
在一些示例性实施方式中，所述人源间充质干细胞膜片的厚度可以为10-300微米，例如50-300微米。
在一些示例性实施方式中，所述人源间充质干细胞膜片中的细胞密度可以为1×105至1×107/cm2，例如3×105至5×106/cm2。
在一些示例性实施方式中，所述人源间充质干细胞细胞膜片可以在贴附后静置15-60分钟，使所述人源间充质干细胞膜片与子宫内膜组织完全贴附。
在一些示例性实施方式中，所述人源间充质干细胞细胞膜片可以在贴附后静置15-30分钟，静置观察期间使用湿润无菌纱布维持膜片贴附区域的湿润。
在一些示例性实施方式中，所述人源间充质干细胞膜片可以通过其分泌的细胞外基质彼此连接，所述细胞外基质富含纤连蛋白和整联蛋白-β1。
在一些示例性实施方式中，所述人源间充质干细胞膜片能够分泌多种细胞因子，所述细胞因子包括HGF、IL-6、IL-8和VEGF中的一种或多种。
本公开具有以下优势：
人源间充质干细胞膜片包含有人源间充质干细胞所分泌的细胞外基质，移植过程中不需要额外的缝合和胶黏，有利于手术过程进行。
人源间充质干细胞膜片包含有人源间充质干细胞所分泌的细胞外基质，与使用人源间充质干细胞悬液治疗方式相比，可以实现间充质干细胞的定点移植，细胞受细胞外基质约束，不会发生流失，有利于细胞在瘢痕部位定向释放细胞因子，发挥功能。
与人源间充质干细胞悬液相比，人源间充质干细胞膜片具有更好的因子分泌功能。
动物体内实验结果显示，与模型对照组相比，人源间充质干细胞膜片能够显著增加子宫损伤部位愈合后的肌层厚度，减少瘢痕形成(纤维化面积减小)。
在阅读并理解了附图和详细描述后，可以明白其他方面。
附图说明
附图用来提供对本公开技术方案的进一步理解，并且构成说明书的一部分，与本公开的实施例一起用于解释本公开的技术方案，并不构成对本公开的技术方案的限制。附图中一个或多个模块的形状和大小不反映真实比例，目的只是示意说明本公开内容。
图1示出了细胞膜片贴附于模型后照片(黑色箭头所指为细胞膜片)。
图2示出了脐带间充质干细胞膜片。
图3示出了术后0天细胞膜片贴附于子宫切口瘢痕处(虚线区域为细胞膜片)。
图4示出了术后实验组与对照组子宫组织苏木精—伊红染色结果(黑色双箭头标记为肌层厚度)。
图5示出了术后实验组与对照组子宫组织马松染色结果(黑色箭头所示蓝色区域为子宫肌层中纤维化区域)。
图6示出了实验组与对照组子宫组织肌层厚度及纤维化统计结果(*p<0.05，***p<0.001)。
图7示出了人脐带间充质干细胞膜片与人脐带间充质干细胞悬液分泌因子的量比较。从中可以看出，三个不同捐献者提供的样本均显示，人脐带间充质干细胞膜片能够分泌更多影响因子。
具体实施方式
为使本公开的目的、技术方案和优点更加清楚明白，下文中将对本公开的实施例进行详细说明。需要说明的是，在不冲突的情况下，本公开中的实施例及实施例中的特征可以相互任意组合。
下面将结合附图对本公开的实施例进行详细说明。实施方式可以以多个不同形式来实施。所属技术领域的普通技术人员可以很容易地理解一个事实，就是方式和内容可以在不脱离本公开的宗旨及其范围的条件下被变换为一种或多种形式。因此，本公开不被解释为仅限定在下面的实施方式所记载的内容中。在不冲突的情况下，本公开中的实施例及实施例中的特征可以相互任 意组合。
除非另有定义，本文使用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常理解的相同含义。尽管与本文描述的那些相似或等效的任何方法和材料都可以用于实践或测试本公开，但描述了优选的方法和材料。为了本公开的目的，在下文中定义了以下术语。
在本申请中，除非另外明确说明，否则单数的使用包括复数。必须注意，除非上下文另外清楚地指示，否则如本说明书中使用的，单数形式“一(a)”、“一(an)”和“所述/该(the)”包括复数指代。在本申请中，除非另外说明，否则“或”的使用意指“和/或”。
术语“约(about)”或“约(approximately)”意指在如由本领域普通技术人员确定的特定值的可接受误差范围以内，其将部分地取决于值如何被测量或确定，即，测量系统的局限性。例如，“约”可以根据本领域的实践意指1个或多于1个标准差以内。可选地，“约”可以意指特定值的最多20％、最多10％、最多5％或最多1％的范围。在其他实例中，“约10”的量包括10和9至11的任何量。
在又其他的实例中，涉及参考数值的术语“约”也可以包括该值的正或负10％、9％、8％、7％、6％、5％、4％、3％、2％或1％的值范围。可选地，特别是涉及生物系统或过程，术语“约”可以意指在值的一个数量级以内。当在本申请和权利要求书中描述特定值时，除非另外说明，否则术语“约”被假定意指特定值的可接受的误差范围以内。
如本说明书和一项或更多项权利要求(claim(s))中使用的，词语“包含/包括(comprising)”(和包含/包括(comprising)的任何形式，诸如“包含/包括(comprise)”和“包含/包括(comprises)”)、“具有(having)”(和具有(having)的任何形式，诸如“具有(have)”和“具有(has)”)、“包括/包含(including)”(和包括/包含(including)的任何形式，诸如“包括/包含(includes)”和“包括/包含(include)”)或“包含/含有(containing)”(和“包含/含有(containing)”的任何形式，诸如“包含/含有(contains)”和“包含/含有(contain)”)是包含性的或开放式的，并不排除另外的、未列举的要素或方法步骤。设想本说明书中讨论的任何实施方案可以参考本公开内容的任何方法或组合来实现，并且反之亦然。此外，本公开内 容的组合可以用于实现本公开内容的方法。
人源脐带间充质干细胞：人源脐带华通氏胶中分离提取间充质干细胞(MSC)，MSC体外容易扩增，表达干细胞表面标志物，并具有多向分化潜能，同时分泌细胞因子和多种生长因子。
细胞外基质(ECM)：由细胞分泌到细胞外间质中的生物大分子物质，ECM构成复杂的蛋白网架结构，起到支撑、连接和调节细胞间生理活动和信号传导作用。
实施例
本公开提供了制作子宫内膜瘢痕动物模型；选用兔(或大鼠)作为实验动物；建立间充质干细胞膜片药效学评价模型；研发具有可重复性、稳定性、易推广性的子宫切口瘢痕模型。本公开的人源间充质干细胞膜片促进瘢痕子宫模型修复(体现为愈合后肌层厚度增加，且纤维化面积减少)
实施例1
培养体系包括无血清培养基及与培养基匹配的包被基质，其中
无血清培养基成分可以为(1)基础培养基+营养添加剂：
基础培养基可以是1640，DMEM，α-MEM，DMEM/F12，F12等；
营养添加剂包括，维生素C、硒酸钠、氢化可的松、胰岛素、转铁蛋白、人血清白蛋白(植物表达)、孕酮、腐胺、生物素、丙酮酸钠、乙醇胺、肉毒碱、氨基酸、维生素、谷胱甘肽、亚油酸、亚麻酸等中的多种。
也可为(2)商用无血清培养基包括但不限于：CTSTM StemProTM MSC SFM Kit、MesenCultTM-ACF Medium、MesenCultTM-ACF Plus Medium、MesenCultTM-XF Medium等。
与培养基配套的包被基质包括但不限于胶原、明胶、纤连蛋白、黏连蛋白、玻连蛋白等。
本培养体系适用于各组织来源的间充质干细胞的培养、细胞膜片制备及检测，包括但不限于脐带间充质干细胞、胎盘间充质干细胞、脂肪间充质干 细胞、骨髓间充质干细胞。本实施例中所使用的为脐带间充质干细胞。
脐带间充质干细胞先经过原代分离培养和传代培养，进行细胞数量扩增，程序如下：
1.脐带间充质干细胞原代培养
将得到的新生儿脐带(来源于北京明德医院，由孕产妇签署知情同意书捐赠，知情同意书已通过明德医院伦理委员会批准)用与人体体液渗透压相等的生理溶液洗净后去除动脉、静脉、外膜，分离出华通氏胶，剪碎成0.1-2mm的小组织块，将组织块均匀铺在包被有基质的培养容器中，组织块之间的间距为2-30mm。将培养容器置于细胞培养箱中，2-7天后加入适量完全培养基覆盖组织块，脐带间充质干细胞在8-21天爬出，爬出细胞贴壁生长，呈成纤维状，形态均一。待爬出细胞长至70-100％汇合时，移除组织块，对细胞进行传代操作。
2.脐带间充质干细胞传代
待细胞长至70-100％汇合时，对细胞进行传代操作：将细胞与培养容器分离，方法包括但不限于胰酶及类似物质消化、细胞刮等。将细胞通过搅拌、涡旋等方法(包括但不限于)分散于培养基中，并按照500-100000/cm2密度将细胞接种于培养容器中。加入适量培养基，根据细胞生长状态每1-5天更换适量新鲜的培养基，待细胞长至70-100％汇合时，重复传代操作。进行培养的脐带间充质干细胞贴壁生长，呈成纤维状，形态均一。
对所得的脐带间充质干细胞进行检测和鉴定，方法如下：
1.脐带间充质干细胞生长曲线检测
生长曲线的测定方法包括但不限于MTT法、WST法、DNA含量检测法、ATP检测法等，此处以WST法为例，进行进一步说明。将脐带间充质干细胞分散，参考试剂说明书以一定密度接种至培养孔板中，按照正常培养条件进行换液操作。在一段时间的每天的固定时间，根据说明书对细胞活性进行检测，得到细胞活性或数量的数据。在7天范围内每天利用WST试剂检测细胞的活性，其方法为将WST试剂按照说明书推荐比例加入正在培养的细胞孔板中，在细胞培养箱内孵育固定时间后用酶标仪或紫外分光光度计检测 该孔版小室液体在450nm波长处的吸光度值，该值与细胞数量成正相关。从其结果可看到随培养时间延长，小室内细胞活性增加，即可推断细胞数量随培养时间延长而增加。
2.脐带间充质干细胞鉴定
脐带间充质干细胞鉴定方法包括但不限于流式细胞术检测细胞表面标记蛋白、脐带间充质干细胞三向分化、PCR法检测细胞表达基因等，此处以流式细胞术、三向分化为例进行说明。
2.1脐带间充质干细胞表面标志物鉴定
将脐带间充质干细胞，将细胞分散于培养基中后离心，用血清或血清蛋白含量为1-20％的与人体体液渗透压相等的生理溶液中，根据所购买的试剂说明书对细胞表面标记蛋白进行染色，包括但不限于CD73、CD90、CD105、CD34、CD11B、CD19、CD45、HLA-DR。其中CD73、CD90、CD105的表型为阳性，其比率应不小于95％，CD34、CD11B、CD19、CD45、HLA-DR的表型为阴性，其比率应不大于2％。
2.2脐带间充质干细胞三向诱导分化
脐带间充质干细胞拥有向骨、软骨、脂肪方向诱导分化的能力。进行成骨和成脂肪分化检测的具体操作为，将细胞按照三向诱导分化试剂说明书的比例接种于合适的培养器皿中，待成骨诱导检测的细胞生长至50-90％汇合，成脂肪诱导检测的细胞生长至90％以上汇合时，分别加入成骨和成脂肪诱导培养基。成软骨诱导时将一定数量的细胞离心至离心管底部，而后加入成软骨诱导培养基，待细胞团成小球后，使细胞小球离开管底，保证与诱导培养基完全接触，
诱导培养7天以上时对细胞进行检测。成骨诱导可用包括但不限于茜素红、anti-hOsteocalcin染色，成脂肪诱导可用包括但不限于油红O、anti-mFABP4染色，成软骨诱导可以用包括但不限于阿尔新蓝、番红O、anti-hAggrecan染色。
脐带间充质干细胞膜片制备程序如下
1.温敏智能培养皿包被
利用无血清培养体系及间充质干细胞在膜片制备前，应预先在温敏智能培养皿表面包被一层有利于间充质干细胞贴附的基质，基质包括但不限于胶原、明胶、纤连蛋白，玻连蛋白，层粘连蛋白，多聚鸟氨酸，多聚赖氨酸等。包被时使用生理盐水、PBS等生理缓冲液将包被基质进行稀释，而后加入到温敏智能培养皿内，盖上培养皿盖。而后将温敏智能培养皿置于37℃，饱和湿度，5％CO2培养箱中进行包被，时间为0.5-48h。
2.脐带间充质干细胞膜片制备
包被完成后将包被基质从智能培养皿中吸除，将间充质干细胞悬液加入温敏智能培养皿中。对本实施例而言，使用直径为35mm温敏智能培养皿，加入细胞量为1.8×105-1.8×107(最优选为1×107)，加入培养液量为1-4ml(最优选为3ml)。而后将温敏智能培养皿置于37℃，饱和湿度，5％CO2培养箱中培养，培养时间为2-48h。培养完成后，将温敏智能培养皿移至4-32℃环境，细胞将成片层状从温敏智能培养皿底部脱离，无需添加任何额外的试剂或材料，成为细胞膜片。细胞膜片呈灰白色，结构致密，表面光滑平整。尺寸为直径为3-30mm的圆形，厚度为50-1000μm。组织切片观察可以看出该细胞膜片包含多层细胞结构，并含有细胞所分泌的细胞外基质成分。
对所形成的细胞膜片进行功能检测如下：
1.酶联免疫法检测细胞膜片分泌因子
按照酶联免疫试剂盒说明书对细胞膜片制备过程中的上清液进行检测，测定细胞膜片分泌的各因子含量，此处所指因子包括但不限于白细胞介素-6(interleukin-6，IL-6)、转化生长因子-β(transforming growth factor-β，TGF-β)、前列腺素E2(prostaglandin E2，PGE2)、肝细胞生长因子(hepatocyte growth factor，HGF)、表皮生长因子、成纤维细胞生长因子、血小板衍生生长因子、血管内皮生长因子(vascular endothelial growth factor，VEGF)、胰岛素生长因子、基质细胞衍生生长因子-1、色氨酸代谢酶(indoleamine 2,3-dioxygenase，IDO)和一氧化氮合酶(inducible nitric oxide synthase，iNOS)等。此处以白细胞介素-6(interleukin-6，IL-6)、肝细胞生长因子(hepatocyte growth factor，HGF)、血管内皮生长因子(vascular endothelial growth factor，VEGF)为例进行某脐带间充质干细胞膜片分泌因子的检测。
2.组织切片观察细胞膜片中纤黏连蛋白
将上述得到的细胞膜片用多聚甲醛或福尔马林固定液固定，而后通过石蜡切片法或冰冻切片法将细胞膜片制成厚度为4-10μm的组织切片进行染色，观察细胞膜片中细胞外基质所含蛋白情况，其中蛋白包括但不限于纤连蛋白(Fibronectin)、整联蛋白族、玻连蛋白等，通常在染色同时用DAPI或Hoechst33258染色等荧光染料对细胞核进行染色，用于辅助定位。除免疫荧光方法外，还可采用包括但不限于免疫组化方法等进行染色观察。此处以纤连蛋白和整联蛋白-β1为例进行说明，纤连蛋白和整联蛋白-β1均用有荧光素标记的抗体染料进行染色，细胞核用DAPI进行染色，可见制备得到的细胞膜片含有大量的纤连蛋白和整联蛋白-β1.
3.扫描电子纤维镜观察细胞膜片的表面结构
将上述得到的细胞膜片就2.5％的戊二醛固定液固定，而后通过梯度酒精进行脱水，自然晾干后得到干燥的细胞膜片用于扫描电子显微镜分析。将干燥的细胞膜片用导电双面胶粘贴在样品台表面，而后用真空磁控溅射法进行表面喷金处理，使被观察样品表面导电。将样品台放入扫描电子显微镜内(所用为Hitachi S-4800)进行观察。
4.制备完成的膜片4℃暂存。
实施例2
制作子宫切口瘢痕动物模型步骤为：(1)吸入或静脉内注射诱导进行动物麻醉；(2)剃毛备皮、消毒后腹部切开暴露子宫；(3)制作子宫贯穿伤口构建损伤模型；(4)间断或连续缝合子宫；(5)将子宫还纳回腹腔；(6)消毒后缝合关闭关腹腔。
上述步骤中(1)麻醉方式可以采用气体(异氟烷，鲁南贝特制药有限公司)麻醉，或静脉内注射(戊巴比妥钠，30mg/kg剂量)麻醉；
步骤(3)制作子宫贯穿伤口时，使用锐利手术刀贯穿子宫浆膜层、肌层和内膜层切开形成切口；切口方向与子宫角延伸方向一致，长度为1-20mm。
步骤(4)缝合子宫。应将子宫内膜层、肌层、浆膜层与相应层对齐逐层缝合。
步骤(6)腹腔关闭缝合。应将腹部肌肉、按照顺序逐层缝合，关闭腹腔。
实施例3
应用动物模型验证脐带间充质干细胞细胞膜片的有效性。其操作为上述步骤(4)，模型建立后，将间充质干细胞细胞膜片移植贴附移植至子宫浆膜层的患处(即缝合处)，并维持30-60分钟，待细胞膜片与组织器官表面完全贴附之后继续上述步骤(5)并将子宫还纳回腹腔(6)缝合关闭腹腔。
本发明中“间充质干细胞膜片”为利用温度敏感型培养皿培养间充质干细胞，该种培养皿表面为一层温敏高分子材料，当温度降低时，培养皿表面张力改变，培养皿与间充质干细胞接触的结合力降低，使细胞从培养皿表面成膜层状自然脱落后收获细胞膜片。
脐带间充质干细胞膜片的制备方法：脐带间充质干细胞消化成为单细胞、之后种植到培养皿表面连接有聚丙烯酰胺类温敏高分子材料的温敏培养皿内、置于37℃，5％CO2，95％湿度环境的培养箱(Thermo Fisher，型号371)内培养。待细胞增殖并充分融合后，将其移至20℃环境下，细胞将迅速成片层状脱离培养皿底部，即可收获脐带间充质干细胞细胞膜片(图2所示)。
上述间充质干细胞来源可以是脐带、胎盘、羊膜、子宫内膜、脂肪、骨髓、牙髓等。
间充质干细胞治疗子宫切口瘢痕机理为：间充质干细胞膜片移植后在创伤局部分泌多种生长因子和细胞因子，如HGF、VEGF、IL-8等。这些因子能够对子宫瘢痕部位微环境进行调节，减低炎症反应，促进局部血管新生，减少纤维化进展改善子宫内膜肌层的修复。
实施例4
子宫切口瘢痕动物模型(实验动物兔子，新西兰白兔，雌性，体重1.8-2.5kg)的制备方法，步骤：
(1)静脉内注射麻药进行动物麻醉；
(2)剔除腹部兔毛，碘伏(山东利尔康)腹部皮肤消毒后切开腹暴露子宫；
(3)使用手术刀制作子宫贯穿伤口模型。伤口方向与子宫角延伸方向一 致，长度为1-20mm，
以长度为8-15mm为宜；
(4)使用可吸收性缝合线(上海金环，型号7-0)缝合子宫。缝合过程中内膜层、肌肉层、浆膜层的层与层之间
应对接齐整进行缝合；
(5)将子宫还纳回腹腔；
(6)不可吸收缝合线逐层缝合腹部肌肉关闭腹腔。
(7)将动物送至饲养区域，待其麻醉后苏醒。术后1小时内仅提供饮用水，稳定后可常规提供饲料，必要时给予保暖。术后三天连续口服青链霉素预防术后感染。
应用动物模型验证脐带来源间充质干细胞细胞膜片的有效性，其操作为在上述步骤(4)之后，裁剪脐带间充质干细胞细胞膜片至直径约3-30mm半圆形或近似面积的适当形状，贴附在子宫内膜缝合切口患处，脐带间充质干细胞直径13-20mm的半圆形为宜，该形状大小有利于膜片贴附，不易脱落，膜片贴附后表面平整(图1所示)。充质干细胞膜片的厚度为10-300微米，例如50-300微米。充质干细胞膜片中间充质干细胞的细胞比例为至少90％，例如至少95％。充质干细胞膜片中的细胞密度为1×105至1×107/cm2，例如3×105至5×106/cm2。充质干细胞膜片是使用传代数为P0-P20，例如P2-P10的间充质干细胞制备的。
脐带间充质干细胞膜片贴附后静置30-60分钟，使脐带间充质干细胞膜片与子宫内膜组织完全贴附。其中最佳时间为15-30分钟，静置观察过程中可使用湿润无菌纱布维持膜片贴附区域的湿润。
之后继续上述(5)(6)(7)等操作步骤，将器官还纳回腹腔后关闭腹腔。术后，进行抗感染等基础护理。
应用动物模型验证脐带间充质干细胞细胞膜片的有效性中，采用未贴附细胞膜片组为试验对照组。
实施例5
应用动物模型验证脐带间充质干细胞细胞膜片的有效性，术后0天、2 周和4周，实验组及对照组动物的子宫样本进行取材固定，苏木精—伊红染色、马松染色。显微镜下观察对照两组动物子宫内膜组织。
术后0天实验组子宫组织切片(苏木精—伊红染色(伊红苏木染液，赛维尔生物))可观察到：细胞膜片贴附与子宫外侧(图3所示)。
术后2周、术后4周子宫组织切片(苏木精—伊红染色)(图4)观察、子宫肌层厚度测量分析结果显示：实验组脐带间充质干细胞膜片移植对子宫切口瘢痕治疗效果，移植后不同时间点，子宫肌层厚度与对照组(未治疗动物相比膜片移植组)相比子宫肌层厚度增大(图4中黑色双箭头所示为子宫肌层厚度)。结论：膜片移植组显示了术后愈合时间加快，子宫肌层厚度增加速率提高(图6)。
术后2周、4周子宫组织切片(马松染色结果(Masson三色染色液，赛维尔生物))(图5)观察、纤维化面积测量分析(图5中黑色箭头所蓝色区域指为肌层纤维化区域)结果显示：膜片移植组术后不同时间点子宫内膜纤维化面积比较对照组有所减小(图6)。

Claims (16)

  1. 人源间充质干细胞膜片在治疗受试者中子宫瘢痕中的用途，其中，所述人源间充质干细胞膜片局部应用于所述受试者的子宫。
  2. 人源间充质干细胞膜片在制备用于治疗受试者中子宫瘢痕的组合物中的用途，其中，所述人源间充质干细胞膜片局部应用于所述受试者的子宫。
  3. 一种用于治疗受试者中子宫瘢痕的方法，其中，所述方法包括将所述人源间充质干细胞膜片局部应用于所述受试者的子宫。
  4. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞细胞膜片的直径为约3-55mm半圆形或近似面积的适当形状。
  5. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞细胞膜片贴附在子宫内膜缝合切口患处。
  6. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞膜片的直径为13-20mm、22-28mm或35-55mm的圆形，或者直径为13-20mm、22-28mm或35-55mm半圆形。
  7. 根据权利要求6所述的用途或所述的方法，其中，所述人源间充质干细胞膜片被适当裁剪、分割，使其具有适于应用的其他厚度或形状。
  8. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞细胞膜片的间充质干细胞来源于脐带、胎盘、羊膜、子宫内膜、脂肪、骨髓、牙髓。
  9. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞是脐带间充质干细胞。
  10. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞膜片是使用传代数为P0-P20，例如P2-P10的间充质干细胞制备的。
  11. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞膜片的厚度为10-300微米，例如50-300微米。
  12. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞膜片中的细胞密度为1×105至1×107/cm2，例如3×105至5×106/cm2。
  13. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞细胞膜片贴附后静置15-60分钟，使所述人源间充质干细胞膜片与子宫内膜组织完全贴附。
  14. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞细胞膜片贴附后静置15-30分钟，静置观察期间使用湿润无菌纱布维持膜片贴附区域的湿润。
  15. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞膜片通过其分泌的细胞外基质彼此连接，所述细胞外基质富含纤连蛋白和整联蛋白-β1。
  16. 根据权利要求1-2中任一项所述的用途或权利要求3所述的方法，其中，所述人源间充质干细胞膜片能够分泌多种细胞因子，所述细胞因子包括HGF、IL-6、IL-8和VEGF中的一种或多种。
PCT/CN2023/106157 2022-07-25 2023-07-06 人源间充质干细胞膜片在子宫瘢痕治疗中的用途 WO2024022079A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210877855.7A CN114984052A (zh) 2022-07-25 2022-07-25 人源间充质干细胞膜片在子宫瘢痕治疗中的用途
CN202210877855.7 2022-07-25

Publications (2)

Publication Number Publication Date
WO2024022079A1 WO2024022079A1 (zh) 2024-02-01
WO2024022079A9 true WO2024022079A9 (zh) 2024-04-18

Family

ID=83020964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/106157 WO2024022079A1 (zh) 2022-07-25 2023-07-06 人源间充质干细胞膜片在子宫瘢痕治疗中的用途

Country Status (2)

Country Link
CN (1) CN114984052A (zh)
WO (1) WO2024022079A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114984052A (zh) * 2022-07-25 2022-09-02 京东方科技集团股份有限公司 人源间充质干细胞膜片在子宫瘢痕治疗中的用途

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102311942A (zh) * 2011-07-26 2012-01-11 王松灵 一种维生素c诱导的间充质干细胞膜片及其制备方法
CN105983133A (zh) * 2015-01-30 2016-10-05 南京鼓楼医院 用于修复子宫内膜损伤的干细胞复合胶原支架试剂盒及其制备方法
CN105169485A (zh) * 2015-07-29 2015-12-23 西安芙金细胞科技有限公司 一种组织工程子宫内膜及其制备方法
US20210386790A1 (en) * 2019-01-16 2021-12-16 University Of Utah Research Foundation Use of mesenchymal stem cell sheets for preventing uterine scar formation
CN111621474A (zh) * 2019-02-28 2020-09-04 京东方科技集团股份有限公司 间充质干细胞膜片及其制备方法
CN113498434A (zh) * 2020-01-22 2021-10-12 京东方科技集团股份有限公司 间充质干细胞膜片及其用途
CN113181218A (zh) * 2021-03-22 2021-07-30 中国福利会国际和平妇幼保健院 人羊膜上皮细胞在制备修复子宫瘢痕细胞制剂中的应用
CN114984052A (zh) * 2022-07-25 2022-09-02 京东方科技集团股份有限公司 人源间充质干细胞膜片在子宫瘢痕治疗中的用途

Also Published As

Publication number Publication date
WO2024022079A1 (zh) 2024-02-01
CN114984052A (zh) 2022-09-02

Similar Documents

Publication Publication Date Title
CN111394299B (zh) 一种肝脏类器官的体外构建方法及应用
Campo et al. Tissue-specific decellularized endometrial substratum mimicking different physiological conditions influences in vitro embryo development in a rabbit model
JP5946046B2 (ja) ヒト角膜内皮細胞シート
BR112015028193B1 (pt) Suporte para cicatrização de feridas
WO2024022079A9 (zh) 人源间充质干细胞膜片在子宫瘢痕治疗中的用途
JPH07504323A (ja) 腎臓の細管幹細胞を単離し，生育するための方法と組成物，試験管内腎臓細管形成および生体外における尿細管の構築
JP2019163295A (ja) 哺乳動物種における誘導組織再生のための組成物および方法
CN112870228B (zh) 一种多功能微环境保护外泌体水凝胶及其制备方法和应用
Rogovaya et al. Reconstruction of rabbit urethral epithelium with skin keratinocytes
CN106492194A (zh) 一种干细胞外泌体制剂及其制备方法和应用
Li et al. Therapeutic potential of adipose-derived stem cell-based microtissues in a rat model of stress urinary incontinence
CA3174384A1 (en) Kidney organoids having a nephron-like structure and methods of preparing the same
US9011837B2 (en) Tissue-engineered endothelial and epithelial implants repair multilaminate tubular airway structure
JP5280451B2 (ja) 脱落膜由来幹細胞または脂肪由来幹細胞を含有する尿失禁の細胞治療剤
Yu et al. Enhanced angiogenic potential of adipose-derived stem cell sheets by integration with cell spheroids of the same source
CN118526567A (zh) 基于子宫内膜干细胞的阴道修复凝胶、制备方法及应用
Zhu et al. Chorionic villi-derived nanofibers enhanced mesenchymal stem cell extracellular vesicle secretion and bioactivity for endometrium regeneration toward intrauterine adhesion treatment
WO2021148000A1 (zh) 间充质干细胞膜片及其用途
Merguerian et al. Grafting of cultured uroepithelium and bladder mucosa into de-epithelialized segments of colon in rabbits
CN109125805A (zh) 一种干细胞复合plga支架试剂盒及其应用
US20200390937A1 (en) Sheet-shaped cell culture for regeneration of digestive tract
Cao et al. Injectable “Homing‐Like” Bioactive Short‐Fibers for Endometrial Repair and Efficient Live Births
Cui et al. Effects of cartilage-derived morphogenetic protein 1 (CDMP1) transgenic mesenchymal stem cell sheets in repairing rabbit cartilage defects
CN116162590A (zh) 一种基于plga包裹的人脐带间充质干细胞外泌体及其应用
CN116426469A (zh) LAP2α在间充质干细胞成脂向分化中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23845292

Country of ref document: EP

Kind code of ref document: A1