WO2021148000A1 - 间充质干细胞膜片及其用途 - Google Patents

间充质干细胞膜片及其用途 Download PDF

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WO2021148000A1
WO2021148000A1 PCT/CN2021/073295 CN2021073295W WO2021148000A1 WO 2021148000 A1 WO2021148000 A1 WO 2021148000A1 CN 2021073295 W CN2021073295 W CN 2021073295W WO 2021148000 A1 WO2021148000 A1 WO 2021148000A1
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mesenchymal stem
stem cell
stem cells
use according
cell membrane
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PCT/CN2021/073295
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English (en)
French (fr)
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常德华
马建林
高爽
王娟
王静
靳新
刘帅
刘东华
赵玉菲
刘洋
谭玉琴
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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
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Priority to US17/430,086 priority Critical patent/US20220347346A1/en
Priority to CN202180003593.5A priority patent/CN113891933A/zh
Publication of WO2021148000A1 publication Critical patent/WO2021148000A1/zh

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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • C12N5/0662—Stem cells
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0375—Animal model for cardiovascular diseases
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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    • C12N2539/00—Supports and/or coatings for cell culture characterised by properties
    • C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating

Definitions

  • the present disclosure relates to the fields of tissue engineering and regenerative medicine, and in particular to the use of mesenchymal stem cell membranes, such as umbilical cord mesenchymal stem cell membranes, for treating cardiac tissue damage or diseases related to cardiac insufficiency in a subject.
  • mesenchymal stem cell membranes such as umbilical cord mesenchymal stem cell membranes
  • the results of the cardiovascular disease survey show that the current prevalence of heart failure in adults in my country is 0.9%. According to the 2019 China Heart Failure Blue Book Report, it is estimated that the number of heart failure patients in my country is as high as 6.5-8.75 million, and the number is increasing by 200,000 per year. An aging population, high blood pressure, high blood lipids and other cardiovascular risk factors are the main causes of heart failure. The prevalence of heart failure in people over 70 years old is >10%, the 5-year mortality rate in patients with heart failure is up to 50%, and the 1-year mortality rate in patients with severe heart failure is up to 50%.
  • stem cells of various sources and types can effectively treat acute and chronic myocardial infarction, ischemic heart failure, etc. Through the paracrine action of stem cells, it improves the contraction and relaxation of the heart, prevents myocardial fibrosis, and improves the prognosis.
  • stem cell therapy still has the following problems in clinical applications: embryonic stem cells face ethical, legal, and immune rejection issues; iPS has potential tumorigenicity and requires HLA matching; skeletal myoblasts can cause ventricular heart rhythm Disorders; bone marrow or peripheral blood mononuclear cells have complex components, mainly immune cells, and contain few stem cells with multidirectional differentiation potential; the source of bone marrow mesenchymal stem cells (MSCs) is limited, and the cell proliferation and stemness vary with age Increase and decrease significantly.
  • MSCs bone marrow mesenchymal stem cells
  • Mesenchymal stem cells are widely present in human tissues, have the ability to expand in vitro, have multiple differentiation potentials and immunomodulatory effects, and have been used in the treatment of self-tissue repair and immune-related diseases.
  • mesenchymal stem cells for self-tissue repair most of the methods use direct injection of cells or transplantation after the combination of cells and tissue engineering scaffold materials, but both of them have certain limitations. sex.
  • Direct injection of cells will cause a large number of cells to be lost, the treatment efficiency is low, and the function of stem cells to perform tissue repair is limited; while the transplantation of cells and tissue engineering scaffolds solves the problem of cell loss, the scaffold materials may cause different degrees in the body Inflammation, and the degradation process and degradation products of the scaffold material may cause local tissue lesions.
  • Cell membrane is a new method of cell therapy in recent years.
  • Cell membrane can form two-dimensional and three-dimensional structures only through the connection between cells.
  • cell membranes can better achieve local fixation and reduce cell loss, thereby improving cell utilization and avoiding the introduction of scaffold materials.
  • the inventors prepared mesenchymal stem cell membranes and evaluated them in the constructed animal model of heart failure. The results show that the mesenchymal stem cell membrane of the present invention has a good therapeutic effect on heart failure, improves the movement and ejection ability of the heart, and reduces the degree of cardiac remodeling and fibrosis.
  • the present disclosure relates to a method for treating a disease related to cardiac tissue damage or cardiac insufficiency in a subject, the method comprising locally applying mesenchymal to the heart of the subject Steps of stem cell patch.
  • the disease may be selected from ischemic heart disease, rheumatic heart disease, congenital heart disease, cardiomyopathy, coronary heart disease, and valvular heart disease.
  • the cardiomyopathy is dilated cardiomyopathy.
  • the disease may be ischemic heart failure, such as acute ischemic heart failure, chronic ischemic heart failure, or end-stage ischemic heart failure.
  • the disease is chronic heart failure caused by ischemic heart disease and dilated cardiomyopathy, for example: one type is heart failure caused by myocardial ischemia, including chronic ischemic heart failure and end-of-life End-stage ischemic heart failure; the other is heart failure caused by dilated cardiomyopathy.
  • the mesenchymal stem cell membrane is attached to the anterior or sidewall of the left ventricle of the heart.
  • the mesenchymal stem cell patch is attached to the damaged or defective part of the heart, or its adjacent part, to treat the disease related to heart tissue damage or cardiac insufficiency.
  • the mesenchymal stem cell membrane is implanted into the damaged or defective part of the heart, or its adjacent part. Compared with traditional single-cell suspensions or methods that combine cells with tissue engineering scaffold materials, cell membranes can better achieve local fixation and reduce cell loss, thereby improving cell utilization and avoiding the introduction of scaffold materials. A heterologous substance that may trigger a larger immune response.
  • the cell proportion of the mesenchymal stem cells in the mesenchymal stem cell membrane sheet may be at least 90%.
  • the cell proportion of the mesenchymal stem cells of the patch is at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99%, for example, by detecting cell surface markers by flow cytometry, Three-way differentiation assay or PCR method to detect the expression of genes identified by the cells.
  • the mesenchymal stem cells may be derived from a tissue selected from the group consisting of amniotic fluid, amniotic membrane, chorion, chorionic villi, decidua, placenta, umbilical cord blood, Wharton’s gum, umbilical cord, Adult bone marrow, dental pulp, adult peripheral blood and adult adipose tissue.
  • the mesenchymal stem cells may be selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and bone marrow mesenchymal stem cells.
  • the mesenchymal stem cells may be umbilical cord mesenchymal stem cells.
  • the mesenchymal stem cell membrane sheet may be prepared using mesenchymal stem cells with a passage number of P0-P20.
  • mesenchymal stem cells with passage numbers P2-P15, P2-P8, or P2-P10 can be used to prepare cell membrane sheets.
  • the thickness of the mesenchymal stem cell membrane sheet may be about 10-300 ⁇ m, for example, 30-300 ⁇ m, 50-300 ⁇ m, 100-300 ⁇ m, 80-300 ⁇ m, 100-300 ⁇ m, 120-300 ⁇ m, 150-300 ⁇ m. 300 ⁇ m or 200-300 ⁇ m thickness. In some embodiments, the thickness of the mesenchymal stem cell membrane may be about 15-250 ⁇ m.
  • the mesenchymal stem cell membrane sheet of the present disclosure may have different cell layers.
  • the mesenchymal stem cell membrane may have 1-15 layers of cells, such as 2-15 layers, 3-15 layers, 5-15 layers, 8-15 layers, or 10-15 layers of cells.
  • the cell density in the mesenchymal stem cell membrane sheet may be about 1 ⁇ 10 5 to 5 ⁇ 10 7 /cm 2 , for example, about 1 ⁇ 10 5 to 1 ⁇ 10 7 /cm 2 , 8 ⁇ 10 5 to 5 ⁇ 10 7 /cm 2 , 3 ⁇ 10 5 to 5 ⁇ 10 6 /cm 2 .
  • the size and shape of the mesenchymal stem cell membrane used can be determined according to actual needs, for example, according to the size and shape of the subject's heart injury or defect.
  • a mesenchymal stem cell membrane sheet in a round shape or a shape that facilitates attachment or implantation may be used.
  • a circular cell membrane with a diameter of 15-55mm and a thickness of 10-300 ⁇ m can be used to attach to the anterior or sidewall of the left ventricle of the heart for the treatment of the cardiac tissue damage or cardiac insufficiency. Disease.
  • the mesenchymal stem cells in the patch are connected to each other through the extracellular matrix secreted by them, and the extracellular matrix is rich in fibronectin and integrin- ⁇ 1.
  • the mesenchymal stem cells in the patch can secrete a variety of cytokines, such as angiogenesis factors and immunoregulatory factors, such as hepatocyte growth factor (HGF), fibroblast growth factor (FGF), Epidermal growth factor (EGF), transforming growth factor- ⁇ (TGF- ⁇ ), prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin 10 (IL-10), interleukin- 8 (IL-8) and one or more of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).
  • HGF hepatocyte growth factor
  • FGF fibroblast growth factor
  • EGF- ⁇ Epidermal growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • PGE2 prostaglandin E2
  • IL-6 interleukin-6
  • IL-10 interleukin 10
  • IL-8 interleukin- 8
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived growth
  • the membrane includes 2 ⁇ 10 7 to 8 ⁇ 10 7 mesenchymal stem cells.
  • 1-4 sheets of the mesenchymal stem cell membrane are locally applied to the heart of the subject.
  • the topical application of the mesenchymal stem cell patch to the heart of the subject is combined with the subject's coronary artery bypass surgery, for example, the subject's coronary artery bypass graft After the operation, the mesenchymal stem cell patch is locally applied to the heart of the subject.
  • the methods of applying the present invention to transplant mesenchymal stem cell membranes include: 1 surgically open the chest, open the pericardium, and apply 1-4 pieces of UMSCCS to the anterior and side walls of the left ventricle of the heart; 2 Coronary After arterial bypass, apply UMSCCS to the anterior wall and sidewall of the left ventricle of the heart; 3Apply UMSCCS to the anterior and sidewall of the left ventricle of the heart through minimally invasive surgery.
  • the mesenchymal stem cells in the mesenchymal stem cell membrane are autologous or allogeneic to the subject.
  • the mesenchymal stem cell membrane sheet may be prepared by a method including the following steps:
  • the mesenchymal stem cells are connected to each other through the extracellular matrix secreted by them, thereby obtaining the mesenchymal stem cell membrane.
  • the mesenchymal stem cells used to prepare the mesenchymal stem cell membrane sheet may be prepared by a method including the following steps:
  • mesenchymal stem cells grow to about 50%-100% confluence, such as about 70%-100% confluence or about 80%-100% confluence, remove the tissue mass, thereby obtaining umbilical cord mesenchymal stem cells; And optional
  • temperature-sensitive petri dish refers to a petri dish coated with a layer of temperature-sensitive polymer material, which has different molecular chain stretches at different temperatures, thus exhibiting hydrophilicity. Or hydrophobicity, so that the hydrophilicity and hydrophobicity of the polymer substance can change with the change of external temperature.
  • the surface of the temperature-sensitive petri dish is hydrophilic, the adhesion to the cells and the extracellular matrix secreted by the cells will become poor, and the cells will fall off in layers.
  • the temperature is lowered below the low critical dissolution temperature of the polymer substance, the surface of the temperature-sensitive petri dish will appear hydrophilic, so that the cells will fall off in layers.
  • the use of a temperature-sensitive petri dish realizes that the lamellar mesenchymal stem cells can be separated from the bottom of the temperature-sensitive petri dish without digestion with enzymes and the like or peeling off by physical methods, leaving the extracellular matrix intact. Connected cell membrane sheet.
  • the cell growth curve can be measured by MTT method, WST method, DNA content detection method, ATP detection method, etc., to evaluate the growth activity of umbilical cord mesenchymal stem cells.
  • the isolated and cultured mesenchymal stem cells can be identified by detecting cell surface markers by flow cytometry, three-way differentiation assay, and PCR method to detect cell expression genes.
  • flow cytometry can be used to detect cell surface marker proteins to identify mesenchymal stem cells.
  • the adhesion matrix used to coat the temperature-sensitive petri dish may include fetal bovine serum, autologous serum, collagen, gelatin, fibronectin, vitronectin, laminin, One or more of polyornithine and polylysine.
  • the serum used to coat the temperature-sensitive petri dish is selected from fetal bovine serum (FBS) or human serum. In some embodiments, 100% serum is used as the coating solution. In other embodiments, a basal medium (for example, 1640, DMEM, ⁇ -MEM, or DMEM/F12) containing at least 10% (v/v) serum is used as the coating solution.
  • FBS fetal bovine serum
  • human serum 100% serum is used as the coating solution.
  • a basal medium for example, 1640, DMEM, ⁇ -MEM, or DMEM/F12
  • the mesenchymal stem cells are detached from the temperature-sensitive petri dish by lowering the temperature, thereby forming a mesenchymal stem cell membrane sheet.
  • the mesenchymal stem cells are detached from the temperature-sensitive petri dish by lowering the temperature to 4-32°C.
  • a buffer such as HBSS, PBS, or physiological saline pre-cooled at 4°C is added to detach the mesenchymal stem cells from the temperature-sensitive culture dish.
  • the mesenchymal stem cell membrane has an upper surface that does not contact the culture dish and a base surface that contacts the culture dish during the preparation process, and the base surface is relatively rough. Due to its structural characteristics, the basal surface of the mesenchymal stem cell membrane can provide greater friction, which is beneficial for the cell membrane to better adhere to the application site during application.
  • the basal surface of the mesenchymal stem cell membrane may be attached to the damaged or defective part of the heart, or its adjacent part.
  • the basal surface of the mesenchymal stem cell membrane may be attached to the anterior wall and side wall of the left ventricle of the heart.
  • the present disclosure relates to the use of mesenchymal stem cell membranes in the treatment of diseases related to cardiac tissue damage or cardiac insufficiency in a subject, wherein the mesenchymal stem cell membranes are locally applied to all diseases.
  • the heart of the subject The heart of the subject.
  • the present disclosure relates to the use of a mesenchymal stem cell patch in the preparation of a composition for treating diseases related to cardiac tissue damage or cardiac insufficiency in a subject, wherein the mesenchymal stem cell The diaphragm is applied locally to the heart of the subject.
  • the disease may be selected from ischemic heart disease, rheumatic heart disease, congenital heart disease, cardiomyopathy, coronary heart disease, and valvular heart disease.
  • the cardiomyopathy is dilated cardiomyopathy.
  • the disease may be ischemic heart failure, such as acute ischemic heart failure, chronic ischemic heart failure, or end-stage ischemic heart failure Exhaustion.
  • ischemic heart failure such as acute ischemic heart failure, chronic ischemic heart failure, or end-stage ischemic heart failure Exhaustion.
  • the mesenchymal stem cell patch is attached to the damaged or defective part of the heart, or its adjacent part.
  • the mesenchymal stem cell membrane is implanted into the damaged or defective part of the heart, or its adjacent part.
  • the mesenchymal stem cell membrane is attached to the anterior or sidewall of the left ventricle of the heart.
  • the cell proportion of the mesenchymal stem cells in the mesenchymal stem cell patch may be at least 90%.
  • the cell proportion of the mesenchymal stem cells of the patch is at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99%, for example, by detecting cell surface markers by flow cytometry, Three-way differentiation assay or PCR method to detect the expression of genes identified by the cells.
  • the mesenchymal stem cells may be derived from a tissue selected from the group consisting of amniotic fluid, amniotic membrane, chorion, chorionic villi, decidua, placenta, umbilical cord blood, Wharton’s gum, umbilical cord, Adult bone marrow, dental pulp, adult peripheral blood and adult adipose tissue.
  • the mesenchymal stem cells may be selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, and bone marrow mesenchymal stem cells.
  • the mesenchymal stem cells may be umbilical cord mesenchymal stem cells.
  • the mesenchymal stem cell membrane sheet may be prepared using mesenchymal stem cells with a passage number of P0-P20.
  • mesenchymal stem cells with passage numbers P2-P15, P2-P8, or P2-P10 can be used to prepare cell membrane sheets.
  • the thickness of the mesenchymal stem cell membrane may be about 10-300 ⁇ m. In some embodiments, the thickness of the mesenchymal stem cell membrane sheet may be about 20-300 ⁇ m, for example, 30-300 ⁇ m, 50-300 ⁇ m, or 100-300 ⁇ m.
  • the mesenchymal stem cell membrane disclosed in the present invention may have different cell layers. In some embodiments, the mesenchymal stem cell membrane may have 1-15 layers of cells, such as 2-15 layers, 3-15 layers, or 5-15 layers of cells.
  • the cell density in the mesenchymal stem cell membrane sheet may be about 1 ⁇ 10 5 to 5 ⁇ 10 7 /cm 2 , for example, about 8 ⁇ 10 5 to 5 ⁇ 10 7 /cm 2 , 3 ⁇ 10 5 to 5 ⁇ 10 6 /cm 2 .
  • the mesenchymal stem cells in the patch are connected to each other through the extracellular matrix secreted by them, and the extracellular matrix is rich in fibronectin and integrin- ⁇ 1.
  • the mesenchymal stem cells in the patch can secrete a variety of cytokines, such as angiogenesis factors and immunoregulatory factors, such as HGF, EGF, FGF, TGF- ⁇ , PGE2, IL-6, IL -8, one or more of IL-10, PDGF and VEGF.
  • cytokines such as angiogenesis factors and immunoregulatory factors, such as HGF, EGF, FGF, TGF- ⁇ , PGE2, IL-6, IL -8, one or more of IL-10, PDGF and VEGF.
  • the membrane includes 2 ⁇ 10 7 to 8 ⁇ 10 7 mesenchymal stem cells.
  • 1-4 sheets of the mesenchymal stem cell membrane are locally applied to the heart of the subject.
  • the topical application of the mesenchymal stem cell patch to the heart of the subject is combined with the subject's coronary artery bypass surgery, for example, the subject's coronary artery bypass graft After the operation, the mesenchymal stem cell patch is locally applied to the heart of the subject.
  • the mesenchymal stem cells in the mesenchymal stem cell patch are heterologous or homologous to the subject.
  • the mesenchymal stem cell membrane sheet may be prepared by a method including the following steps:
  • the mesenchymal stem cells are connected to each other through the extracellular matrix secreted by them, thereby obtaining the mesenchymal stem cell membrane.
  • the mesenchymal stem cells used to prepare the mesenchymal stem cell membrane sheet may be prepared by a method including the following steps:
  • mesenchymal stem cells grow to about 50%-100% confluence, such as about 70%-100% confluence or about 80%-100% confluence, remove the tissue mass, thereby obtaining umbilical cord mesenchymal stem cells; And optional
  • the adhesion matrix used to coat the temperature-sensitive petri dish may include fetal bovine serum, autologous serum, collagen, gelatin, fibronectin, vitronectin, laminin, One or more of polyornithine and polylysine.
  • the serum used to coat the temperature-sensitive petri dish is selected from fetal bovine serum (FBS) or human serum. In some embodiments, 100% serum is used as the coating solution. In other embodiments, a basal medium (for example, 1640, DMEM, ⁇ -MEM, or DMEM/F12) containing at least 10% (v/v) serum is used as the coating solution.
  • FBS fetal bovine serum
  • human serum 100% serum is used as the coating solution.
  • a basal medium for example, 1640, DMEM, ⁇ -MEM, or DMEM/F12
  • the mesenchymal stem cells are detached from the temperature-sensitive petri dish by lowering the temperature, thereby forming a mesenchymal stem cell membrane sheet.
  • the mesenchymal stem cells are detached from the temperature-sensitive petri dish by lowering the temperature to 4-32°C.
  • a buffer pre-cooled at 4°C is added to detach the mesenchymal stem cells from the temperature-sensitive petri dish.
  • the mesenchymal stem cell membrane sheet has an upper surface that does not contact the culture dish and a base surface that contacts the culture dish during the preparation process, and the base surface is relatively rough.
  • the basal surface of the mesenchymal stem cell membrane is attached to the damaged or defective part of the heart, or its adjacent part.
  • the basal surface of the mesenchymal stem cell membrane may be attached to the anterior wall and side wall of the left ventricle of the heart.
  • the mesenchymal stem cell membrane is a single-dose mesenchymal stem cell membrane, wherein the membrane comprises 2 ⁇ 10 7 to 8 ⁇ 10 7 mesenchymal stem cells.
  • the diameter of the single-dose mesenchymal stem cell membrane is 15-55 mm, and the thickness of the membrane is 10-300 ⁇ m.
  • the present invention relates to a mesenchymal stem cell membrane used to treat diseases related to cardiac tissue damage or cardiac insufficiency in a subject.
  • the cell density in the mesenchymal stem cell membrane sheet is 8 ⁇ 10 5 to 5 ⁇ 10 7 /cm 2 .
  • the present invention relates to a single-dose mesenchymal stem cell membrane sheet, wherein the membrane sheet contains 2 ⁇ 10 7 to 8 ⁇ 10 7 mesenchymal stem cells, and the mesenchymal stem cells pass between The extracellular matrix secreted by them is connected to each other.
  • the "single dose” mentioned in this application refers to the dose that can exert a therapeutic effect every time one mesenchymal stem cell membrane is used.
  • the single-dose mesenchymal stem cell membrane according to the embodiment of the present invention can be used at a time, and 1-4 pieces can be applied locally to the heart of the subject, and applied to treat diseases related to heart tissue damage or cardiac insufficiency of the subject , For example, can be attached to the anterior wall and side wall of the left ventricle of the heart.
  • the diameter of the single-dose mesenchymal stem cell membrane is 15-55 mm, and the thickness of the membrane is 10-300 ⁇ m.
  • the cell density in the mesenchymal stem cell membrane sheet is 8 ⁇ 10 5 to 5 ⁇ 10 7 /cm 2 .
  • the present invention relates to a composition comprising mesenchymal stem cell membranes, the composition being used to treat diseases related to cardiac tissue damage or cardiac insufficiency in a subject.
  • the disease may be selected from ischemic heart disease, rheumatic heart disease, congenital heart disease, cardiomyopathy, coronary heart disease Heart disease and heart valve disease.
  • the disease may be ischemic heart failure, such as acute ischemic heart failure, chronic ischemic heart failure Or end-stage ischemic heart failure.
  • the mesenchymal stem cell has one or more of the first, second, and third aspects of the present disclosure. Item characteristics.
  • Figure 1 shows a photograph of an umbilical cord mesenchymal stem cell membrane obtained according to an embodiment of the present invention.
  • Figure 2 shows the test results of the adipogenic and osteogenic differentiation of umbilical cord mesenchymal stem cells.
  • Figure 1A Results of Alizarin Red staining
  • Figure 1B Results of Oil Red O staining.
  • Figure 3 shows a scanning electron microscopic image of a membrane of umbilical cord mesenchymal stem cells.
  • Figure 3A Surface (upper surface) of cell membrane sheet.
  • Figure 3B Basal surface of cell membrane sheet.
  • Figure 4 shows an immunofluorescence imaging photograph of a membrane of umbilical cord mesenchymal stem cells.
  • Figure 4A Fibronectin.
  • Figure 4B Integrin ⁇ 1.
  • Figure 5 shows the results of using the ELISA method to detect the cytokine expression in the culture supernatant of the optic cord mesenchymal stem cell patch.
  • Figure 6 shows a photograph of a mesenchymal stem cell patch transplanted to the mouse heart.
  • Figure 7 shows the characterization of the constructed mouse disease model of heart failure.
  • Fig. 7A Heart photos of disease model mice;
  • Fig. 7B ECG results of disease model mice.
  • Figure 8 shows the use of umbilical cord mesenchymal stem cell patch to treat disease model mice.
  • Fig. 8A shows an exemplary photograph of the mesenchymal stem cell membrane used;
  • Fig. 8B shows a photograph of the cell membrane attached to the surface of a mouse heart.
  • Figure 9 shows the mouse echocardiogram results at different time points.
  • Figure 9A before modeling
  • Figure 9B 1 week after modeling
  • Figure 9C 4 weeks after modeling.
  • Left side control group animals
  • right side cell patch transplantation group animals.
  • Figure 10 shows the curve of the left ventricular ejection fraction of mice in the cell patch treatment group and the control group over time.
  • Figure 11 shows the curve of the left ventricular short axis shortening index of mice in the cell patch treatment group and the control group over time.
  • Figure 12 shows the curve of the left ventricular diameter of the mice in the cell patch treatment group and the control group over time.
  • Figure 13 shows the curve of the left ventricular volume of mice in the cell patch treatment group and the control group over time.
  • Figure 14 shows the results of Masson staining of mouse heart tissue sections 28 days after cell patch treatment. Left side: control group animals; right side: cell patch treatment group animals.
  • Figure 15 shows a photograph of transplanting 1-2 sheets of the obtained mesenchymal stem cell membrane to the anterior wall of the left ventricle of a miniature pig.
  • Figure 16 shows the ECG before and after the model of myocardial infarction in mini-pigs and after the treatment of the transplanted membrane.
  • Figure 17 shows a statistical chart of changes in EF, SV, LVFS, EDV, ESV and other indicators in the patch transplantation group compared with the model control group.
  • Figure 18 shows the pathological examination results of the myocardial infarction rate of animals in the model control group and the patch transplantation group.
  • Figure 19 shows the pathological examination results of the degree of myocardial fibrosis in the model control group and the patch transplantation group.
  • the technical problem to be solved by the present invention is to apply the human umbilical cord mesenchymal stem cell membrane to treat patients with heart failure caused by ischemic heart disease, dilated cardiomyopathy and the like.
  • the transplantation of cell membranes improves myocardial function, improves the quality of life, and reduces the mortality of patients with heart failure.
  • the membrane transplantation method will overcome the inability to ensure that a sufficient number of stem cells reach the diseased site caused by the injection of mesenchymal stem cell suspension to give full play to the biology.
  • the shortcomings of scientific efficacy overcome the shortcomings of stem cells + biological scaffolds that induce inflammatory reactions due to incomplete absorption of biological materials.
  • the "umbilical cord mesenchymal stem cell membrane" referred to in the present invention can be prepared according to the following method: take P2-P8 generation umbilical cord mesenchymal stem cells to prepare a single cell suspension, with 3 ⁇ 10 5 -1.2 ⁇ 10 6 cells /cm 2 is added to a temperature-sensitive culture dish coated with a substrate (such as fetal bovine serum, autologous serum, gelatin, fibronectin, vitronectin, laminin, polylysine) before cell culture To the supersaturated state, place the temperature-sensitive petri dish under 20°C, and the cells will spontaneously form a film and fall off.
  • a substrate such as fetal bovine serum, autologous serum, gelatin, fibronectin, vitronectin, laminin, polylysine
  • a circular sheet-shaped cell membrane sheet with a diameter of 15-55mm and a thickness of 10-300 ⁇ m is obtained (as shown in Figure 1), and the number of cells contained is 2 ⁇ 10 7 -8 ⁇ 10 7 , which can be used immediately or cryopreserved for later use.
  • the umbilical cord mesenchymal stem cell membrane of the present invention is a round sheet-shaped cell junction body, and cells are closely connected to each other, maintaining a complete extracellular matrix (Extracellular matrix).
  • ECM extracellular matrix
  • the ingredients include fibronectin, laminin, collagen, mucopolysaccharides and so on.
  • ECM is an important basic material for stem cells to maintain several physiological functions, helps the secretion and signal transmission of cytokines, and is conducive to the colonization and survival of stem cells in the body.
  • the umbilical cord mesenchymal stem cell patch of the present invention can secrete a variety of cytokines after being applied to the lesion site, mainly including epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), blood vessel Endothelial growth factor (VEGF), transforming growth factor- ⁇ (TGF- ⁇ ), prostaglandin E2 (PGE2), platelet-derived growth factor (PDGF), interleukin 6 (IL-6), interleukin 10 (IL- 10), etc., to repair the ischemic damaged tissues and organs or promote their self-repair, so as to achieve the therapeutic effect.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF blood vessel Endothelial growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • PGE2 prostaglandin E2
  • PDGF platelet-derived growth factor
  • IL-6 interleukin 6
  • the “chronic heart failure caused by ischemic heart disease and dilated cardiomyopathy” referred to in the present invention includes two categories: one is heart failure caused by myocardial ischemia, including chronic ischemic heart failure and end-stage Ischemic heart failure; the other is heart failure caused by dilated cardiomyopathy.
  • the mechanism of the umbilical cord mesenchymal stem cell patch transplantation of the present invention to treat chronic heart failure caused by ischemic heart disease and dilated cardiomyopathy a large number of umbilical cord mesenchymal stem cell patch secretes a variety of growth factors and cytokines at the transplantation site, Mainly include HGF, VEGF, IL-6, IL-10, etc., which regulate the microenvironment of myocardial ischemia, inhibit myocardial inflammatory response, and prevent the progression of myocardial infarction fibrosis; promote local angiogenesis, and provide blood supply to the myocardium through compensatory methods. Improve myocardial function.
  • the umbilical cord mesenchymal stem cell membrane sheet of the present invention is derived from foreign bodies, has excellent performance, and can replace autologous stem cells for the treatment of heart failure.
  • Umbilical cord mesenchymal stem cell membranes show significant advantages: 1The umbilical cord is widely used, convenient, and non-invasive; 2Uniform quality standards can be established, and the best selection of materials can be used to break through the constraints of autologous stem cells due to their own objective conditions; 3Multi-level cells can be prepared in advance Library and spot products greatly shorten the in vitro cell culture and patient waiting time; 4Umbilical cord mesenchymal stem cell membranes have low immunogenicity, and allogeneic applications will hardly cause immune rejection; 5Umbilical cord mesenchymal stem cell membranes are safer , No tumorigenesis, no toxic reaction; 6Improve myocardial ischemia in patients with heart failure through the paracrine effect of stem cells, and prolong the survival period of patients.
  • the umbilical cord of a human newborn is taken, and the outer membrane and blood vessels are removed to obtain the Wharton's gel-like tissue in the umbilical cord tissue. Cut the Wharton's gel-like tissue with sterile scissors into tissue pieces of about 1-2 mm 3 , and spread them in a petri dish for culture. After the umbilical cord mesenchymal stem cells crawl out, remove the tissue mass, and add fresh medium to continue the culture. When the cells grow to about 70-100% confluence, the cells are subcultured. Under the microscope, the umbilical cord mesenchymal stem cells were observed to grow adherently, fibrous and uniform in shape.
  • the following cell surface markers were detected by flow cytometry to identify isolated mesenchymal stem cells: CD105, CD34, CD31 and CD117, where CD105 is a positive marker; CD34, CD31 and CD117 are negative markers.
  • CD105 was 99.64%
  • CD34 was 0.02%
  • CD31 was 0.00%
  • CD117 was 0.51%.
  • the above results indicate that the obtained umbilical cord mesenchymal stem cells have high purity.
  • umbilical cord mesenchymal stem cells The ability of umbilical cord mesenchymal stem cells to differentiate into bone and adipocytes was further tested. Specifically, the proportion of umbilical cord mesenchymal stem cells was seeded in a petri dish. For osteogenic induction, add osteoinduction medium when the cells grow to about 50-90% confluence, and stain the cells with Alizarin Red after 7 days of culture; for adipogenic induction, add when the cells grow to more than 90% confluence Adipogenic induction medium, after 7 days of culture, the cells were stained with Oil Red O.
  • the mesenchymal stem cells can be stained with Alizarin Red (Figure 2A) or Oil Red O ( Figure 2B) after osteogenic induction or adipogenic induction, indicating that they have the ability to differentiate into bone and adipocytes. ability.
  • the above-mentioned P2-P8 generation umbilical cord mesenchymal stem cells are digested into single cells, and after making a single cell suspension, it is inoculated into the pre-packaged at a density of 3 ⁇ 10 5 -1.2 ⁇ 10 6 In a temperature-sensitive petri dish of substrate (such as fetal bovine serum, autologous serum, gelatin, fibronectin, vitronectin, laminin, polylysine), at 37°C, 5% CO 2 and 95% Cultivate in an incubator in a humid environment.
  • substrate such as fetal bovine serum, autologous serum, gelatin, fibronectin, vitronectin, laminin, polylysine
  • the cells After the cells are cultured to a supersaturated state, move them to an environment of about 20°C or add 4°C pre-cooled HBSS solution, PBS solution or physiological saline.
  • the cells detached from the bottom of the temperature-sensitive petri dish in a layered form to form a complete cell membrane connected by the extracellular matrix.
  • the obtained membrane is a round sheet with a diameter of 15-55mm and a thickness of 10-300 ⁇ m. It is off-white, with a dense structure, smooth and flat surface.
  • the rate of viable cells in the membrane is high, and the cell condition is good (Figure 1).
  • the number of cells contained is 2 ⁇ 10 7 -8 ⁇ 10 7 , for immediate use or cryopreservation for later use.
  • the structure of the prepared umbilical cord mesenchymal stem cell membrane was characterized by scanning electron microscopy and immunofluorescence imaging.
  • the cell membrane was fixed by 2.5% glutaraldehyde, alcohol gradient dehydration, and air-dried to prepare samples and then photographed by scanning electron microscopy.
  • the cell membrane has a surface that is not in contact with the culture dish (upper surface, Figure 3A) and a base surface that is in contact with the culture dish (lower surface, Figure 3B).
  • the surface is due to the natural sedimentation of cells ,
  • the formed surface is relatively smooth;
  • the base surface is relatively rough in contact with the warm dish material. Due to its structural characteristics, the base surface can provide greater friction, which is conducive to better adhesion of the cell membrane to the application site during application.
  • the expression of fibronectin and integrin ⁇ 1 in the membrane of umbilical cord mesenchymal stem cells was detected by immunofluorescence method.
  • the membrane was fixed in the fixative and then frozen sectioned, stained with fluorescein-labeled fibronectin and integrin ⁇ 1 antibody, and subjected to immunofluorescence imaging analysis.
  • the result is shown in Figure 4, the cell membrane sheet prepared by the method of the present disclosure contains a large amount of fibronectin ( Figure 4A) and integrin ⁇ 1 ( Figure 4B).
  • Fibronectin is widely present in animal tissues and tissue fluids, and has the function of promoting the adhesion and growth of cells, and the adhesion and growth of cells is a necessary condition for maintaining and repairing the tissue structure of the body.
  • Integrin ⁇ 1 is an important member of the integrin family. It plays an important role in mediating cell-to-cell, cell-to-extracellular matrix (ECM) adhesion and two-way signal transduction, and is involved in tissue repair and fibrosis. Form a close correlation.
  • ECM cell-to-extracellular matrix
  • HGF hepatocyte growth factor
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • VEGF vascular endothelial growth factor
  • the culture supernatant was taken, and the cytokine in the supernatant was detected by the ELISA method.
  • the detection result is shown in FIG. 5.
  • the results showed that the above four cytokines were all expressed in the supernatant, and the expression levels of HGF and IL-8 were high.
  • the above results indicate that the umbilical cord mesenchymal stem cell membrane of the present disclosure can secrete a variety of cytokines, including angiogenic factors and immunoregulatory factors, proving that it has high biological activity and functions, and can promote local angiogenesis and tissue repair processes.
  • the high level of IL-8 expression indicates that the cell membrane has the function of promoting immune response and inhibiting bacteria during use, which is beneficial to the cell membrane to better perform its biological functions.
  • Example 2 Take out the frozen mesenchymal stem cell membrane sheet obtained in Example 1 from the cryopreserved container, and quickly put it into a 37°C incubator or a water bath to quickly thaw it within 2 minutes. Transfer the thawed mesenchymal stem cell membrane to a common petri dish, wash the membrane twice with physiological saline, add 10 mL of fresh membrane protection solution, and perform aseptic sealing and packaging.
  • allogeneic umbilical cord mesenchymal stem cells belongs to allogeneic stem cell transplantation, and immune rejection needs to be considered. To this end, the inventors performed in vitro immunogenicity identification of umbilical cord mesenchymal stem cells.
  • the inventors used flow cytometry to detect umbilical cord mesenchymal stem cells and found that umbilical cord mesenchymal stem cells do not express HLA-II antigens HLA-DR and co-stimulatory antigens CD80 and CD86; the inventors combined umbilical cord mesenchymal stem cells with human peripheral blood Mononuclear cells were mixed and co-cultured in a complete medium containing 10% fetal bovine serum at a ratio of 1:2-1:30. After 2 days, the suspended cells in the supernatant were collected for counting and flow cytometry. None was observed Abnormal proliferation of lymphocytes, no changes in T cell subsets CD3 and CD8 were observed.
  • Balb/c nude mice were subcutaneously inoculated with human lung cancer A549 cells to establish a solid tumor model, and NPG nude mice were subcutaneously inoculated with human lymphoma Raji cells to establish a hematoma model.
  • Sufficient doses of umbilical cord mesenchymal stem cells were injected through the tail vein, and the tumor volume was measured for 4 to 8 weeks, and then gross dissection, tumor weight, and pathological examination were performed.
  • the tumor volume and weight of the nude mice in the umbilical cord mesenchymal stem cell injection group were not significantly different. There was no tumor occurrence in all major organs of the body, and the lymphocyte infiltration and the degree of infiltration to the surrounding tissues were not found in pathological examination. Significant differences.
  • balb/c nude mice were subcutaneously transplanted with sufficient doses of the mesenchymal stem cell membranes obtained in Examples 1, 3, or 4 of the application, raised in SPF clean environment mouse cages, and continued observation for 20 weeks. There was no tumor formation at the transplantation site. Anatomy, the main organs of the nude mice were taken for pathological examination, no tumor foci were found.
  • the mesenchymal stem cell membrane of the present invention is different from the traditional injection and administration method of stem cell suspension. It is directly applied to the surface of the heart without entering the blood and transferring. It only plays the role of nourishing the myocardium and regulating the microenvironment locally. The scope of influence is under certain control. In order to further clarify its safety, the inventors tested mesenchymal stem cell membrane toxicity in mice.
  • NPG mice aged 6-8 weeks were selected, half male and half male, and randomly divided into three groups: cell membrane group, cell suspension group, and negative control group, each with 24 mice and 12 females.
  • the cell patch group was transplanted 10 times the human dose of mesenchymal stem cell patch into the mouse heart (Figure 6), and the cell suspension group was injected with umbilical cord mesenchymal stem cells via the tail vein at a dose of 1 ⁇ 10 6 cells per mouse ,
  • the negative control group was injected with an equal volume of 0.9% sodium chloride injection. Body weight was measured once a week after administration, and observation was performed next to the cage once a day. The observation period was 4 weeks.
  • mice of each sex in each group were dissected for gross anatomy observation.
  • a mouse model of ischemic heart failure was constructed by coronary artery ligation.
  • sutures were used to ligate the left anterior descending branch, which hindered the blood supply of the left ventricle, and caused the apoptosis of myocardial cells in the infarct area, resulting in a decrease in the ejection function of the left ventricle.
  • the ventricular structure is remodeled and eventually develops into heart failure. It includes the following steps:
  • mice with isoflurane mixed with oxygen concentration of isoflurane is about 3.5-5%
  • perform hair removal treatment with depilatory cream
  • the maintenance anesthetic gas is about 3% isoflurane, the tidal volume is 0.3ml, the frequency is ⁇ 124 times/min, and the breathing ratio is 50:50.
  • a Medlab two-lead physiological signal collection line system was used to monitor mouse ECG, where the right upper limb was subcutaneously connected to the positive electrode, the left lower limb was subcutaneously connected to the negative electrode, and the right lower limb was subcutaneously grounded.
  • the thoracic cavity is sutured. After the model is completed, the chest expander is removed, the intercostal muscle tissue is returned to its place, and then the epidermis is sutured.
  • the air in the thoracic cavity can be removed by squeezing the thoracic cavity before the suture is completed or after the suture is completed to suck the thoracic cavity with a syringe to avoid the death of the animal caused by pneumothorax.
  • mice were subjected to echocardiography.
  • the parasternal short-axis view was taken at the level of the left ventricular papillary muscle. Mark points can be observed echocardiogram. From the results in Fig. 9B and Fig. 9C, it can be seen that the heart of the heart failure model animal has obvious weakening of motion after modeling.
  • the cell patch treatment group animals (right panel) had stronger heart movements.
  • the curve of the left ventricular ejection fraction with time before and after the operation ( Figure 10) and the curve of the left ventricular short axis shortening index with time were calculated and drawn ( Figure 11).
  • Left ventricular ejection fraction is an important index to evaluate left ventricular function.
  • the left ventricular ejection fraction value of the heart failure model animals significantly decreased after modeling, but the ejection fraction of the cell patch treatment group was significantly higher than that of the control group.
  • the left ventricular short axis shortening index refers to the ratio of the short axis of the left ventricle during contraction and diastole. The larger the ratio, the stronger the systolic function of the heart.
  • the left ventricular short axis shortening index value of the heart failure model animals decreased significantly after modeling, but the left ventricular short axis shortening index value of the cell patch treatment group was significantly higher than that of the control group.
  • the left ventricular diameter versus time curve ( Figure 12) and the left ventricular volume versus time curve ( Figure 13) were also calculated and drawn based on echocardiograms, both of which can be used to describe the left ventricular volume.
  • the left ventricle undergoes compensatory remodeling and the ventricular volume becomes larger.
  • the left ventricular diameter and volume (systolic and diastolic) of the cells in the cell patch treatment group were significantly lower than those in the control group, indicating that the use of cell patches has no effect on inhibition.
  • Left ventricular remodeling caused by bloody heart failure has a significant effect and can significantly improve heart function.
  • mice were sacrificed and heart tissues were taken for fixation, sectioning and staining.
  • the section results ( Figure 14) showed that compared with the control animals, the left ventricular wall of the mice in the mesenchymal stem cell patch treatment group was thicker, the ventricular remodeling was lighter, and the degree of fibrosis was less (Masson stain, collagen The fibers appear blue).
  • the above results indicate that the degree of fibrosis of the left ventricle of the mice treated with the cell membrane sheet is significantly lower than that of the control animals.
  • the Bama miniature pig was selected as the experimental animal, and the myocardial infarction model was established by ligating the anterior descending coronary artery.
  • the modeling method is as follows:
  • Animals were injected intramuscularly with Shutai to induce anesthesia, and the operation area was skin-prepared and fixed on a constant temperature animal operating table.
  • the anesthetized animal is intubated with a tracheal tube, connected to an anesthesia ventilator, and anesthesia is maintained with isoflurane, changing from autonomous breathing to passive breathing.
  • the operation area is wiped and disinfected with 4.5g/L ⁇ 5.5g/L iodophor and 75% alcohol.
  • a high-frequency electrocoagulation knife to open between the 4th and 5th ribs on the left side of the chest, separate layer by layer into the chest cavity, and expose the heart with a chest dilator. Free the left circumflex branch of the left coronary artery, implant and fix the coronary artery blocking reperfusion device. The outer end of the reperfusion device is blocked, and it is subcutaneously pulled to the chest or back of the neck and fixed. After that, the pericardium was sutured and the chest cavity was closed after injecting an appropriate amount of antibiotics. After the operation, the animals were injected with penicillin sodium intramuscularly.
  • the animal with the coronary artery blocking reperfusion device is placed in the cloth bag of the fixed frame, and the ECG of the animal before the coronary artery blocking to the reperfusion is detected through the vest-type physiological signal telemetry system.
  • Myocardial infarction model is successfully judged: the coronary blood flow of the animal is blocked by injecting air or water into the coronary occlusion reperfusion device to confirm the myocardium by increasing the ST segment voltage of the electrocardiogram after the blood flow is blocked by more than 0.1mV Ischemia.
  • the ST segment in the electrocardiogram is continuously elevated, the T wave is towering, or the pathological Q wave appears. Reperfusion injury.
  • the heart was removed and placed in the sodium chloride injection solution and rinsed gently. Infuse the heart with 1% TTC solution heated at 37°C, about 20 mL/head, through the coronary entrance of the aortic root, and then ligate the left circumflex branch.
  • the echocardiogram showed that compared with the blank control group, the D21, D28, D42, and D78 patch transplantation groups (product transplantation group) showed a significant increase in EF, SV, LVFS and other indicators, and ESV indicators showed a significant increase Decreasing trend, no obvious changes in other indicators (Figure 17).
  • the pathological examination results showed that the myocardial infarction rate of the animals in the patch transplantation group was significantly reduced ( Figure 18, Table 1), and the degree of myocardial fibrosis was reduced (Figure 19).
  • transplantation of mesenchymal stem cell membrane can effectively improve heart function and significantly reduce the occurrence of myocardial fibrosis and myocardial infarction volume.
  • Example 9 Surgical method for thoracic transplantation of mesenchymal stem cell membrane
  • the mesenchymal stem cell membrane package of Example 3 or 4 was opened, and the membrane was washed twice with physiological saline, and the amount of each washing was 20 ml.
  • the patient underwent thoracotomy, cut the pericardial capsule to expose the left ventricle, and optionally apply the mesenchymal stem cell membrane to the anterior and sidewall of the left ventricle.
  • the dosage for each patient is 1-4 sheets of mesenchymal stem cell membranes, and there may be some overlapping areas during application. Observe for 5-10 minutes after transplantation. After confirming that the mesenchymal stem cell membrane is stable on the surface of the heart, the pericardium can be sutured and the chest cavity can be closed.
  • Example 1 After the patient underwent coronary artery bypass grafting, the mesenchymal stem cell patch of Example 1, 3, or 4 was applied to the site after the bypass, and the rest of the surgical method was the same as that of Example 9.
  • the patient underwent minimally invasive surgery entered the thoracic cavity under the guidance of endoscopy, cut the pericardial capsule, accurately found the ischemic lesion of the left ventricular anterior wall, and released the curled mesenchymal stem cell membrane of Example 1, 3 or 4 to the lesion Position, and then spread it flat. It can be used in multiple stacks, the total is 1-4 tablets/person.

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Abstract

提供了一种治疗受试者中的心脏组织损伤或心功能不全相关的疾病的方法，该方法包括对受试者的心脏局部应用间充质干细胞膜片，例如脐带间充质干细胞膜片的步骤。还提供了间充质干细胞膜片的相关用途和组合物。

Description

间充质干细胞膜片及其用途
本申请要求于2020年1月22日递交的申请号为PCT/CN2020/073765，发明名称为“间充质干细胞膜片及其用途”的PCT申请的优先权，在此全文引用上述PCT申请公开的内容作为本申请的一部分。
发明领域
本公开涉及组织工程与再生医学领域，尤其涉及间充质干细胞膜片例如脐带间充质干细胞膜片用于治疗受试者中的心脏组织损伤或心功能不全相关的疾病的用途。
技术背景
心血管病调查结果显示，目前我国成人心力衰竭患病率为0.9％，根据2019年中国心力衰竭蓝皮书报告，推算我国心力衰竭患者高达650-875万，并以每年20万的数量增加。人口老龄化和高血压，高血脂等心血管危险因素为心力衰竭的主要原因。70岁以上人群的心衰患病率>10％，心衰患者5年死亡率达50％，重度心衰患者1年死亡率可达50％。
几乎所有的心血管疾病最终将导致心力衰竭。心肌梗死、心肌病等由于缺血，血流动力学负荷过重、炎症等引起心肌损伤，继而造成心肌结构和功能的变化，最后导致心室泵血和充盈功能低下，引起心力衰竭。虽然化学药物治疗、介入技术和外科手术治疗使急性心肌梗死等病死率明显下降，但临床观察发现，即使早期成功接受再血管化治疗的急性心肌梗死患者中，仍有超过30％的患者最终发展为缺血性心力衰竭。目前，针对终末期心衰有效治疗手段仅有两个：心脏移植和植入人工心脏(心室辅助装置)。心脏移植因为供体严重短缺、无法满足众多等待接受心脏移植患者的需要。人工心脏则仅作为桥梁适用于短期过渡的心力衰竭患者，且费用昂贵，术后易发生感染、出血等并发症。因此，目前国际医学界尚无有效方法彻底解决重症心力衰竭患者的病情。
近年来，干细胞与再生医学的发展为重症心衰患者带来了希望。既往的动物试验与临床研究发现，多种来源和类型的干细胞均可有效地治疗急慢性心肌梗死、缺血性心力衰竭等。通过干细胞的旁分泌作用提高心脏的收缩与 舒张功能、阻止心肌纤维化、改善预后。然而，上述干细胞疗法在临床实际应用中仍然存在以下问题：胚胎干细胞面临伦理、法律、免疫排斥的问题；iPS具有潜在的致瘤性，需要HLA配型；骨骼肌成肌细胞会导致室性心律失常；骨髓或外周血单个核细胞成分复杂，以免疫细胞为主，所含具备多向分化潜能的干细胞较为稀少；骨髓间充质干细胞(MSCs)来源受限，细胞增殖能力与干性随年龄增长而明显降低。
间充质干细胞广泛存在于人体的组织中，具有体外扩增能力，多项分化潜能和免疫调节作用，已经被应用于自身组织修复和免疫相关疾病的治疗中。目前在间充质干细胞用于自身组织修复的基础研究和临床应用中，大部分方法采用了细胞直接注射或者细胞与组织工程支架材料结合后移植的方法，但这两者也均具有一定的局限性。细胞直接注射会导致大量细胞丢失，治疗效率低，干细胞发挥组织修复的功能有限；而细胞与组织工程支架结合后进行移植尽管解决了细胞丢失的问题，但是支架材料在生物体内可能会引起不同程度的炎症反应，且支架材料的降解过程及降解产物可能使局部组织发生病变。
因此，目前尚无一种理想的干细胞产品能够满足重症心衰患者的临床需求。
细胞膜片是近年来一种新的细胞治疗方式，细胞膜片可以仅通过细胞之间的连接形成二维和三维的结构。与传统的单细胞悬液或将细胞与组织工程支架材料结合的方法相比，细胞膜片可以更好地实现局部固定、减少细胞流失，从而提高了细胞利用率，同时避免了支架材料所引入的可能引发较大免疫反应的异源物质。
发明内容
本发明人制备了间充质干细胞膜片，并在构建的心力衰竭动物模型中进行了评估。结果表明，本发明的间充质干细胞膜片对于心力衰竭具有良好的治疗效果，提高了心脏的运动和射血能力，并降低了心脏重构和纤维化程度。
相应的，在第一个方面，本公开涉及一种治疗受试者中与心脏组织损伤或心功能不全相关的疾病的方法，所述方法包括对所述受试者的心脏局部应用间充质干细胞膜片的步骤。
在一些实施方案中，所述疾病可以选自缺血性心脏病、风湿性心脏病、 先天性心脏病、心肌病、冠心病和心脏瓣膜病。
在一些实施方案中，所述心肌病为扩张型心肌病。
在一些实施方案中，所述疾病可以是缺血性心力衰竭，例如急性缺血性心力衰竭、慢性缺血性心力衰竭或终末期缺血性心力衰竭。
在一些实施方案中，所述疾病是因缺血性心脏病及扩张型心肌病引起的慢性心力衰竭，例如：一类是因心肌缺血引起的心力衰竭，包括慢性缺血性心力衰竭及终末期缺血性心力衰竭；另一类是扩张型心肌病引起的心力衰竭。
在一些实施方案中，将所述间充质干细胞膜片贴附于心脏的左室前壁或侧壁。
在一些实施方案中，将所述间充质干细胞膜片贴附于心脏的损伤或缺陷部位，或其邻近部位，以治疗所述与心脏组织损伤或心功能不全相关的疾病。在另一些实施方案中，将所述间充质干细胞膜片植入心脏的损伤或缺陷部位，或其邻近部位。与传统的单细胞悬液或将细胞与组织工程支架材料结合的方法相比，细胞膜片可以更好地实现局部固定、减少细胞流失，从而提高了细胞利用率，同时避免了支架材料所引入的可能引发较大免疫反应的异源物质。
在一些实施方案中，所述间充质干细胞膜片中间充质干细胞的细胞比例可以为至少90％。在一些方案中，所述膜片中间充质干细胞的细胞比例为至少95％，例如至少96％，至少97％，至少98％或至少99％，例如通过流式细胞术检测细胞表面标志物、三向分化测定或PCR法检测细胞表达基因鉴定的。
在上述方法的一些实施方案中，所述间充质干细胞可以来源于选自以下的组织：羊水、羊膜、绒毛膜、绒毛膜绒毛、蜕膜、胎盘、脐带血、华通氏胶、脐带、成人骨髓、牙髓、成人外周血和成人脂肪组织。
在上述方法的一些实施方案中，所述间充质干细胞可以选自脐带间充质干细胞、胎盘间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞和骨髓间充质干细胞。例如，所述间充质干细胞可以是脐带间充质干细胞。
在一些实施方案中，所述间充质干细胞膜片可以是使用传代数为P0-P20的间充质干细胞制备的。例如，可以使用传代数为P2-P15、P2-P8或P2-P10的间充质干细胞来制备细胞膜片。
在一些实施方案中，所述间充质干细胞膜片的厚度可以是约10-300μm， 例如30-300μm、50-300μm、100-300μm、80-300μm、100-300μm、120-300μm、150-300μm或200-300μm的厚度。在一些实施方案中，所述间充质干细胞膜片的厚度可以是约15-250μm。
此外，本公开的间充质干细胞膜片可以具有不同的细胞层数。在一些实施方案中，所述间充质干细胞膜片可以具有1-15层细胞，例如2-15层，3-15层、5-15层细胞、8-15层或10-15层细胞。
在一些实施方案中，所述间充质干细胞膜片中的细胞密度可以是约1×10 5至5×10 7/cm 2，例如约1×10 5至1×10 7/cm 2,8×10 5至5×10 7/cm 2，3×10 5至5×10 6/cm 2。
在本公开的方法中，使用的间充质干细胞膜片的大小和形状可以根据实际需要来确定，例如根据受试者的心脏损伤或缺陷部位的大小和形状。在一些实施方案中，可以使用圆形或有利于贴附或植入的形状的间充质干细胞膜片。在一些实施方案中，可以使用直径15-55mm、厚度10-300μm的圆形细胞膜片，贴附于心脏的左室前壁或侧壁，用于治疗所述与心脏组织损伤或心功能不全相关的疾病。
在一些实施方案中，所述膜片中的间充质干细胞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。
在一些实施方案中，所述膜片中的间充质干细胞能够分泌多种细胞因子，例如血管生成因子和免疫调节因子，例如肝细胞生长因子(HGF)、成纤维细胞生长因子(FGF)、表皮生长因子(EGF)、转化生长因子-β(TGF-β)、前列腺素E2(PGE2)、白细胞介素-6(IL-6)、白细胞介素10(IL-10)、白细胞介素-8(IL-8)和血管内皮生长因子(VEGF)、血小板衍生生长因子(PDGF)中的一种或多种。
在一些实施方案中，所述膜片中包括2×10 7至8×10 7的间充质干细胞。
在一些实施方案中，将1-4片所述间充质干细胞膜片局部应用于所述受试者的心脏。
在一些实施方案中，将所述间充质干细胞膜片局部应用于所述受试者的心脏与所述受试者施行冠状动脉搭桥术联用，例如，所述受试者施行冠状动脉搭桥术后，将所述间充质干细胞膜片局部应用于所述受试者的心脏。
在一些实施方案中，应用本发明移植间充质干细胞膜片的方式包括：①手术开胸，打开心包膜，将1-4片UCMSCS贴敷到心脏左室前壁和侧壁；② 冠状动脉搭桥术后，将UCMSCS贴敷到心脏左室前壁和侧壁；③通过微创手术操作，将UCMSCS贴敷到心脏左室前壁和侧壁。
在一些实施方案中，所述间充质干细胞膜片中的间充质干细胞与所述受试者是自体的或同种异体的。
在上述方法的一些实施方案中，所述间充质干细胞膜片可以是通过包括以下步骤的方法制备的：
a.在粘附基质和/或血清预包被的温敏培养皿中培养间充质干细胞；
b.通过降低温度使间充质干细胞从所述温敏培养皿脱离，
其中所述间充质干细胞通过其分泌的细胞外基质彼此连接，从而获得所述间充质干细胞膜片。
在一些实施方案中，用于制备间充质干细胞膜片的间充质干细胞可以是通过包括以下步骤的方法制备的：
a.从脐带分离华通氏胶；
b.将华通氏胶剪碎成小组织块，并培养所述组织块足够的时间段，使得间充质干细胞从组织块爬出；
c.待所述间充质干细胞生长至约50％-100％汇合时，例如约70％-100％汇合或约80％-100％汇合时移除组织块，从而获得脐带间充质干细胞；和任选的
d.培养和传代所述脐带间充质干细胞。
本文所用的术语“温敏培养皿”是指表面涂覆了一层温度敏感性高分子物质的培养皿，该高分子物质在不同温度下分子链段的伸展情况不同，从而表现出亲水性或疏水性，使得该高分子物质的亲疏水性能够随外部温度变化而变化。当温敏培养皿表面呈现亲水性时，与细胞及其分泌的细胞外基质粘合性变差，细胞将成层状脱落。在具体应用中，当将温度降低至该高分子物质的低临界溶解温度之下，该温敏培养皿表面呈现亲水性，从而使得细胞将成层状脱落。
利用温敏培养皿实现了在不使用酶及类似物消化也不使用物理方法剥离的情况下的将形成片层状的间充质干细胞从温敏培养皿底部脱离，成为保留有细胞外基质完整连结的细胞膜片。
在培养获得间充质干细胞之后，可以通过MTT法、WST法、DNA含量检测法、ATP检测法等测定细胞生长曲线，以评估脐带间充质干细胞的生长 活性。另外，可通过流式细胞术检测细胞表面标志物、三向分化测定以及PCR法检测细胞表达基因来鉴定所分离培养的间充质干细胞。在一些实施方案中，可以使用流式细胞术检测细胞表面标记蛋白来鉴定间充质干细胞。
在上述方法的一些实施方案中，用于包被所述温敏培养皿的所述粘附基质可以包括胎牛血清、自体血清、胶原、明胶、纤连蛋白，玻连蛋白，层粘连蛋白，多聚鸟氨酸和多聚赖氨酸中的一种或多种。
在上述方法的一些实施方案中，用于包被所述温敏培养皿的所述血清选自胎牛血清(FBS)或人血清。在一些实施方案中，使用100％的血清作为包被液。在另一些实施方案中，使用包含至少10％(v/v)血清的基础培养基(例如1640、DMEM、α-MEM或DMEM/F12)作为包被液。
在上述方法的一些实施方案中，通过降低温度使得间充质干细胞从温敏培养皿脱离，从而形成间充质干细胞膜片。例如，在培养温度为约37℃的情况下，通过将温度降低至4-32℃从而使间充质干细胞从所述温敏培养皿脱离。在一些实施方案中，加入4℃预冷的缓冲液(例如HBSS、PBS或生理盐水)从而使间充质干细胞从所述温敏培养皿脱离。
在一些实施方案中，所述间充质干细胞膜片具有在制备过程中不接触培养皿的上表面和接触培养皿的基底面，所述基底面较粗糙。间充质干细胞膜片的基底面由于其结构特征，基底面可以提供较大的摩擦力，有利于细胞膜片应用时更好地贴附于应用部位。
因此，在一些实施方案中，可以将所述间充质干细胞膜片的基底面贴附于心脏的损伤或缺陷部位，或其邻近部位。
在一些实施方案中，可以将所述间充质干细胞膜片的基底面贴附于心脏的左室前壁和侧壁。
在第二个方面，本公开涉及间充质干细胞膜片在治疗受试者中与心脏组织损伤或心功能不全相关的疾病中的用途，其中将所述间充质干细胞膜片局部应用于所述受试者的心脏。
在第三个方面，本公开涉及间充质干细胞膜片在制备用于治疗受试者中与心脏组织损伤或心功能不全相关的疾病的组合物中的用途，其中将所述间充质干细胞膜片局部应用于所述受试者的心脏。
在本公开第二方面和第三方面的用途的一些实施方案中，所述疾病可以选自缺血性心脏病、风湿性心脏病、先天性心脏病、心肌病、冠心病和心脏 瓣膜病。
在一些实施方案中，所述心肌病为扩张型心肌病。
在本公开第二方面和第三方面的用途的一些实施方案中，所述疾病可以是缺血性心力衰竭，例如急性缺血性心力衰竭、慢性缺血性心力衰竭或终末期缺血性心力衰竭。
在上述用途的一些实施方案中，将所述间充质干细胞膜片贴附于心脏的损伤或缺陷部位，或其邻近部位。在另一些实施方案中，将所述间充质干细胞膜片植入心脏的损伤或缺陷部位，或其邻近部位。在一些实施方案中，将所述间充质干细胞膜片贴附于心脏的左室前壁或侧壁。
在上述用途的一些实施方案中，所述间充质干细胞膜片中间充质干细胞的细胞比例可以为至少90％。在一些方案中，所述膜片中间充质干细胞的细胞比例为至少95％，例如至少96％，至少97％，至少98％或至少99％，例如通过流式细胞术检测细胞表面标志物、三向分化测定或PCR法检测细胞表达基因鉴定的。
在上述用途的一些实施方案中，所述间充质干细胞可以来源于选自以下的组织：羊水、羊膜、绒毛膜、绒毛膜绒毛、蜕膜、胎盘、脐带血、华通氏胶、脐带、成人骨髓、牙髓、成人外周血和成人脂肪组织。
在上述用途的一些实施方案中，所述间充质干细胞可以选自脐带间充质干细胞、胎盘间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞和骨髓间充质干细胞。例如，所述间充质干细胞可以是脐带间充质干细胞。
在上述用途的一些实施方案中，所述间充质干细胞膜片可以是使用传代数为P0-P20的间充质干细胞制备的。例如，可以使用传代数为P2-P15、P2-P8或P2-P10的间充质干细胞来制备细胞膜片。
在上述用途的一些实施方案中，所述间充质干细胞膜片的厚度可以是约10-300μm。在一些实施方案中，所述间充质干细胞膜片的厚度可以是约20-300μm，例如30-300μm、50-300μm或100-300μm的厚度。此外，本发明公开的间充质干细胞膜片可以具有不同的细胞层数。在一些实施方案中，所述间充质干细胞膜片可以具有1-15层细胞，例如2-15层，3-15层或5-15层细胞。
在上述用途的一些实施方案中，所述间充质干细胞膜片中的细胞密度可以是约1×10 5至5×10 7/cm 2，例如约8×10 5至5×10 7/cm 2，3×10 5至5×10 6/cm 2。
在一些实施方案中，所述膜片中的间充质干细胞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。
在一些实施方案中，所述膜片中的间充质干细胞能够分泌多种细胞因子，例如血管生成因子和免疫调节因子，例如HGF、EGF、FGF、TGF-β、PGE2、IL-6、IL-8、IL-10、PDGF和VEGF中的一种或多种。
在一些实施方案中，所述膜片中包括2×10 7至8×10 7的间充质干细胞。
在一些实施方案中，将1-4片所述间充质干细胞膜片局部应用于所述受试者的心脏。
在一些实施方案中，将所述间充质干细胞膜片局部应用于所述受试者的心脏与所述受试者施行冠状动脉搭桥术联用，例如，所述受试者施行冠状动脉搭桥术后，将所述间充质干细胞膜片局部应用于所述受试者的心脏。
在一些实施方案中，所述间充质干细胞膜片中的间充质干细胞与所述受试者异源或同源。
在上述用途的一些实施方案中，所述间充质干细胞膜片可以是通过包括以下步骤的方法制备的：
a.在粘附基质和/或血清预包被的温敏培养皿中培养间充质干细胞；
b.通过降低温度使间充质干细胞从所述温敏培养皿脱离，
其中所述间充质干细胞通过其分泌的细胞外基质彼此连接，从而获得所述间充质干细胞膜片。
在上述用途的一些实施方案中，用于制备间充质干细胞膜片的间充质干细胞可以是通过包括以下步骤的方法制备的：
a.从脐带分离华通氏胶；
b.将华通氏胶剪碎成小组织块，并培养所述组织块足够的时间段，使得间充质干细胞从组织块爬出；
c.待所述间充质干细胞生长至约50％-100％汇合时，例如约70％-100％汇合或约80％-100％汇合时移除组织块，从而获得脐带间充质干细胞；和任选的
d.培养和传代所述脐带间充质干细胞。
在上述用途的一些实施方案中，用于包被所述温敏培养皿的所述粘附基质可以包括胎牛血清、自体血清、胶原、明胶、纤连蛋白，玻连蛋白，层粘连蛋白，多聚鸟氨酸和多聚赖氨酸的一种或多种。
在上述用途的一些实施方案中，用于包被所述温敏培养皿的所述血清选 自胎牛血清(FBS)或人血清。在一些实施方案中，使用100％的血清作为包被液。在另一些实施方案中，使用包含至少10％(v/v)血清的基础培养基(例如1640、DMEM、α-MEM或DMEM/F12)作为包被液。
在上述用途的一些实施方案中，通过降低温度使得间充质干细胞从温敏培养皿脱离，从而形成间充质干细胞膜片。例如，在培养温度为约37℃的情况下，通过将温度降低至4-32℃从而使间充质干细胞从所述温敏培养皿脱离。在一些实施方案中，加入4℃预冷的缓冲液从而使间充质干细胞从所述温敏培养皿脱离。
在上述用途的一些实施方案中，所述间充质干细胞膜片具有在制备过程中不接触培养皿的上表面和接触培养皿的基底面，所述基底面较粗糙。在一些实施方案中，将所述间充质干细胞膜片的基底面贴附于心脏的损伤或缺陷部位，或其邻近部位。在一些实施方案中，可以将所述间充质干细胞膜片的基底面贴附于心脏的左室前壁和侧壁。
在上述用途的一些实施方案中，所述间充质干细胞膜片为单剂量间充质干细胞膜片，其中所述膜片包含2×10 7至8×10 7的间充质干细胞。
一些实施方案中，所述单剂量间充质干细胞膜片的直径为15-55mm，所述膜片的厚度为10-300μm。
在第四个方面，本发明涉及一种间充质干细胞膜片，其用于治疗受试者中与心脏组织损伤或心功能不全相关的疾病。
一些实施方案中，所述间充质干细胞膜片中的细胞密度为8×10 5至5×10 7/cm 2。
在第五个方面，本发明涉及一种单剂量间充质干细胞膜片，其中所述膜片包含2×10 7至8×10 7的间充质干细胞，所述间充质干细胞之间通过其分泌的细胞外基质彼此连接。
需要说明的是,本申请所述的“单剂量”是指每次使用一片充质干细胞膜片，即可发挥治疗效果的剂量。根据本发明实施例的单剂量间充质干细胞膜片，可一次性使用1-4片局部应用于所述受试者的心脏，应用于治疗受试者心脏组织损伤或心功能不全相关的疾病，例如可贴附于心脏的左室前壁和侧壁。
一些实施方案中，所述单剂量间充质干细胞膜片的直径为15-55mm，所述膜片的厚度为10-300μm。
一些实施方案中，所述间充质干细胞膜片中的细胞密度为8×10 5至5×10 7/cm 2。
在第六个方面，本发明涉及一种包含间充质干细胞膜片的组合物，所述组合物用于治疗受试者中与心脏组织损伤或心功能不全相关的疾病。
在本公开的第四个方面、第五个方面和第六个方面的一些实施方案中，所述疾病可以选自缺血性心脏病、风湿性心脏病、先天性心脏病、心肌病、冠心病和心脏瓣膜病。
在本公开的第四个方面、第五个方面的第六个方面的在一些实施方案中，所述疾病可以是缺血性心力衰竭，例如急性缺血性心力衰竭、慢性缺血性心力衰竭或终末期缺血性心力衰竭。
在本公开的第四个方面第五个方面和第六方面的在一些实施方案中，所述间充质干细胞具有本公开的第一、第二和第三方面中所述的一项或多项特征。
附图说明
图1示出了根据本发明实施例获得的脐带间充质干细胞膜片照片。
图2示出了脐带间充质干细胞成脂、成骨分化功能检测结果。图1A：茜素红染色的结果；图1B：油红O染色的结果。
图3示出了脐带间充质干细胞膜片的扫描电镜成像照片。图3A：细胞膜片的表面(上表面)。图3B：细胞膜片的基底面。
图4示出了脐带间充质干细胞膜片的免疫荧光成像照片。图4A：纤粘蛋白。图4B：整联蛋白β1。
图5示出了使用ELISA方法检测视脐带间充质干细胞膜片培养上清液中的细胞因子表达的结果。
图6示出了在小鼠心脏部位移植了间充质干细胞膜片的照片。
图7示出了构建的心力衰竭小鼠疾病模型的表征。图7A：疾病模型小鼠的心脏照片；图7B：疾病模型小鼠的心电图结果。
图8显示了使用脐带间充质干细胞膜片处理疾病模型小鼠的图。图8A显示了使用的间充质干细胞膜片的示例性照片；图8B显示了将细胞膜片贴附于小鼠心脏表面的照片。
图9示出了不同时间点的小鼠超声心动图结果。图9A：建模前；图9B： 建模后1周；图9C：建模后4周。左侧：对照组动物；右侧：细胞膜片移植组动物。
图10示出了细胞膜片治疗组与对照组小鼠左心室射血分数随时间变化的曲线。
图11示出了细胞膜片治疗组与对照组小鼠左心室短轴缩短指数随时间变化的曲线。
图12示出了细胞膜片治疗组与对照组小鼠左心室内径随时间变化的曲线。
图13示出了细胞膜片治疗组与对照组小鼠左心室容积随时间变化的曲线。
图14示出了细胞膜片治疗28天后，小鼠心脏组织切片的Masson染色结果图。左侧：对照组动物；右侧：细胞膜片治疗组动物。
图15示出了将1-2片所获得的间充质干细胞膜片移植至小型猪左心室前壁的照片。
图16示出了小型猪心梗造模前后以及移植膜片治疗后的心电图。
图17示出了与模型对照组比较，膜片移植组动物EF、SV、LVFS、EDV、ESV等指标变化的统计图。
图18示出了模型对照组和膜片移植组动物心肌梗死率的病理检查结果。
图19示出了模型对照组和膜片移植组心肌纤维化程度的病理检查结果。
具体实施方式
本发明要解决的技术问题是，将人源脐带间充质干细胞膜片应用于治疗因缺血性心脏病、扩张型心肌病等引起的心力衰竭患者。通过细胞膜片的移植改善心肌功能，提高生活质量，降低心力衰竭患者的死亡率；同时，膜片移植方法将克服间充质干细胞悬液注射引起的不能确保足够数量的干细胞抵达病变部位充分发挥生物学功效的缺陷，克服干细胞+生物支架因生物材料吸收不完全诱发炎性反应的缺陷。
本发明所称的“脐带间充质干细胞膜片”，可按照以下方法制备：取P2-P8代脐带间充质干细胞制成单细胞悬液，以3×10 5-1.2×10 6个细胞/cm 2的浓度加入预先包被基质(如胎牛血清、自体血清、明胶、纤连蛋白、玻连蛋白、层黏连蛋白、多聚赖氨酸)的温敏培养皿中，待细胞培养至过饱和状态，将 温敏培养皿置于20℃以下条件，细胞会自发成膜脱落。最终获得一圆形片状、直径15-55mm、厚度10-300μm的细胞膜片(如图1)，所含细胞数量为2×10 7-8×10 7，即时使用或冷冻保存备用。
相比于传统胰酶法消化获得的单个干细胞，本发明的脐带间充质干细胞膜片为一圆形片状细胞连接体，细胞与细胞之间紧密联系，保持完整的细胞外基质(Extracellular matrix,ECM)成分，该成分包括纤连蛋白、层黏连蛋白、胶原蛋白、粘多糖等。ECM是干细胞维持若干生理功能的重要基础物质，有助于细胞因子的分泌和信号传递，有利于干细胞在体内定植与存活。将细胞膜片贴敷于病灶部位，可以100％将脐带间充质干细胞移植至病灶，延长干细胞在体内存活时间，从而发挥干细胞更有效更持久的治疗效果。本发明的脐带间充质干细胞膜片贴敷到病灶部位后能够分泌多种细胞因子，主要包括表皮生长因子(EGF)、成纤维细胞生长因子(FGF)、肝细胞生长因子(HGF)、血管内皮生长因子(VEGF)、转化生长因子-β(TGF-β)、前列腺素E2(PGE2)、血小板衍生生长因子(PDGF)、白细胞介素6(IL-6)、白细胞介素10(IL-10)等，修复缺血受损的组织器官或促进其自我修复，从而达到治疗效果。
本发明所称的“因缺血性心脏病及扩张型心肌病引起的慢性心力衰竭”包括两个类别：一类是因心肌缺血引起的心力衰竭，包括慢性缺血性心力衰竭及终末期缺血性心力衰竭；另一类是扩张型心肌病引起的心力衰竭。
本发明的脐带间充质干细胞膜片移植治疗缺血性心脏病和扩张型心肌病所致慢性心力衰竭的机理：大量脐带间充质干细胞膜片在移植部位分泌多种生长因子和细胞因子，主要包括HGF、VEGF、IL-6、IL-10等，调节心肌缺血部位微环境，抑制心肌炎性反应，阻止心肌梗死纤维化进展；促进局部血管新生，通过代偿途径为心肌提供血供，改善心肌功能。
本发明的脐带间充质干细胞膜片取材来自异体，性能卓越，可替代自体干细胞用于治疗心力衰竭。脐带间充质干细胞膜片表现出显著的优势：①脐带取材广泛、便捷、无创；②可以制定统一质量标准，取材时择优录用，突破自体干细胞受自身客观条件限制；③可以提前制备多级细胞库及现货产品，大幅缩短细胞体外培养及病人等待时间；④脐带间充质干细胞膜片免疫原性低，异体应用基本不会发生免疫排斥现象；⑤脐带间充质干细胞膜片安全性较高，无致瘤促瘤作用，无毒性反应；⑥通过干细胞的旁分泌作用改善 心力衰竭患者心肌缺血，延长患者的生存期。
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
实施例1.脐带间充质干细胞膜片的制备
取人新生儿脐带，去除外膜和血管后获得脐带组织内的华通氏胶样组织。将华通氏胶样组织用无菌剪剪碎成约1-2mm 3的组织块，平铺于培养皿中进行培养。待脐带间充质干细胞爬出后去掉组织块，并加入新鲜培养基继续培养。待细胞长至约70～100％汇合时，对细胞进行传代操作。在显微镜下观察到脐带间充质干细胞贴壁生长，呈成纤维状，形态均一。
通过流式细胞术检测以下细胞表面标志物来鉴定分离的间充质干细胞：CD105、CD34、CD31和CD117，其中CD105为阳性标志物；CD34、CD31和CD117位阴性标志物。结果显示，在分离的脐带间充质干细胞中，CD105为99.64％，CD34为0.02％；CD31为0.00％；CD117为0.51％。上述结果表明获得的脐带间充质干细胞具有高纯度。
进一步检测了脐带间充质干细胞向骨和脂肪细胞分化的能力。具体而言，将脐带间充质干细胞比例接种于培养皿中。对于成骨诱导，待细胞生长至约50-90％汇合时加入成骨诱导培养基，培养7天后使用茜素红对细胞进行染色；对于成脂肪诱导，待细胞生长至90％以上汇合时加入成脂肪诱导培养基，培养7天后使用油红O对细胞进行染色。如图2所示，间充质干细胞经成骨诱导或成脂肪诱导后，能够分别被茜素红(图2A)或油红O染色(图2B)，表明其具备分化成骨和脂肪细胞的能力。
对于间充质干细胞膜片的制备，将上述P2-P8代脐带间充质干细胞消化成为单细胞，制成单细胞悬液后，以3×10 5-1.2×10 6的密度接种到预先包被基质(如胎牛血清、自体血清、明胶、纤连蛋白、玻连蛋白、层黏连蛋白、多聚赖氨酸)的温敏培养皿中，于37℃，5％CO 2和95％湿度环境的培养箱内培养，待细胞培养至过饱和状态，将其移至约20℃的环境下或加入4℃预冷的HBSS液，PBS液或生理盐水。细胞成片层状从温敏培养皿底部脱离，形成由细胞外基质连结的完整细胞膜片。获得的膜片呈圆形片状、直径15-55mm、厚度10-300μm，灰白色，结构致密，表面光滑平整，细胞膜片中的活细胞率 很高，且细胞状态良好(如图1)，所含细胞数量为2×10 7-8×10 7，即时使用或冷冻保存备用。
实施例2.间充质干细胞膜片的表征
使用扫描电镜和免疫荧光成像对制备的脐带间充质干细胞膜片的结构进行表征。将细胞膜片经2.5％戊二醛固定、酒精梯度脱水和风干等步骤制样后进行扫描电镜拍摄。如图3所示，细胞膜片具有不与培养皿接触的表面(上表面，图3A)和与培养皿接触的基底面(下表面，图3B)，其结构上存在差异：表面由于细胞自然沉降，形成的表面较光滑；基底面与温皿材料接触，相对较粗糙。由于其结构特征，基底面可以提供较大的摩擦力，有利于细胞膜片应用时更好地贴附于应用部位。
随后，通过免疫荧光的方法检测了脐带间充质干细胞膜片中的纤连蛋白和整合素β1的表达情况。膜片经固定液固定后进行冰冻切片，使用荧光素标记的纤连蛋白和整合素β1抗体染色，并进行免疫荧光成像分析。结果如图4所示，通过本公开的方法制备得到的细胞膜片含有大量的纤连蛋白(图4A)和整合素β1(图4B)。
纤连蛋白广泛存在于动物组织和组织液中，具有促进细胞的黏连生长的功能，而细胞的黏连生长是维持和修复机体组织结构的必要条件。整合素β1是整合素家族的重要成员，其在介导细胞与细胞之间、细胞与细胞外基质(ECM)之间的相互黏附以及双向信号传导方面具有重要作用，并与组织修复和纤维化形成紧密相关。上述结果表明，本发明公开的脐带间充质干细胞膜片并非是细胞简单地堆积而成，而是通过细胞外基质连接形成的具有致密组织性和生物活性的膜片。
进一步检测了间充质干细胞分泌细胞因子的能力，包括肝细胞生长因子(HGF)；白细胞介素-6(IL-6)、白细胞介素-8(IL-8)和血管内皮生长因子(VEGF)。HGF由间充质干细胞产生，其参与上皮-间充质转化(EMT)过程并对多种组织和细胞具有调控作用，能够促进细胞运动和分裂；IL-6和IL-8参与调节机体免疫反应和免疫细胞的多种生理过程；VEGF具有促进内皮细胞增殖和诱导血管新生的功能。上述细胞因子具有促进细胞生长和分化和促进血管生成过程的功能，对于组织修复具有重要作用。
在细胞膜片的制备过程中取培养上清液，通过ELISA方法对上清液中的细胞因子进行检测，检测结果如图5所示。结果表明，上述四种细胞因子在上清液中均有表达，且HGF和IL-8的表达水平较高。上述结果表明，本公开的脐带间充质干细胞细胞膜片能够分泌多种细胞因子，包括血管生成因子和免疫调节因子，证明其具有高生物学活性和功能，能够促进局部血管生成和组织修复过程。并且，高IL-8表达水平表明细胞膜片在使用过程中具有促进免疫反应和抑制细菌的功能，有利于细胞膜片更好地发挥其生物学功能。
实施例3 即时使用间充质干细胞膜片新鲜品的制备
从细胞培养箱取出实施例1长有间充质干细胞膜片的温敏培养皿，吸弃培养基，加入4℃预冷的PBS或生理盐水，10分钟后细胞自动从培养皿边缘开始剥离并逐渐向中央扩展；若细胞未能自动剥离，可用10μL枪头轻轻地沿培养皿壁划一圈促进细胞的剥离。将完全剥离的间充质干细胞膜片转移至普通培养皿，用生理盐水清洗膜片2次，加入10mL新鲜的膜片保护液，进行无菌密封包装。
实施例4 冷冻保存间充质干细胞膜片备用品的复苏
从深低温容器中取出冻存的实施例1获得的间充质干细胞膜片，迅速放入37℃温箱或者水浴锅中，使其在2分钟内快速解冻。将解冻的间充质干细胞膜片转移至普通培养皿，用生理盐水清洗膜片2次，加入10mL新鲜的膜片保护液，进行无菌密封包装。
实施例5
1)间充质干细胞免疫原性鉴定
以异体脐带间充质干细胞作为生产材料，属于同种异体干细胞移植，需考虑免疫排斥问题。为此，发明人对脐带间充质干细胞进行体外免疫原性鉴定。
发明人通过流式细胞术检测脐带间充质干细胞，发现脐带间充质干细胞不表达HLA-Ⅱ类抗原HLA-DR及协同刺激抗原CD80、CD86；发明人将脐带间充质干细胞与人外周血单个核细胞按照1:2-1:30的比例混合共同培养在含10％胎牛血清的完全培养基中，2天后收集上清液中的悬浮细胞进行计数和 流式细胞检测，未观察到淋巴细胞异常增殖现象，未观察到T细胞亚群CD3、CD8变化。
这些体外实验结果表明，脐带间充质干细胞免疫原性较低。
2)脐带间充质干细胞促瘤性评价
balb/c裸鼠皮下接种人肺癌A549细胞建立实体瘤模型，NPG裸鼠皮下接种人淋巴瘤Raji细胞建立血液瘤模型。通过尾静脉注射足够剂量的脐带间充质干细胞，连续4～8周测量肿瘤体积，然后大体解剖、称瘤重、病理检查。与对照组相比，脐带间充质干细胞注射组裸鼠的肿瘤体积和重量均无显著差异，全身各主要器官均未见肿瘤发生，病理检查淋巴细胞浸润以及对周围组织的浸润程度均未见明显差异。
本实验说明脐带间充质干细胞对实体瘤和血液瘤无抑制或促进作用。
3)间充质干细胞膜片成瘤性评价
balb/c裸鼠皮下移植足够剂量的本申请实施例1、3或4所获得的间充质干细胞膜片，在SPF洁净环境鼠笼饲养，持续观察20周，移植部位未见肿瘤形成，大体解剖，取裸鼠全身主要脏器进行病理检查，未发现肿瘤灶。
本实验说明本申请所获得的间充质干细胞膜片不具有成瘤性。
4)间充质干细胞膜片动物体内毒性评价
本发明的间充质干细胞膜片区别于传统干细胞悬液的注射给药方式，是直接贴敷在心脏表面部位，不会入血转移，只在局部发挥营养心肌和调节微环境的作用，其影响范围受到一定的控制。为了进一步阐明其安全性，发明人在小鼠体内检测间充质干细胞膜片毒性反应。
选择6-8周龄NPG小鼠72只，雌雄各半，随机分成三组：细胞膜片组、细胞悬液组、阴性对照组，每组24只，雌性各12只。细胞膜片组在小鼠心脏部位移植相当于10倍人用剂量的间充质干细胞膜片(图6)，细胞悬液组经尾静脉注射脐带间充质干细胞剂量为1×10 6细胞/只，阴性对照组注射等体积的0.9％氯化钠注射液。给药后每周测定一次体重，每天进行一次笼旁观察，观察期4周，观察内容包括裸鼠移植部位肿瘤形成情况、死亡情况、外观、体征、行为活动、腺体分泌、呼吸、粪便性状等。分别在给药后24小时、1周、2周、4周，每组每性别剖杀3只小鼠，进行大体解剖观察。
结果：实验期间各组动物无异常，存活状态良好，体重无差异，饮水进食、躯体活动、呼吸排泄等均无异常，未见明显不良反应。在各时间点进行 大体解剖观察，未发现病理病变或成瘤现象。
结论：在小鼠体内移植本申请所获得的间充质干细胞膜片，未见明显急性毒性反应。
实施例6.心力衰竭动物模型的构建
在本实施例中，通过冠状动脉结扎法构建缺血性心力衰竭小鼠模型。在雄性C57BL/6小鼠(约12周龄)中，使用缝合线对左前降支进行结扎，阻碍左心室心肌血液供给，使梗死区域心肌细胞发生凋亡，从而导致左心室射血功能下降、心室结构重构，并最终发展为心力衰竭。具体包括以下步骤：
(1)使用已异氟烷混合氧气(异氟烷浓度为约3.5-5％)的方式麻醉小鼠，用脱毛膏进行脱毛处理。
(2)经颈透照直视插管。用镊子将小鼠舌头稍拖出，暴露咽部，用22G滞留针经口插入声门，稍向下移进入气管约5mm，退出滞留针针芯。用巴氏吸管或移液器向滞留针内吹/吸气，可以看到小鼠胸腔随之有较大起伏，即表示气管插管成功。
(3)使用呼吸机维持麻醉。维持麻醉气体为约3％的异氟烷，潮气量为0.3ml，频率为～124次/min，呼吸比为50:50。
(4)测心电信号。利用Medlab二导联生理信号采集线系统对小鼠心电进行监测，其中右上肢皮下接正极、左下肢皮下接负极，右下肢皮下接地。
(5)开胸，暴露心脏。用锐器从剑突处向上打开胸部皮肤，剥离皮下组织，用镊子刺破第4-5肋间肌肉组织，用扩胸器撑开肋骨，剪开心包膜，调整扩胸器打开幅度及小鼠体位，暴充分露心脏。
(6)左前降支结扎。使用6-0或7-0手术缝合线，对小鼠左前降支进行结扎，即在距离左心耳下缘约1.5mm处。
(7)胸腔缝合。造模完成后，移除扩胸器，将肋间肌肉组织归位，而后对表皮进行缝合。可以在缝合完成前通过挤压胸腔或缝合完成后利用注射器抽吸胸腔的方法排除胸腔内空气，避免气胸引起动物死亡。
(8)术后护理。停止呼吸机内异氟烷通入，观察小鼠是否有自主呼吸。如有，则将小鼠移至温毯恢复直至完全清醒，能够自行移动。如小鼠尚不能自主呼吸，则继续使用呼吸机为其进行辅助呼吸至其恢复自主呼吸。术后1小时内仅提供饮用水，而后如常提供饲料。必要时给予一定的保暖措施。
在步骤(6)之后，可明显观察到小鼠左心室壁发白(图7A)。从心电图上可明显看到ST段抬高，呈心肌梗死状态心电图(图7B)。该建模方法可以较好地模拟急性缺血型心力衰竭的病程，建模成功率高，稳定性好。
实施例7.间充质干细胞膜片在治疗心力衰竭中的应用
在如实施例6所述的疾病动物模型中评估脐带间充质干细胞的治疗效果。对于脐带间充质干细胞膜片治疗组小鼠，在步骤(6)后将裁剪成约2-5mm的圆形(图8A)或近似面积的适当形状的脐带间充质干细胞细胞膜片贴附于模型动物左心室表面(图8B)。该间充质干细胞膜片能够良好地贴附，不易掉落，贴附后表面平整。静置3-5分钟后进行上述步骤(7)胸腔缝合和(8)术后护理。未贴附细胞膜片的动物作为对照。细胞膜片治疗组和对照组各10只小鼠。
在建模前(图9A)、建模后1周(图9B)和建模后4周(图9C)对小鼠进行超声心动检查，胸骨旁短轴切面以左心室乳头肌水平的切面为标志点可观察到超声心动图。从图9B和图9C中的结果可以看出，建模后心力衰竭模型动物的心脏有明显的运动减弱现象。并且，相比于对照组动物(左侧图)，细胞膜片处理组动物(右侧图)的心脏运动较强。
根据超声心动图计算并绘制了手术前后小鼠左心室射血分数随时间变化的曲线(图10)和左心室短轴缩短指数随时间变化曲线(图11)。左心室射血分数是评价左心室功能的重要指标。如图10中的结果所示，建模后心力衰竭模型动物的左心室射血分数值显著下降，但细胞膜片处理组动物的射血分数显著高于对照组动物。左心室短轴缩短指数是指左心室收缩和舒张时短轴比例，比例越大表明心脏收缩功能越强。如图11中的结果所示，建模后心力衰竭模型动物的左心室短轴缩短指数值有显著下降，但细胞膜片处理组动物的左心室短轴缩短指数值显著高于对照组动物。
还根据超声心动图计算并绘制了左心室内径随时间变化的曲线(图12)和左心室容积随时间变化曲线(图13)，两者均可用于描述左心室容积。在制备动物模型后，由于缺血性心力衰竭，左心室发生代偿性重构，心室体积变大。如图12和图13中的结果所示，建模后，细胞膜片处理组动物的左心室内径及容积(收缩期和舒张期)均显著低于对照组动物，说明细胞膜片的使用对抑制缺血性心力衰竭引起的左心室重构有显著的效果，能够明显提高心脏功 能。
在实验结束时(建模后第28天)，处死小鼠并取心脏组织进行固定、切片和染色。切片结果(图14)显示，与对照组动物相比，间充质干细胞膜片处理组的小鼠左心室壁较厚，心室重构情况较轻，且纤维化程度较小(Masson染色,胶原纤维呈现蓝色)。上述结果表明，处理了细胞膜片的小鼠左心室的纤维化程度与对照组动物相比明显较低。
实施例8间充质干细胞膜片移植对动物模型心肌恢复试验
选择巴马小型猪作为实验动物，通过结扎冠状动脉前降支建立心梗模型，造模方法如下：
动物肌肉注射舒泰实施诱导麻醉，手术区域备皮、固定于恒温动物手术台。麻醉动物气管插管，连接麻醉呼吸机，以异氟烷维持麻醉，由自主呼吸变为被动呼吸。手术区域以4.5g/L～5.5g/L碘伏及75％酒精擦拭消毒。肌肉注射痛立定进行镇痛。
用高频电凝刀于胸部左侧第4，5肋骨间开口，逐层分离进入胸腔，以胸腔扩张器暴露心脏。游离左冠状动脉左旋支，植入冠脉阻断复灌器并固定。阻断复灌器外端，通过皮下牵引至胸背部或颈背部并固定。之后，缝合心包，注入适量抗生素后关闭胸腔。术后，动物肌注青霉素钠。
次日，动物清醒状态下，将埋植冠脉阻断复灌器的动物放置于固定架的布袋中，通过马甲式生理信号遥测系统检测动物冠脉阻断前至复灌后的心电图。
心梗模型成功判断：通过向冠脉阻断复灌器内注入空气或水进行加压，使动物冠脉血流阻断，以血流阻断后心电图ST段电压抬高0.1mV以上确认心肌缺血。冠脉阻断1小时后，抽出冠脉阻断复灌器内空气或水，实施冠脉血流复灌，以心电图形中ST段持续抬高，T波高耸，或出现病理Q波确认出现复灌损伤。
心梗模型形成两周后，将1-2片本申请实施例1、3或4所获得的间充质干细胞膜片移植至左心室前壁(图15)；模型对照组不移植任何材料，只做心梗造模和二次开胸假手术处理。膜片移植后继续观察9周，观察指标包括：生理指标、心电图、血流动力学、超声心动图，并于实验终点安乐死动物做组织病理学检查，检查方法如下：
摘取心脏放置氯化钠注射液溶液中轻柔地冲洗。经主动脉根部冠脉入口向心脏内灌注37℃加热的1％的TTC溶液，约20mL/只，然后结扎左旋支。
横断面切开心脏组织，间隔厚度为5mm切成5-6片心脏组织，放置于37℃TTC溶液中孵育，约5-10min(使TTC充分反应，缺血组织灰白色，正常组织红色或鲜红色)。心脏组织均将后一刀的切片朝上放置，扫描后放入10％中性缓冲福尔马林溶液中固定。照片采用photoshop 7.0选取梗死区域面积，采用NIH Image J软件测定心肌梗死区域的体积(mm 3)、左心室总体积(mm 3)及梗死率(％)。体积计算公式＝(前切片面积+后切片面积)×切片厚度(5mm)/2，梗死率＝总梗死体积/总左室体积×100％。
取第2片心脏组织制作白片，进行Masson三色法染色，并采用光学显微镜进行组织病理学检查。评价心肌组织纤维化程度。采用4分级法对显微镜检查结果进行分级，分别为轻微(+)，轻度(++)，中度(+++)，重度(++++)，以便于进行组间比较。
结果：本实验过程中，动物一般临床观察未见明显异常，模型对照组和膜片移植组动物体重平稳增加，动物摄食及饮水等生理需求正常。心电图变化为心梗造模成功后ST段抬高，T波高耸及病理性Q波(图16)。两组实验动物的左心室收缩末期压(LVESP，mmHg)、左心室舒张末期压(LVEDP，mmHg)及±dp/dt max指标未见明显差异。超声心动图显示，与模型对照组(blank control group)比较，D21、D28、D42、D78膜片移植组(product transplantation group)动物EF、SV、LVFS等指标出现明显的增加趋势，ESV指标出现明显降低趋势，其它指标未见明显变化(图17)。病理检查结果显示，膜片移植组动物心肌梗死率显著降低(图18，表1)，心肌纤维化程度减轻(图19)。
结论：小型猪心梗模型，移植间充质干细胞膜片能够有效改善心脏功能，显著降低心肌纤维化的发生和心肌梗死体积。
表1小型猪心肌梗死对照表
Figure PCTCN2021073295-appb-000001
实施例9 开胸移植间充质干细胞膜片的手术方法
打开实施例3或4的间充质干细胞膜片包装，用生理盐水清洗膜片2次，每次清洗用量为20ml。患者行开胸手术，剪开心包膜，暴露左室部位，任选地将间充质干细胞膜片平整贴敷在左室前壁、侧壁部位。每位患者用量为1-4片间充质干细胞膜片，贴敷时可以有部分重叠区域。移植后观察5-10分钟，确定间充质干细胞膜片与心脏表面粘贴稳定后，即可缝合心包，关闭胸腔。
实施例10 结合冠状动脉塔桥术移植间充质干细胞膜片的方法
在患者行冠状动脉搭桥术后，将实施例1、3或4间充质干细胞膜片贴敷在搭桥后的部位，其余手术方法同实施例9。
实施例11 微创手术移植间充质干细胞膜片的方法
患者行微创手术，在内镜指导下进入胸腔，剪开心包膜，准确找到左室前壁缺血病变部位，将卷曲的实施例1、3或4的间充质干细胞膜片释放至病变部位，然后使其平坦铺展。可以多片叠加使用，总量为1-4片/人。
以上对本发明的具体实施例进行了详细描述，但其只是作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (31)

  1. 间充质干细胞膜片在治疗受试者中与心脏组织损伤或心功能不全相关的疾病中的用途，其中将所述间充质干细胞膜片局部应用于所述受试者的心脏。
  2. 间充质干细胞膜片在制备用于治疗受试者中与心脏组织损伤或心功能不全相关的疾病的组合物中的用途，其中将所述间充质干细胞膜片局部应用于所述受试者的心脏。
  3. 如权利要求1或2所述的用途，其中所述疾病选自缺血性心脏病、风湿性心脏病、先天性心脏病、心肌病、冠心病和心脏瓣膜病。
  4. 如权利要求3所述的用途，所述心肌病为扩张型心肌病。
  5. 如权利要求1或2所述的用途，其中所述疾病是缺血性心力衰竭，例如急性缺血性心力衰竭、慢性缺血性心力衰竭或终末期缺血性心力衰竭。
  6. 如权利要求1-5中任一项所述的用途，其中将所述间充质干细胞膜片贴附于心脏的左室前壁或侧壁。
  7. 如权利要求1-5中任一项所述的用途，其中将所述间充质干细胞膜片贴附于心脏的损伤或缺陷部位，或其邻近部位。
  8. 如权利要求1-5中任一项所述的用途，其中将所述间充质干细胞膜片植入心脏的损伤或缺陷部位，或其邻近部位。
  9. 如权利要求1-8中任一项所述的用途，其中所述间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％。
  10. 如权利要求9所述的用途，其中所述间充质干细胞来源于选自以下的组织：羊水、羊膜、绒毛膜、绒毛膜绒毛、蜕膜、胎盘、脐带血、华通氏胶、脐带、成人骨髓、牙髓、成人外周血和成人脂肪组织。
  11. 如权利要求9所述的用途，其中所述间充质干细胞选自脐带间充质干细胞、胎盘间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞和骨髓间充质干细胞。
  12. 如权利要求11所述的用途，其中所述间充质干细胞是脐带间充质干细胞。
  13. 如权利要求1-12中任一项所述的用途，其中所述间充质干细胞膜片是使用传代数为P0-P20，例如P2-P10或P2-P8的间充质干细胞制备的。
  14. 如权利要求1-13中任一项所述的用途，其中所述间充质干细胞膜片的厚度为10-300μm，例如30-300μm或50-300μm。
  15. 如权利要求1-14中任一项所述的用途，其中所述间充质干细胞膜片中的细胞密度为1×10 5至1×10 7/cm 2，例如3×10 5至5×10 6/cm 2。
  16. 如权利要求1-14中任一项所述的用途，其中所述间充质干细胞膜片中的细胞密度为1×10 5至5×10 7/cm 2，例如8×10 5至5×10 7/cm 2，3×10 5至5×10 6/cm 2。
  17. 如权利要求1-16中任一项所述的用途，其中所述膜片中的间充质干细胞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。
  18. 如权利要求1-17中任一项所述的用途，其中所述膜片中的间充质干细胞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。
  19. 如权利要求1-17中任一项所述的用途，其中所述膜片中的间充质干细胞能够分泌多种细胞因子，所述细胞因子包括EGF、FGF、HGF、TGF-β、PGE2、IL-6、IL-8、IL-10、PDGF和VEGF中的一种或多种。
  20. 如权利要求1-19中任一项所述的用途，其中所述膜片中包括2×10 7至8×10 7的间充质干细胞。
  21. 如权利要求1-20中任一项所述的用途，其中将1-4片所述间充质干细胞膜片局部应用于所述受试者的心脏。
  22. 如权利要求1-21中任一项所述的用途，其中将所述间充质干细胞膜片局部应用于所述受试者的心脏与所述受试者施行冠状动脉搭桥术联用，例如，所述受试者施行冠状动脉搭桥术后，将所述间充质干细胞膜片局部应用于所述受试者的心脏。
  23. 如权利要求1-22中任一项所述的用途，其中所述间充质干细胞膜片中的间充质干细胞与所述受试者是自体的或同种异体的。
  24. 如权利要求1-23中任一项所述的用途，其中所述间充质干细胞膜片是通过包括以下步骤的方法制备的：
    a.在粘附基质或血清预包被的温敏培养皿中培养间充质干细胞；
    b.通过降低温度使间充质干细胞从所述温敏培养皿脱离，
    其中所述间充质干细胞通过其分泌的细胞外基质彼此连接，从而获得所 述间充质干细胞膜片。
  25. 如权利要求24所述的用途，其中所述粘附基质包括胶原、明胶、纤连蛋白，玻连蛋白，层粘连蛋白，多聚鸟氨酸和多聚赖氨酸的一种或多种。
  26. 如权利要求24所述的用途，其中所述粘附基质包括胎牛血清、自体血清、胶原、明胶、纤连蛋白，玻连蛋白，层粘连蛋白，多聚鸟氨酸和多聚赖氨酸的一种或多种。
  27. 如权利要求24-26中任一项所述的用途，其中所述间充质干细胞膜片具有在制备过程中不接触培养皿的上表面和接触培养皿的基底面，所述基底面较粗糙。
  28. 如权利要求27所述的用途，其中将所述间充质干细胞膜片的基底面贴附于心脏的损伤或缺陷部位，或其邻近部位。
  29. 如权利要求27所述的用途，其中将所述间充质干细胞膜片的基底面贴附于心脏的左室前壁和侧壁。
  30. 如权利要求1-29任一项所述的用途，其中所述间充质干细胞膜片为单剂量间充质干细胞膜片，所述膜片包含2×10 7至8×10 7的间充质干细胞。
  31. 如权利要求30所述的用途，其中所述膜片的直径为15-55mm，所述膜片的厚度为10-300μm。
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