WO2021146994A1 - 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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WO2021146994A1
WO2021146994A1 PCT/CN2020/073765 CN2020073765W WO2021146994A1 WO 2021146994 A1 WO2021146994 A1 WO 2021146994A1 CN 2020073765 W CN2020073765 W CN 2020073765W WO 2021146994 A1 WO2021146994 A1 WO 2021146994A1
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mesenchymal stem
stem cells
use according
stem cell
cell membrane
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PCT/CN2020/073765
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English (en)
French (fr)
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高爽
常德华
靳新
谭玉琴
王娟
赵玉菲
刘洋
刘帅
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京东方科技集团股份有限公司
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Priority to PCT/CN2020/073765 priority Critical patent/WO2021146994A1/zh
Priority to CN202080000065.XA priority patent/CN113498434A/zh
Priority to US17/430,086 priority patent/US20220347346A1/en
Priority to PCT/CN2021/073295 priority patent/WO2021148000A1/zh
Priority to CN202180003593.5A priority patent/CN113891933A/zh
Publication of WO2021146994A1 publication Critical patent/WO2021146994A1/zh

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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
  • 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.
  • 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.
  • Heart failure is one of the main causes of human death and disability. It is seen in people of all ages, and the risk increases with age, and is the most common among the elderly.
  • Heart failure is a syndrome or clinical condition caused by the inability of the heart to maintain adequate blood circulation. It can be chronic or acute, and can be caused by many types of diseases, including coronary heart disease, myocardial infarction, valvular heart disease, and myocardium. Disease, congenital heart disease, rheumatic heart disease, arrhythmia, high blood pressure, severe lung disease and severe anemia.
  • 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 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 a mesenchymal stem cell membrane to the heart of the subject Steps to slices.
  • the disease may be selected from ischemic heart disease, rheumatic heart disease, congenital heart disease, cardiomyopathy, coronary heart disease, and valvular heart disease.
  • the disease may be ischemic heart failure, such as acute ischemic heart failure.
  • 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, 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-derived 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 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 15-250 ⁇ m, for example, 50-300 ⁇ m or 100-300 ⁇ m. In addition, the mesenchymal stem cell membrane sheet of the present disclosure 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 1 ⁇ 10 7 /cm 2 , for example, about 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.
  • 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 immunomodulatory factors, such as hepatocyte growth factor (HGF), interleukin-6 (IL-6) ), one or more of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF).
  • cytokines such as angiogenesis factors and immunomodulatory factors, such as hepatocyte growth factor (HGF), interleukin-6 (IL-6) ), one or more of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF).
  • HGF hepatocyte growth factor
  • IL-6 interleukin-6
  • IL-8 interleukin-8
  • VEGF vascular endothelial growth factor
  • 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 determined 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 be selected from collagen, gelatin, fibronectin, vitronectin, laminin, polyornithine and One or more of 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 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 rich in cell junction proteins and 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 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 disease may be ischemic heart failure, such as acute ischemic heart failure.
  • 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 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, 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, 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 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, 50-300 ⁇ m or 100-300 ⁇ m. In addition, the mesenchymal stem cell membrane sheet of the present disclosure 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 1 ⁇ 10 7 /cm 2 , for example, about 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 angiogenic factors and immunoregulatory factors, such as one or more of HGF, IL-6, IL-8, and VEGF. kind.
  • cytokines such as angiogenic factors and immunoregulatory factors, such as one or more of HGF, IL-6, IL-8, and VEGF. kind.
  • 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 be selected from collagen, gelatin, fibronectin, vitronectin, laminin, polyornithine and One or more of 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 rich in cell junction proteins and 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 present disclosure relates to a mesenchymal stem cell membrane sheet, which is used to treat diseases related to cardiac tissue damage or cardiac insufficiency in a subject.
  • the present disclosure relates to a composition
  • a composition comprising a membrane of mesenchymal stem cells, which is 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, and valvular heart disease .
  • the disease may be ischemic heart failure, such as acute ischemic heart failure.
  • the mesenchymal stem cell has one or more of the characteristics described in the first, second, and third aspects of the present disclosure.
  • Figure 1 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 2 shows a scanning electron microscopic image of a membrane of umbilical cord mesenchymal stem cells.
  • Figure 2A Surface (upper surface) of cell membrane sheet.
  • Figure 2B Basal surface of cell membrane sheet.
  • Figure 3 shows an immunofluorescence imaging photograph of a membrane of umbilical cord mesenchymal stem cells.
  • Figure 3A Fibronectin.
  • Figure 3B Integrin ⁇ 1.
  • Figure 4 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 5 shows the characterization of the constructed mouse disease model of heart failure.
  • Figure 5A Photos of the heart of disease model mice;
  • Figure 5B ECG results of disease model mice.
  • Figure 6 shows the use of umbilical cord mesenchymal stem cell patch to treat disease model mice.
  • Fig. 6A shows an exemplary photograph of the mesenchymal stem cell membrane used;
  • Fig. 6B shows a photograph of the cell membrane attached to the surface of a mouse heart.
  • Figure 7 shows the mouse echocardiogram results at different time points.
  • Figure 7A before modeling
  • Figure 7B 1 week after modeling
  • Figure 7C 4 weeks after modeling.
  • Left side control group animals
  • right side cell patch transplantation group animals.
  • Fig. 8 shows the curve of the ejection fraction of the left ventricle of the mouse before and after modeling with time.
  • Figure 9 shows the curve of the short axis shortening index of the left ventricle of mice before and after modeling with time.
  • Figure 10 shows the curve of the left ventricle diameter of the mouse before and after modeling with time.
  • Figure 11 shows the curve of the volume of the left ventricle of the mouse before and after modeling with time.
  • Figure 12 shows the results of Masson staining of mouse heart tissue sections at the end of the experiment. Left side: control group animals; right side: cell patch treatment group animals.
  • 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.
  • mesenchymal stem cells can be stained with Alizarin Red (Figure 1A) or Oil Red O ( Figure 1B) after osteogenic induction or adipogenesis induction, indicating that they have the ability to differentiate into bone and adipocytes. ability.
  • the above-mentioned umbilical cord mesenchymal stem cells are digested into single cells, and then seeded at a suitable density into a temperature-sensitive culture dish with polyacrylamide temperature-sensitive polymer materials attached to the surface of the culture dish. Cultivate in an incubator at 37°C, 5% CO 2 and 95% humidity. After the cells proliferate for a period of time, move them to an environment of about 20°C or add 4°C pre-cooled HBSS solution. 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 diaphragm is off-white, with a dense structure, and a smooth and flat surface. The viable cell rate in the prepared cell membrane sheet is high, and the cell condition is good.
  • 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 sheet has a surface that is not in contact with the petri dish (upper surface, Figure 2A) and a base surface that is in contact with the petri dish (lower surface, Figure 2B).
  • 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 7, the cell membrane sheet prepared by the method of the present disclosure contains a large amount of fibronectin ( Figure 3A) and integrin ⁇ 1 ( Figure 3B).
  • 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. 4.
  • 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.
  • 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. 7B and Fig. 7C, it can be seen that the heart failure model animals have obvious weakening of the heart after modeling.
  • the cell patch treatment group animals had stronger heart movements.
  • Left ventricular ejection fraction is an important index to evaluate left ventricular function.
  • 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 10) and the left ventricular volume versus time curve ( Figure 11) 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 membrane treatment group were significantly lower than those in the control animals, indicating that the use of cell membranes 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 12) 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.

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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 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
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，这两者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，对于间充质干细胞的新的应用方式以及相关产品，本领域中仍然存在需要。
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。
在世界范围内，心力衰竭是造成人类死亡和病残的主要原因之一，其见于所有年龄段的人群中，且风险随着年龄增长而增加，在老年人中最为常见。心力衰竭是心脏不能维持充足的血液循环所导致的综合征或临床状况，其可以是慢性或是急性的，并可以由多种类型的疾病导致，包括冠心病、心肌梗死、心脏瓣膜病、心肌病、先天性心脏病、风湿性心脏病、心律失常、高血压、严重的肺部疾病和严重贫血等。对于心力衰竭，目前的治疗方法主要包括利尿剂、血管扩张药和正性肌力药等，但这些疗法并未完全解决心力衰竭 的高死亡率和伤残率的问题，并具有相当的副作用。因此，本领域中对于新的、有效的心力衰竭治疗方法仍然存在需要。
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，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，在一方面，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，所述疾病可以是缺血性心力衰竭，例如急性缺血性心力衰竭。
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、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。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，例如50-300μm或100-300μm的厚度。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层或5-15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1×10 7/cm 2，例如约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，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)、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)中的一种或多种。
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选自胶原、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，所述基底面富含细胞连接蛋白且较粗糙。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。
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、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50-300μm或100-300μm的厚度。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1×10 7/cm 2，例如约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、IL-6、IL-8和VEGF中的一种或多种。
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 质可以选自胶原、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，所述基底面富含细胞连接蛋白且较粗糙。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。
在第四个方面，本公开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。
在第五个方面，本公开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和第五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和第五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。
在本公开的第四个方面和第五个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2示出了脐带间充质干细胞膜片的扫描电镜成像照片。图2A：细胞膜 片的表面(上表面)。图2B：细胞膜片的基底面。
图3示出了脐带间充质干细胞膜片的免疫荧光成像照片。图3A：纤粘蛋白。图3B：整联蛋白β1。
图4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5示出了构建的心力衰竭小鼠疾病模型的表征。图5A：疾病模型小鼠的心脏照片；图5B：疾病模型小鼠的心电图结果。
图6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6A显示了使用的间充质干细胞膜片的示例性照片；图6B显示了将细胞膜片贴附于小鼠心脏表面的照片。
图7示出了不同时间点的小鼠超声心动图结果。图7A：建模前；图7B：建模后1周；图7C：建模后4周。左侧：对照组动物；右侧：细胞膜片移植组动物。
图8示出了建模前后小鼠左心室射血分数随时间变化的曲线。
图9示出了建模前后小鼠左心室短轴缩短指数随时间变化的曲线。
图10示出了建模前后小鼠左心室内径随时间变化的曲线。
图11示出了建模前后小鼠左心室容积随时间变化的曲线。
图12示出了实验结束时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。
具体实施方式
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1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1A)或油红O染色(图1B)，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，将上述脐带间充质干细胞消化成为单细胞后，以适合的密度接种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中，于37℃，5％CO 2和95％湿度环境的培养箱内培养。待细胞增殖一段时间后，将其移至约20℃的环境下或加入4℃预冷的HBSS液。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。获得的膜片呈灰白色，结构致密，表面光滑平整。制备的细胞膜片中的活细胞率很高，且细胞状态良好。
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2所示，细胞膜片具有不与培养皿接触的表面(上表面，图2A)和与培养皿接触的基底面(下表面，图2B)，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7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3A)和整合素β1(图3B)。
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4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.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5A)。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5B)。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4.间充质干细胞膜片在治疗心力衰竭中的应用
在如实施例3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6A)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6B)。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7A)、建模后1周(图7B)和建模后4周(图7C)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7B和图7C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8)和左心室短轴缩短指数随时间变化曲线(图9)。左心室射血分数是评价左心室功能的重要指标。如图8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9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10)和左心室容积随时间变化曲线(图11)，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10和图11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12)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。
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 (20)

  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。
  5. 如权利要求1-4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。
  6. 如权利要求1-4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。
  7. 如权利要求1-6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％。
  8. 如权利要求7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、成人外周血和成人脂肪组织。
  9. 如权利要求7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、脂肪间充质干细胞和骨髓间充质干细胞。
  10. 如权利要求9所述的用途，其中所述间充质干细胞是脐带间充质干细胞。
  11. 如权利要求1-10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的间充质干细胞制备的。
  12. 如权利要求1-11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50-300μm。
  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。
  14. 如权利要求1-13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。
  15. 如权利要求1-14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。
  16. 如权利要求1-15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。
  17. 如权利要求16所述的用途，其中所述间充质干细胞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50％-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。
  18. 如权利要求16或17所述的用途，其中所述粘附基质选自胶原、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。
  19. 如权利要求16-18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，所述基底面富含细胞连接蛋白且较粗糙。
  20. 如权利要求19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。
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