Preparation method of high-efficiency allogenic mesenchymal stem cells, high-efficiency allogenic mesenchymal stem cells and application of high-efficiency allogenic mesenchymal stem cells

Abstract

The invention discloses a preparation method of efficient allogenic mesenchymal stem cells, the efficient allogenic mesenchymal stem cells and application of the efficient allogenic mesenchymal stem cells, the method comprises the steps that raw material mesenchymal stem cells and statins are mixed and cultured for 12-36 hours, a mixed solution is prepared, and the molar concentration of the statins in the mixed solution is 1-2 mu M; and separating the raw material allogenic mesenchymal stem cells in the mixed solution, and washing the raw material allogenic mesenchymal stem cells with a buffer solution for at least two times to prepare the high-efficiency allogenic mesenchymal stem cells. The efficient allogenic mesenchymal stem cells prepared by the method disclosed by the embodiment of the invention can be used for myocardial repair after acute myocardial infarction and have an obvious repair effect.

Description

Technical Field

[0001] The present application belongs to the field of stem cell technology, and in particular relates to a method for preparing highly efficient allogeneic mesenchymal stem cells and highly efficient allogeneic mesenchymal stem cells and applications thereof. Background Art

[0002] Acute myocardial infarction (AMI) is the number one killer that endangers human health. After ischemia, a large number of cardiomyocytes undergo necrosis and apoptosis, and fibroblasts proliferate and activate, which are important causes of myocardial fibrosis and ventricular remodeling, and then end-stage heart failure and even death. The emergence of stem cell transplantation technology has made exogenous myocardial repair possible, but the low efficacy makes this method have obvious limitations.

[0003] In addition, most clinical trials currently prefer autologous stem cell transplantation, but it still takes 2 to 3 weeks of in vitro culture for autologous stem cells to be obtained and expanded to the number required for transplantation, which cannot meet the actual needs of clinical emergency treatment. Therefore, how to break through the bottleneck of the efficacy of stem cell transplantation and enable it to be used in the treatment of AMI patients in a timely manner is the focus of research. Summary of the invention

[0004] The embodiments of the present application provide a method for preparing highly efficient allogeneic mesenchymal stem cells and highly efficient allogeneic mesenchymal stem cells. The highly efficient allogeneic mesenchymal stem cells prepared by the method of the embodiments of the present application can be used for myocardial repair after acute myocardial infarction and have obvious repair effects.

[0005] In a first aspect, the present invention provides a method for preparing highly efficient allogeneic mesenchymal stem cells, comprising:

[0006] The raw material mesenchymal stem cells and the statin are mixed and cultured for 12 to 36 hours to prepare a mixed solution, wherein the molar concentration of the statin in the mixed solution is 1 μM to 2 μM;

[0007] The raw material allogeneic mesenchymal stem cells in the mixed solution are separated and washed with a buffer solution at least twice to obtain the high-efficiency allogeneic mesenchymal stem cells.

[0008] According to an embodiment of one aspect of the present application, the statin includes one or more of atorvastatin, simvastatin, and rosuvastatin.

[0009] According to an embodiment of one aspect of the present application, the raw material mesenchymal stem cells are derived from one or more of pig bone marrow, human bone marrow, and human umbilical cord.

[0010] According to an embodiment of one aspect of the present application, the method for preparing the raw material mesenchymal stem cells comprises:

[0011] Puncture the pig's iliac crest and extract 10-20 mL of bone marrow fluid, and use density gradient centrifugation to separate the raw cells;

[0012] The raw material cells are cultured in vitro until the colony of the raw material mesenchymal stem cells grows to 80-90% confluence, thereby obtaining the raw material mesenchymal stem cells.

[0013] According to an embodiment of one aspect of the present application, the buffer is one or more of a phosphate buffer having a mass fraction of 10% to 20% or a Hepsin buffer having a mass fraction of 10% to 20%.

[0014] According to an embodiment of one aspect of the present application, the concentration of the raw mesenchymal stem cells in the mixed solution is 10 5 to 10 6 cells / mL.

[0015] According to an embodiment of one aspect of the present application, the raw material allogeneic mesenchymal stem cells in the mixed solution are separated and washed with a buffer solution at least twice until the concentration of the statin in the solution is less than 10 ppm.

[0016] In a second aspect, an embodiment of the present application provides a highly efficient allogeneic mesenchymal stem cell prepared by the method of the first aspect.

[0017] Use of the high-efficiency allogeneic mesenchymal stem cells prepared by the preparation method of the first aspect or the high-efficiency allogeneic mesenchymal stem cells of the second aspect in the preparation of a drug for myocardial repair after acute myocardial infarction.

[0018] According to an embodiment of one aspect of the present application, the concentration of the highly efficient allogeneic mesenchymal stem cells is 10 7 to 10 8 cells / mL.

[0019] The embodiments of the present application have at least the following beneficial effects:

[0020] The preparation method provided in the embodiment of the present application uses statins to treat raw mesenchymal stem cells to obtain highly efficient allogeneic mesenchymal stem cells. The highly efficient allogeneic mesenchymal stem cells are infused into the infarcted myocardium via the coronary artery route for myocardial repair treatment after acute myocardial infarction, and have a good repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0022] Figure 1 The results of identification of highly efficient allogeneic mesenchymal stem cells in the examples of the present application are shown.

[0023] Figure 2 The graph shows that the highly efficient allogeneic porcine bone marrow-derived mesenchymal stem cell transplantation according to the embodiment of the present application can significantly improve cardiac function after myocardial infarction at the large animal level.

[0024] Figure 3 The graph shows that the highly efficient allogeneic porcine bone marrow-derived mesenchymal stem cell transplantation according to the embodiment of the present application can reduce the infarct area and alleviate myocardial fibrosis and collagen deposition at the large animal level.

[0025] Figure 4 The diagram shows the effect of highly efficient allogeneic porcine bone marrow-derived mesenchymal stem cell transplantation without immune rejection reaction at the large animal level in the embodiment of the present application.

[0026] Figure 5 It is shown that the highly efficient allogeneic porcine bone marrow-derived mesenchymal stem cells of the present application example can inhibit the expression of Col1α1 and Col3α1 in fibroblasts through exosomes in vitro. DETAILED DESCRIPTION

[0027] In order to make the invention purpose, technical solution and beneficial technical effect of the present application clearer, the present application is described in detail below in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are only for explaining the present application, not for limiting the present application.

[0028] For simplicity, only some numerical ranges are explicitly disclosed herein. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range, and any upper limit can be combined with any other upper limit to form an unambiguous range. In addition, although not explicitly stated, each point or single value between the range endpoints is included in the range. Thus, each point or single value can be combined with any other point or single value as its own lower limit or upper limit or with other lower limits or upper limits to form an unambiguous range.

[0029] In the description of this article, it should be noted that, unless otherwise specified, "above" and "below" are inclusive of the number, and "several" in "one or several" means two or more.

[0030] The above-mentioned summary of the invention of the present application is not intended to describe each disclosed embodiment or each implementation in the present application. The following description more specifically illustrates exemplary embodiments. In many places throughout the application, guidance is provided by a series of examples, which can be used in various combinations. In each example, enumeration is only used as a representative group and should not be interpreted as exhaustive.

[0031] In a first aspect, the present invention provides a method for preparing highly efficient allogeneic mesenchymal stem cells, comprising:

[0032] The raw material mesenchymal stem cells and the statin are mixed and cultured for 12 to 36 hours to prepare a mixed solution, wherein the molar concentration of the statin in the mixed solution is 1 μM to 2 μM;

[0033] The raw material allogeneic mesenchymal stem cells in the mixed solution are separated and washed with a buffer solution at least twice to obtain the high-efficiency allogeneic mesenchymal stem cells.

[0034] Optionally, the raw mesenchymal stem cells are mixed with the statin and cultured for any value of 12 h, 16 h, 20 h, 24 h, 30 h, 36 h or a range of their composition.

[0035] According to the embodiments of the present application, raw mesenchymal stem cells treated with statins can significantly improve the cardiac function of miniature pigs after AMI and improve ventricular remodeling; can significantly reduce the infarct area, alleviate myocardial fibrosis and collagen deposition; and at the same time do not cause immune rejection reactions.

[0036] In addition, MSC ATV Exosomes from MSC ATV -Exo) intervention can more significantly reduce the expression levels of fibrosis markers Col1α1 and Col3α1 in fibroblasts, preliminarily indicating that allogeneic MSCs ATV The efficient myocardial repair ability is related to the secretion of exosomes. In some optional embodiments, the efficient allogeneic mesenchymal stem cells include exosomes.

[0037] In some optional embodiments, the statin includes one or more of atorvastatin, simvastatin, and rosuvastatin.

[0038] Statins are a class of lipid-lowering drugs widely used in clinical practice, mainly used to reduce low-density lipoprotein cholesterol (LDL-C) in the blood, thereby preventing and treating cardiovascular diseases. Atorvastatin is a commonly used statin that reduces cholesterol synthesis by inhibiting HMG-CoA reductase (3-hydroxy-3-methylglutaryl coenzyme A reductase) in the liver. In the embodiments of the present application, mesenchymal stem cells are treated with statins such as atorvastatin, which can significantly improve the cardiac function of miniature pigs after AMI and improve ventricular remodeling after transplantation; can significantly reduce the infarct area, reduce myocardial fibrosis and collagen deposition; and at the same time do not cause immune rejection.

[0039] In some optional embodiments, the raw material mesenchymal stem cells are derived from one or more of pig bone marrow, human bone marrow, and human umbilical cord.

[0040] The mesenchymal stem cells from the above sources have the ability to differentiate into adipogenesis, osteogenesis and chondrogenesis.

[0041] In some optional embodiments, the method for preparing raw mesenchymal stem cells comprises:

[0042] Puncture the pig's iliac crest and extract 10-20 mL of bone marrow fluid, and use density gradient centrifugation to separate the raw cells;

[0043] The raw material cells are cultured in vitro until the colony of the raw material mesenchymal stem cells grows to 80-90% confluence, thereby obtaining the raw material mesenchymal stem cells.

[0044] In some optional embodiments, the method for preparing raw mesenchymal stem cells comprises subculturing the raw cells to generations P3 to P5.

[0045] In some optional embodiments, puncturing the pig iliac crest and extracting 10-20 mL of bone marrow fluid, and separating the raw material cells by density gradient centrifugation specifically includes:

[0046] After diluting the bone marrow fluid with an equal amount of preheated 1× PBS buffer, transfer it to 60% Percoll separation solution at a volume ratio of 1:1, centrifuge at 600×g (acceleration rate (ACC) = 5 rank, deceleration rate (DEC) = 5 rank) at room temperature for 20 minutes, then carefully aspirate the cells between the serum and the separation solution, and add complete culture medium for culture.

[0047] Percoll is a density gradient medium widely used for separation of cells, biological particles and macromolecules. It is made of silica gel particles in the form of micelles suspended in a non-ionic hydrophilic solvent PVP (polyethylene glycol).

[0048] 60% Percoll separation solution refers to a solution of Percoll in a buffer (such as PBS or culture medium) with a concentration of 60%.

[0049] In some optional embodiments, in order to reduce the removal of statins contained in the high-efficiency allogeneic mesenchymal stem cell solution or its cell fluid, the solution is washed with a buffer solution at least twice.

[0050] In some optional embodiments, the buffer comprises one or more of phosphate buffer with a mass fraction of 10% to 20% or Hepsin buffer with a mass fraction of 10% to 20%. The above buffer can improve cell activity and help cells to exert their repair effect.

[0051] In some optional embodiments, in order to reduce the impact of statins when using highly efficient allogeneic mesenchymal stem cells, the raw allogeneic mesenchymal stem cells in the mixed solution are separated and washed with buffer at least twice until the concentration of the statin in the solution is less than 10 ppm.

[0052] The concentration of statins can be detected by enzyme-linked immunosorbent assay.

[0053] In some optional embodiments, in order to improve the survival rate and vitality of cells, the concentration of raw mesenchymal stem cells in the mixed solution is 10 5 to 10 6 cells / mL.

[0054] In a second aspect, an embodiment of the present application provides a highly efficient allogeneic mesenchymal stem cell prepared by the method of the first aspect.

[0055] According to the embodiments of the present application, the processed highly efficient allogeneic mesenchymal stem cells can significantly improve the cardiac function of miniature pigs after AMI and improve ventricular remodeling; can significantly reduce the infarct area, alleviate myocardial fibrosis and collagen deposition; and at the same time do not cause immune rejection reactions.

[0056] In a third aspect, the embodiments of the present application provide the use of the highly efficient allogeneic mesenchymal stem cells of the second aspect in the preparation of a drug for myocardial repair after acute myocardial infarction.

[0057] In some optional embodiments, in the drug, the concentration of the highly efficient allogeneic mesenchymal stem cells is 10 7 to 10 8 cells / mL.

[0058] Example

[0059] The following examples more specifically describe the disclosure of the present application, which are intended for illustrative purposes only, as it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the disclosure of the present application. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0060] Example 1

[0061] Preparation method of highly efficient allogeneic porcine bone marrow-derived mesenchymal stem cells:

[0062] Chinese miniature pigs (weighing about 30 kg) from the Animal Experiment Center of Fuwai Hospital, Chinese Academy of Medical Sciences were anesthetized, and a bone marrow puncture needle was used to puncture the bone marrow cavity at the iliac crest to extract 20-30 mL of bone marrow fluid and transfer it to a 50 mL sterile centrifuge tube; the bone marrow was diluted with an equal amount of PBS buffer preheated to 36-37°C, and then transferred to 60% Percoll separation solution (1.077 g / mL) at a volume ratio of 1:1 using a dropper, and centrifuged at 600×g (ACC=5rank, DEC=5rank) for 20 min at room temperature (after this step, four liquid phases can be seen in the centrifuge tube, which are serum phase, bone marrow mononuclear cell phase, Percoll separation liquid phase and precipitation phase from top to bottom); the cells in the bone marrow mononuclear cell phase were aspirated and transferred to a 15 mL sterile centrifuge tube, washed with PBS buffer and centrifuged at 3-5×10 5 / mL, and inoculated into a T175 culture flask, and cultured to P3-5 generations using Iscove's Modified Dulbecco's Medium (IMDM) complete medium from Thermo Fisher Scientific (supplemented with 10% Thermo Fisher Scientific's premium fetal bovine serum, 1% Thermo Fisher Scientific's glutamine storage solution, and 1% Thermo Fisher Scientific's penicillin / streptomycin solution). At this time, the cell density was 10 5 to 10 6 cells / mL (cell colonies grow to 80-90% confluence);

[0063] Atorvastatin (ATV, MedChemExpress, USA) at a final concentration of 1 μmol / L was added to the IMDM medium for treatment for 24 h. The cells were washed three times with 10-20% PBS and then replaced with fresh IMDM medium for continued culture. After 48 h, the cells were collected for subsequent stem cell identification and transplantation experiments.

[0064] Comparative Example 1

[0065] Preparation method of mesenchymal stem cells:

[0066] Chinese miniature pigs (weighing about 30 kg) from the Animal Experiment Center of Fuwai Hospital, Chinese Academy of Medical Sciences were anesthetized, and a bone marrow puncture needle was used to puncture the bone marrow cavity at the iliac crest to extract 20-30 mL of bone marrow fluid and transfer it to a 50 mL sterile centrifuge tube; the bone marrow was diluted with an equal amount of PBS buffer preheated to 36-37°C, and then transferred to 60% Percoll separation solution (1.077 g / mL) at a volume ratio of 1:1 using a dropper, and centrifuged at 600×g (ACC=5rank, DEC=5rank) for 20 min at room temperature (after this step, four liquid phases can be seen in the centrifuge tube, which are serum phase, bone marrow mononuclear cell phase, Percoll separation liquid phase and precipitation phase from top to bottom); the cells in the bone marrow mononuclear cell phase were aspirated and transferred to a 15 mL sterile centrifuge tube, washed with PBS buffer and centrifuged at 3-5×10 5 / mL, and inoculated into a T175 culture flask, and cultured to P3-5 generations using Iscove's Modified Dulbecco's Medium (IMDM) complete medium from Thermo Fisher Scientific (supplemented with 10% Thermo Fisher Scientific's premium fetal bovine serum, 1% Thermo Fisher Scientific's glutamine storage solution, and 1% Thermo Fisher Scientific's penicillin / streptomycin solution). At this time, the cell density was 10 5 to 10 6 cells / mL (cell colonies grow to 80-90% confluence); wash three times with 10-20% PBS and then switch to fresh IMDM medium for continued culture; collect the cells after 48 hours to obtain mesenchymal stem cells.

[0067] Test Section

[0068] 1) Observation and identification of highly efficient allogeneic mesenchymal stem cells:

[0069] Highly efficient allogeneic mesenchymal stem cells (MSC) derived from bone marrow obtained after ATV treatment ATV ) appear spindle-shaped, star-shaped or polygonal under the microscope. Highly efficient allogeneic mesenchymal stem cells have the ability to differentiate into adipogenesis, osteoblastogenesis and chondrogenesis. The results of flow cytometry detection are as follows Figure 1 . Figure 1 Figure A shows stem cells under an optical microscope, and Figures B-D show the detection of high-efficiency allogeneic mesenchymal stem cells with adipogenesis, osteogenic and chondrogenic states by flow cytometry. OriCell Multipurpose Bone Marrow Mesenchymal Stem Cell Differentiation Induction Kit (Cyagen, China) was used for staining, and the test results are shown in Figure E, where FL4-A+, FL4-A-, FL2-A+, FL2-A-, FL1A+, and FL1-A- are the channel parameters displayed by the flow cytometer. The figure shows the ratio of CD105, CD29, CD90, CD14, and CD45 negative and positive, red is positive, blue is negative, and the results show that the three positive markers CD105, CD29, and CD90 are in MSC ATV The surface expression is obvious, and the expression of CD14 and CD45 is negative; the above meets the criteria of the International Society for Cellular Therapy (ISCT) for defining multipotent mesenchymal stem cells, namely MSC. ATV It still possesses the corresponding biological characteristics of multipotent mesenchymal stem cells.

[0070] 2) High-efficiency allogeneic mesenchymal stem cell transplantation experiment

[0071] Application method of highly efficient allogeneic porcine bone marrow-derived mesenchymal stem cells: The left anterior descending branch of the coronary artery was occluded by balloon occlusion to establish a Chinese miniature pig acute myocardial infarction model. Thirty minutes after the coronary artery was opened, the 5×10 7 A number of highly efficient allogeneic bone marrow-derived mesenchymal stem cells (Example 1) or mesenchymal stem cells not treated with ATV (Comparative Example 1) were used. The effectiveness and safety of allogeneic highly efficient bone marrow-derived mesenchymal stem cells transplantation for the treatment of acute myocardial infarction were preclinically evaluated at the large animal level. The effectiveness indicators included cardiac function after myocardial infarction and the size of the infarcted myocardial area, and the safety indicators included the occurrence of immune rejection after transplantation and whether the functions of major organs were affected.

[0072] Several Bama miniature pigs with similar physical conditions were placed in a supine position on the operating table. After routine disinfection and draping, femoral artery puncture was performed in the right groin area. After successful puncture, a 6F arterial sheath was inserted through the femoral artery, and then 12,500U of heparin was injected intravenously. Thereafter, 1,250U of heparin was injected every 1 hour. The piglets were placed in a left anterior oblique position (45°). Under the fluoroscopy of a C-arm X-ray machine, a 6F JR4.0 right coronary artery guiding catheter was inserted through the femoral artery sheath. The guiding catheter was guided to the ascending aorta through a 0.035" InQwire angiography guidewire. After the guidewire was withdrawn, the guiding catheter was operated again to enter the left coronary sinus, left main trunk and LAD opening in turn, so that the catheter opening was coaxial with the coronary artery opening; then iohexol injection was injected for left coronary angiography. After locating the first diagonal branch, a 0.014" Balance Middleweight finger guidewire was inserted through the guiding catheter into the distal end of the first diagonal branch. Under the guidance of the guidewire, a 2.5mm×8mm The PTCA balloon dilatation catheter was placed 2-5 mm distal to the first diagonal branch of LAD, and the balloon was opened with 6 atmospheres to occlude LAD. Angiography showed that the blood flow at the distal end of the balloon was interrupted. After balloon occlusion, the electrocardiogram and hemodynamic changes were closely monitored. A 2-mg / kg loading dose of lidocaine was injected intravenously 2 minutes before occlusion to prevent arrhythmias, and then it was continuously pumped in at a rate of 2-3 mg / (kg·h) and maintained until the end of modeling. Once frequent ventricular premature beats or paroxysmal ventricular tachycardia occurred during the operation, 5 mg / kg of amiodarone was injected intravenously, and then maintained at a pump rate of 0.5-1 mg / (kg·h). If ventricular tachycardia or ventricular fibrillation occurred, direct current cardioversion was performed for defibrillation, and cardiac compression was performed when necessary. At the same time, dopamine, epinephrine and other drugs were used for rescue.

[0073] After 90 minutes of occlusion, the balloon was released and blood flow was restored by reperfusion for 30 minutes. The pigs were divided into 4 groups according to the experimental groups, namely SHAM, AMI, MSC and MSC. ATV The piglets that remained in the operation were the SHAM group. During balloon dilation, an equal volume of IMDM medium was injected through the over-the-wire (OTW) balloon central catheter as a control, which was the AMI group.

[0074] Under balloon dilatation, MSCs were placed via an over-the-wire (OTW) balloon central catheter. ATV 5×10 7The above-mentioned mesenchymal stem cells (Example 1) or the mesenchymal stem cells not treated with ATV (Comparative Example 1) were pretreated, wherein the mesenchymal stem cells of Example 1 and Comparative Example 1 were cultured using an equal volume of the same IMDM culture medium. In order to minimize the coronary ischemia caused by balloon dilation during the transplantation process, the transplantation process was carried out in two stages, with a blood flow recovery period in between, during which the balloon was in a deflated state to restore coronary blood flow. Subsequently, 3 mL of normal saline was injected in the same manner to ensure that allogeneic BM-MSCs were completely transplanted. After 90 minutes of occlusion, the balloon was released, and the catheter and sheath were withdrawn after angiography showed blood flow recovery again. The operation was terminated after the femoral artery puncture point was compressed to stop bleeding. Penicillin was injected intramuscularly within 3 days after the operation to prevent infection.

[0075] SHAM, AMI, MSC and MSC ATV In the four groups, SHAM was treated with angiographic guidewire only, AMI was treated with IMDM medium, MSC was treated with mesenchymal stem cells from comparative example 1, and MSC ATV The mesenchymal stem cells of Example 1 were used for treatment, and the test results were as follows: Figure 2 ;in, Figure 2 The left ventricular long axis section is shown. The Simpson's Method, also known as the biplane Simpson method, is used to evaluate the left ventricular function of the heart. The left ventricular ejection fraction, left ventricular shortening fraction, left ventricular end-diastolic volume and left ventricular end-systolic volume are evaluated from four dimensions. The evaluation shows that compared with mesenchymal stem cells (MSCs) not treated with ATV, allogeneic MSCs transplanted through the coronary artery ATV It can significantly improve cardiac function and ventricular remodeling after AMI in miniature pigs at the large animal level.

[0076] Left Ventricular Ejection Fraction (LVEF) is an important indicator for evaluating the function of the left ventricle of the heart. It is used to measure the efficiency of the heart in pumping blood from the left ventricle to the whole body during each contraction.

[0077] Left ventricular fractional shortening is another indicator used to assess left ventricular function, which mainly reflects the changes in the wall of the left ventricle during cardiac contraction.

[0078] Left Ventricular End-Diastolic Volume refers to the total amount of blood contained in the left ventricle at the end of diastole (when the heart is full of blood and has not yet begun to contract). It reflects the heart's filling capacity and preload, that is, the load the heart bears before each contraction.

[0079] Left Ventricular End-Systolic Volume refers to the amount of blood remaining in the left ventricle after a cardiac contraction, and is an important indicator of the heart's contractile ability. If LVESV increases, it means that the heart's pumping efficiency decreases, which may mean weakened myocardial function or heart failure.

[0080] 3) Detection of cardiac-related parameters after efficient allogeneic mesenchymal stem cell transplantation: Pathological evaluation showed that allogeneic MSC ATV Transplantation can significantly reduce infarct size and alleviate myocardial fibrosis and collagen deposition ( Figure 3 ).

[0081] 4) Detection of relevant indicators in the blood and other organ conditions after the high-efficiency allogeneic mesenchymal stem cell transplantation experiment: In the safety evaluation, the results are as follows Figure 4 During the follow-up period, no allogeneic MSC transplantation ATV A decrease in complement levels was observed in the group, while liver and kidney function remained within the normal range at each time point. Further HE staining results showed that the recipients of allogeneic MSCs ATV There was no immune cell infiltration in the major internal organs of the transplanted individuals, indicating that MSC ATV The transplantation did not lead to immune rejection. The above results show that intracoronary transplantation of allogeneic MSCs is effective in large animals. ATV Treatment of AMI is efficient and safe.

[0082] The highly efficient allogeneic bone marrow-derived mesenchymal stem cells and mesenchymal stem cells not treated with ATV in the culture medium were collected, and then the exosomes were extracted by differential ultracentrifugation to obtain MSC-derived exosomes (MSC-Exo) and MSC ATV Exosomes from MSC ATV -Exo).

[0083] The in vitro fibrosis model was established by induction of human ventricular fibroblasts using transforming growth factor-β (TGF-β). 5 Up to 2×10 6 , about 5×10 5cells) were added with MSC or MSC ATV Exosomes from MSC or MSC ATV The concentration of exosomes in this system was 10 μg / mL.

[0084] The treated fibroblasts were subjected to real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) detection, and the results were as follows: Figure 5 , Figure 5 Figure A is a schematic diagram of the experimental process. In Figures B and C, control is a control group, which is a normal cell without any treatment on the fibroblasts; the TGF-β group added 10ngTGF-β to the normal cells to induce fibrosis; the MSC-Exo group added 20μg of MSC-derived exosomes to the prepared fibroblasts; the MSC ATV -Exo group added 20 μg of MSC to fibroblasts ATV Compared with MSC-derived exosomes (MSC-Exo), MSC ATV Exosomes from MSC ATV -Exo) intervention can more significantly reduce the expression levels of fibrosis markers Col1α1 and Col3α1 in fibroblasts, preliminarily indicating that allogeneic MSCs ATV The efficient myocardial repair ability is related to its secretion of exosomes which exert paracrine effects.

[0085] In summary, compared with ordinary MSCs, transcoronary artery transplantation of allogeneic MSCs ATV It has significant therapeutic effects on AMI at the animal level, which can be manifested in improving cardiac function, improving ventricular remodeling and reducing infarct size. Its mechanism is related to the secretion of exosomes acting on fibroblasts, reducing the expression of fibrosis markers and thus inhibiting myocardial fibrosis. In addition, allogeneic MSC ATV Transplantation does not cause immune rejection. ATV It is expected to be a second-generation "highly efficient" stem cell preparation with broad application prospects in the clinical treatment of AMI.

[0086] It should also be noted that the exemplary embodiments mentioned in this application describe some methods or systems based on a series of steps or devices. However, this application is not limited to the order of the above steps, that is, the steps can be performed in the order mentioned in the embodiment, or in a different order from the embodiment, or several steps can be performed simultaneously.

[0087] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A method for preparing highly efficient allogeneic mesenchymal stem cells, comprising: The raw material mesenchymal stem cells and the statin are mixed and cultured for 12 to 36 hours to prepare a mixed solution, wherein the molar concentration of the statin in the mixed solution is 1 μM to 2 μM; The raw material allogeneic mesenchymal stem cells in the mixed solution are separated and washed with a buffer solution at least twice to obtain the high-efficiency allogeneic mesenchymal stem cells.

2. The method according to claim 1, characterized in that The statins include one or more of atorvastatin, simvastatin, and rosuvastatin.

3. The method according to claim 1, characterized in that The raw material mesenchymal stem cells are derived from one or more of pig bone marrow, human bone marrow and human umbilical cord.

4. The method according to claim 1, characterized in that: The method for preparing the raw material mesenchymal stem cells comprises: Puncture the pig's iliac crest and extract 10-20 mL of bone marrow fluid, and use density gradient centrifugation to separate the raw cells; The raw material cells are cultured in vitro until the colony of the raw material mesenchymal stem cells grows to 80-90% confluence, thereby obtaining the raw material mesenchymal stem cells.

5. The method according to claim 1, characterized in that The buffer is one or more of a phosphate buffer having a mass fraction of 10% to 20% or a Hepsin buffer having a mass fraction of 10% to 20%.

6. The method according to claim 1, characterized in that The concentration of the raw material mesenchymal stem cells in the mixed solution is 10 5 to 10 6 cells / mL.

7. The method according to claim 1, characterized in that The raw material allogeneic mesenchymal stem cells in the mixed solution are separated and washed with a buffer solution at least twice until the concentration of the statin in the solution is less than 10 ppm.

8. A highly efficient allogeneic mesenchymal stem cell, characterized in that: It is prepared by the method according to any one of claims 1 to 7.

9. Use of the highly efficient allogeneic mesenchymal stem cells obtained by the preparation method according to any one of claims 1 to 7 or the highly efficient allogeneic mesenchymal stem cells according to claim 8 in the preparation of a drug for myocardial repair after acute myocardial infarction.

10. The use according to claim 9, characterized in that: In the drug, the concentration of the highly efficient allogeneic mesenchymal stem cells is 10 7 to 10 8 cells / mL.

Images