CN110669727A - Preparation method and application of mesenchymal stem cell membrane - Google Patents

Preparation method and application of mesenchymal stem cell membrane Download PDF

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CN110669727A
CN110669727A CN201911080070.1A CN201911080070A CN110669727A CN 110669727 A CN110669727 A CN 110669727A CN 201911080070 A CN201911080070 A CN 201911080070A CN 110669727 A CN110669727 A CN 110669727A
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mesenchymal stem
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包凤鸣
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Shenzhen Kekanggan Cell Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • 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
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

Abstract

The invention discloses a preparation method of a mesenchymal stem cell membrane and application of the mesenchymal stem cell membrane in treating myocardial ischemia-reperfusion injury. The method for extracting the expanded cells by using density gradient centrifugation, cell adherence and cell cryopreservation can obtain enough undifferentiated single MSCs, has strong multiplication capacity and high purity, can be used as seed cells of tissue engineering, and provides a basis for the construction of tissue engineering blood vessels. Compared with the Reyes method, the method eliminates the step of immunomagnetic bead selection, reduces the expensive cost in the practical application, and reduces the pollution chance in the operation.

Description

Preparation method and application of mesenchymal stem cell membrane
Technical Field
The invention relates to the technical field of bioengineering, in particular to a preparation method and application of a mesenchymal stem cell membrane.
Background
According to the statistics of the cardiovascular disease report in 2017, the morbidity and mortality of the cardiovascular disease are still increased, which are higher than those of tumors and other diseases, and are the top. The myocardial ischemia reperfusion injury is myocardial ischemia (such as cardiac surgery, cardiac transplantation, myocardial infarction and the like) caused by various reasons, oxygen imbalance is caused by no blood flow to cardiac tissue, cardiac dysfunction or myocardial tissue damage is generated, blood perfusion of the myocardial ischemia part is effectively recovered in time, the ischemic myocardial damage and necrosis can be reduced, and the phenomenon of further tissue damage is caused. The occurrence mechanism of myocardial ischemia reperfusion injury comprises oxygen free radical outbreak, rapid recovery of physiological pH value, insulin resistance, cell calcium overload, mitochondrial injury, inflammatory reaction and the like; at present, no effective method is available for treating myocardial ischemia-reperfusion injury, some medicines and cell therapy are used as research targets, common intervention medicines comprise sufentanil, atorvastatin and the like, and the medicine treatment of myocardial ischemia-reperfusion injury has the defects that the time for achieving the treatment effect is long, and most medicines are time-dependent.
In recent years, with the rapid development of stem cell therapy techniques, some researchers have also applied mesenchymal stem cells to the treatment of myocardial ischemia-reperfusion injury. The bone marrow mesenchymal stem cells show strong myocardial tissue regeneration potential, and the bone marrow mesenchymal stem cells are transplanted after myocardial infarction for treatment, so that the myocardial infarction area can be obviously reduced, the scar formation is reduced, the number of new vessels is increased, and the obvious curative effect is shown.
The bone marrow mesenchymal stem cell is derived from the bone marrow stroma of mammals, except hematopoietic stem cells, and has the potential of self-replication and multi-differentiation in vitro. Under standard culture conditions, the cell surface markers CD105, CD73 and CD90 can be expressed, but the cell surface markers CD45, CD34, CD14 or CD11b, CD79, CD19 or HLA-DR are not expressed, and the stem cell biological characteristics of the stem cell include self-renewal, multi-directional differentiation potential, and in vitro large-scale expansion. The transplantation of the mesenchymal stem cells after myocardial infarction can increase the number of new blood vessels, reduce the infarction area and reduce scars. The bone marrow mesenchymal stem cells are induced and differentiated into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, neurons, hepatocytes and the like under certain conditions. Direct evidence of Liuyuhao and the like indicates that the human bone marrow mesenchymal stem cells can differentiate into myocardial cells and vascular endothelial cells. Research shows that the mesenchymal stem cells have low immunogenicity. The bone marrow mesenchymal stem cells and extracellular matrix molecules expressed by differentiated cells thereof, such as adhesion molecules and integrins, participate in forming a microenvironment supporting hematopoiesis and help the adhesion and homing of hematopoietic stem cells and the mutual adhesion among cells. Cytokines secreted by mesenchymal stem cells play an important regulatory role in the self-renewal and differentiation of hematopoietic stem/progenitor cells.
Disclosure of Invention
The invention mainly aims to provide a preparation method and application of a mesenchymal stem cell membrane, aiming at improving the viability and treatment effect of mesenchymal stem cells.
In order to achieve the above objects, a first object of the present invention is to provide a method for preparing a mesenchymal stem cell membrane, comprising the steps of:
s1: sucking 10ml of bone marrow liquid by using a 20ml syringe, and slowly injecting the cell suspension into a test tube which is preset with human lymphocyte separation liquid with the equal volume density of 1.077kg/L to form a clear interface between the two;
s2: centrifuging at 2500r/min for 20min, sucking the middle milky cloudy mononuclear cell layer, rinsing with PBS, centrifuging at 1500r/min for 10min, discarding supernatant, and adding 10ml of culture solution containing 15% FBSDMEM-LG to obtain single cell suspension;
s3: inoculating the cells into a 50ml culture dish at the density of 3 multiplied by 105/cm2, culturing the cells under the conditions of 37 ℃ and 5% CO2 saturated humidity, changing the liquid for the first half after 4 days, discarding all cells which are not attached to the wall, and changing the liquid for the full amount every 2-3 days later;
s4: after the cells are confluent into a monolayer, digesting with 0.25% trypsin (containing 0.02% EDTA) to obtain primary MSCs suspension, and adding 1 × 104Density of/cm 2 in a ratio of 1: 2 subculturing to form a cell patch;
s5: observing cell growth by an inverted microscope;
s6: drawing a cell growth curve;
s7: the anchorage rate is determined and an anchorage rate curve is plotted.
Preferably, in the S1, the syringe contains 2500U/ml of diluted heparin sodium about 0.5 ml.
Preferably, in S1, the method for extracting bone marrow fluid comprises selecting a posterior superior iliac spine puncture point, extracting a core after a successful puncture of a bone marrow puncture needle after local anesthesia under aseptic conditions, and puncturing and extracting 10ml of bone marrow with a 10ml syringe containing 5ml of saline and heparin (100U/ml).
Preferably, in said S2, the centrifuge for centrifugation is a table-top high-speed centrifuge.
Preferably, in said S5, the growth and morphological change characteristics of the cells are observed day by day using an inverted phase contrast microscope and photographed.
Preferably, in S6, the 3 rd to 5 th generation cells are selected at 5X 10 th per well4One of the cells was inoculated into a 24-well plate, cultured under the above conditions, and the number of cells in each well was counted by a hemocytometer counting method in 3 wells per day for 7 days continuously.
Preferably, in said S7, the 3 rd generation cells are taken at 5X 10 cells per bottle4Inoculating the cells into a 50ml culture bottle for culture, taking 2 bottles every 2h, pouring out the culture solution (containing non-adherent cells), digesting the adherent cells by 0.25% trypsin, counting, and calculating the adherence rate.
The second purpose of the invention is to provide the application of the mesenchymal stem cell membrane in treating myocardial ischemia-reperfusion injury.
The invention has the beneficial effects that:
the method for extracting the expanded cells by using density gradient centrifugation, cell adherence and cell cryopreservation can obtain enough undifferentiated single MSCs, has strong multiplication capacity and high purity, can be used as seed cells of tissue engineering, and provides a basis for the construction of tissue engineering blood vessels. Compared with the Reyes method, the method eliminates the step of immunomagnetic bead selection, reduces the expensive cost in the practical application, and reduces the pollution chance in the operation.
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FIG. 1: MSCS primary culture day 7 (inverted phase contrast under microscope × 100);
FIG. 2: 3 rd generation MSCs culture (inverted phase contrast microscope × 100);
FIG. 3: cell growth profile;
FIG. 4: a graph of cell anchorage rate;
FIG. 5: the bone marrow mesenchymal stem cells have the effect target point and the effect mechanism chart for treating the myocardial ischemia reperfusion injury.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
In the embodiment of the invention, the preparation method of the mesenchymal stem cell membrane comprises the following steps:
in-vitro isolation and culture of the mesenchymal stem cells:
s1: 10ml of bone marrow fluid was aspirated using a 20ml syringe containing 2500U/ml of diluted heparin sodium about 0.5 ml. Slowly injecting the cell suspension into a test tube which is preset with human lymphocyte separation liquid with the equal volume density of 1.077kg/L to ensure that a clear interface is formed between the two.
After separation with 1.077kg/L of human lymphocyte separation medium, the intratubular fluid was seen to have distinct 4 layers. 1/2 at the uppermost layer of the liquid column, which is a red culture liquid layer; the 3 rd layer occupies about 1/2 of the liquid column and is a milky Percol1 separation layer; the boundary of the two layers is an off-white cloud layer with the height of about 5.0mm, which is a mononuclear cell layer (the layer is the required cell layer); the bottom layer is closely attached to the wall of the test tube, and is a thin red cell layer with high density of red blood cells and the like.
In the step S1, the method for sucking the bone marrow fluid comprises selecting a posterior superior iliac spine puncture point, pulling out the needle core after the successful puncture of the bone marrow puncture needle after local anesthesia under aseptic conditions, and puncturing and extracting 10ml of bone marrow by a 10ml syringe containing 5ml of physiological saline and heparin (100U/ml).
S2: centrifuging at 2500r/min for 20min by using a desktop high-speed centrifuge, sucking the middle milky cloudy mononuclear cell layer, rinsing with PBS, centrifuging at 1500r/min for 10min, discarding the supernatant, and adding 10ml of culture solution containing 15% FBSDMEM-LG to prepare single cell suspension;
s3: inoculating the cells into a 50ml culture dish at the density of 3 multiplied by 105/cm2, culturing the cells under the conditions of 37 ℃ and 5% CO2 saturated humidity, changing the liquid for the first half after 4 days, discarding all cells which are not attached to the wall, and changing the liquid for the full amount every 2-3 days later;
s4: after the cells are confluent into a monolayer, digesting with 0.25% trypsin (containing 0.02% EDTA) to obtain primary MSCs suspension, and adding 1 × 104Density of/cm 2 in a ratio of 1: 2 subculture to form cell patches.
After primary MSCs are inoculated, the primary MSCs are scattered at the bottom of a culture flask and are distributed in a circular shape, the cell body is transparent, and the refractivity is good. Partial cells adhere to the wall after 24-48 h, the cell shape gradually changes into a short fusiform or triangular shape, a few cells are polygonal, and cell protrusions with different lengths exist. The nucleus is large and oblate. Half the liquid change for the first time in 72h, the adherent cells begin to divide and proliferate, and cell clusters are formed in partial areas. After 7d, the cells formed dispersed fibroblast colonies, and the cells in the colonies expanded radially around. 14d cell colonies fused to each other to form a monolayer that grew to the bottom of the flask. And (3) allowing the MSCs after passage to adhere to the wall rapidly within 2-4 h, allowing most cells to adhere to the wall within 10h, allowing all cells to adhere to the wall within 12h, and starting division and proliferation. The cells are uniformly distributed, do not form colony-like growth any more, are in a shape of slender spindle and are arranged along a certain direction. And (5) paving the bottom of the bottle with the cells for 3-5 days, and then digesting and passaging. At the beginning, the cells are mostly in a slender fusiform I type, and when the cells are transmitted to the 7 th and 8 th generations, the cells are spread widely, flatly and thinly, so that the cells are in a type II type, the proliferation speed is slow, the cell passage cycle is prolonged, and the particulate matters in the cells are increased (fig. 1 and 2).
S5: cell growth was observed by inverted microscope.
The growth condition and morphological change characteristics of the cells are observed day by using an inverted phase contrast microscope, and the images are photographed.
S6: and (6) drawing a cell growth curve.
Taking 3 rd-5 th generation cells, inoculating 5 × 104 cells per well in a 24-well culture plate, culturing under the above conditions, taking 3 wells per day, continuously taking 7 days, and counting the number of cells per well by adopting a blood counting plate counting method. Calculating the formula: cell count/ml stock solution (sum of 4 large cells/4) × 104。
In the experiment, the passage cells grow faster than the primary cells, and the whole cell growth curve is S-shaped. The cell growth curves after passaging were essentially identical. When 1 bottle is transferred to 3 bottles, the cells can be transferred for 1 generation in 4-5 days, and the growth curves of the cells of each generation are basically the same. The growth curve is S-shaped, the cell growth number does not change greatly on days 1 and 2 of subculture, the cells increase greatly from day 3d, and the cells decrease slightly after reaching the top from days 5 to 6d (FIG. 3).
S7: the anchorage rate is determined and an anchorage rate curve is plotted.
The 3 rd generation cells were sampled at 5X 10 cells per vial4Inoculating the cells into a 50ml culture bottle for culture, taking 2 bottles every 2h, pouring out the culture solution (containing non-adherent cells), digesting the adherent cells by 0.25% trypsin, counting, and calculating the adherence rate. Calculating the formula: the anchorage rate (%) -%, number of adherent viable cells/number of seeded cells × 100%.
The anchorage rate of each generation of cells is not obviously different, the cells are attached to the wall faster after digestion and passage, the anchorage rate of the cells exceeds 50% in 4h, and more than 90% of the cells are attached to the wall in 10h (figure 4).
The second purpose of the invention is to provide the application of the mesenchymal stem cell membrane in treating myocardial ischemia-reperfusion injury.
The invention detects cultured cells through a flow cytometer after multiple passages, can express cell surface markers CD105, CD73 and CD90 under standard culture conditions, but does not express cell surface markers CD45, CD34, CD14 or CD11b, CD79, CD19 or HLA-DR, and has stem cell biological characteristics of self-renewal, multidirectional differentiation potential, in-vitro large-scale expansion and the like. The transplantation of the mesenchymal stem cells after myocardial infarction can increase the number of new blood vessels, reduce the infarction area and reduce scars. The bone marrow mesenchymal stem cells are induced and differentiated into osteoblasts, chondrocytes, adipocytes, cardiomyocytes, neurons, hepatocytes and the like under certain conditions. Research shows that the mesenchymal stem cells have low immunogenicity. The bone marrow mesenchymal stem cells and extracellular matrix molecules expressed by differentiated cells thereof, such as adhesion molecules and integrins, participate in forming a microenvironment supporting hematopoiesis and help the adhesion and homing of hematopoietic stem cells and the mutual adhesion among cells. Cytokines secreted by mesenchymal stem cells play an important regulatory role in the self-renewal and differentiation of hematopoietic stem/progenitor cells.
Homing and field planting characteristics:
hematopoietic stem cells are considered to have the ability to migrate from the blood to different organs and return to the bone marrow stroma under the navigation of chemical signals. Bone marrow mesenchymal stem cells are believed to have similar characteristics that facilitate their migration and transplantation into ischemic myocardium and mediate repair. Experimental research results show that the homing capability of the mesenchymal stem cells is seriously damaged after in vitro amplification. Transplanting the mesenchymal stem cells which are not subjected to in vitro culture and are genetically marked by the green fluorescent protein into a sublethal radiation mouse, wherein 55-65% of the cells marked by the green fluorescent protein survive in a bone marrow stroma, 4-7% of the cells survive in a spleen, and the survival rate of the mesenchymal stem cells marked by the green fluorescent protein after 24 hours of in vitro culture is reduced to 10%. Bone marrow mesenchymal stem cells in late passage lose the surface expression of chemokine receptors CCR1, CCR9, CXCR6, CXCR5 and CXCR4, and also lose the corresponding chemotactic response. Therefore, there is a need to optimize the culture conditions of mesenchymal stem cells to maintain homing expression of chemokine receptors. The bone marrow mesenchymal stem cells can be expanded and differentiated into osteoblasts, chondrocytes, cardiomyocytes, adipocytes and the like under certain induction conditions. For example, bone marrow mesenchymal stem cells are differentiated into cardiomyocyte-like cells under the induction of 5-azacytidine. The differentiation capacity of the mesenchymal stem cells under different culture conditions is different. Bone marrow mesenchymal stem cells are transplanted at the position of myocardial injury through homing and directionally differentiated into myocardial cells, thereby achieving the effect of treating myocardial ischemia reperfusion injury.
Further, mesenchymal stem cells rely on their paracrine action for their effects of improving organ function. Research shows that bone marrow mesenchymal stem cell transplantation utilizes paracrine effect to improve cardiac function after myocardial infarction. In addition, paracrine effects may explain many repair-related mechanisms of action, including promoting neovascularization, reducing infarct size and scarring, improving myocardial contractile function, and the like. There is evidence to suggest that bone marrow mesenchymal stem cells improve cardiac function by paracrine related factors, see table 1, rather than by direct differentiation into cardiomyocytes. The bone marrow mesenchymal stem cell exosomes also carry miRNA genes, and participate in the regulation of anti-apoptosis and angiogenesis proteins after transcription, as shown in table 2. In this context, exosomes have gained widespread interest as a vector for transporting cardioprotective molecules. Exosomes secreted by bone marrow mesenchymal stem cells can protect the heart and brain through anti-apoptotic, anti-inflammatory, pro-angiogenic and immunomodulatory effects. The mesenchymal stem cells probably secrete FSTL 1(FSTL1) to reduce the myocardial infarction area of rats, thereby playing a role in myocardial protection. Therefore, the paracrine effect of mesenchymal stem cells is another important mechanism for treating myocardial ischemia-reperfusion injury.
TABLE 1 bone marrow mesenchymal stem cell paracrine action and related factors
TABLE 2 bone marrow mesenchymal stem cell exosomes miRNA
Figure BDA0002263676980000092
The bone marrow mesenchymal stem cells can form a specific microenvironment by secreting various soluble factors, cell surface molecules and extracellular matrixes to play a therapeutic role. The research shows that the mesenchymal stem cells of the bone marrow exert the biological effect by producing a plurality of cytokines, such as vascular endothelial growth factor, stem cell factor, hepatocyte growth factor, tumor necrosis factor alpha, insulin-like growth factor 1 and the like. The indirect anti-apoptosis, anti-inflammatory and anti-fibrosis effects of the cytokines promote the endogenous repair of tissues. The anti-inflammatory and anti-apoptosis characteristics of the bone marrow mesenchymal stem cells are the basis for treating myocardial ischemia reperfusion injury, and can reduce inflammation and myocardial cell apoptosis and relieve myocardial injury.
The low immunogenicity of mesenchymal stem cells is proved by studying the interaction between mesenchymal stem cells and various immune cells and the interaction between mesenchymal stem cells and allogeneic T cells, wherein the mesenchymal stem cells have a lower stimulation threshold for T cell response and cannot induce the activation of allogeneic T cells, 2 signaling pathways are required for the sufficient activation of T cells, ① T receptor has a lower stimulation threshold for Major Histocompatibility Complex (MHC) molecules and antigen presenting cell surface antigens, ② T cell activation requires a co-stimulation signal including CD28 activated in T cells and CD80 or CD86 in antigen presenting cells, the surface of mesenchymal stem cells can express low-level MHC class I molecules, but lack MHC class II molecules and co-stimulatory molecules CD80 or CD86, the expression of CD40 is beneficial to the formation of mesenchymal stem cells with low immunogenicity, although the MHC class I molecules and the co-stimulatory molecules can express low-immune response, the mesenchymal stem cells can stimulate the MHC class II molecules, and the co-stimulatory molecules CD80 or CD86, the expression of the CD40 can reduce the expression of the MHC class I molecules, and the immune cells can provide a low-immune response to the immune cells, the ischemia of the marrow mesenchymal stem cells, the immune cells can reduce the immune response of the immune cells, and the immune system can reduce the immune response of the immune cells.
The bone marrow mesenchymal stem cells have the action target and action mechanism for treating myocardial ischemia reperfusion injury:
this example combines the results of the lab and the current study to summarize the potential mechanism of action of mesenchymal stem cells in treating myocardial ischemia reperfusion injury, as shown in fig. 5.
Role of mitochondrial fusion protein 2:
it was found that mitochondrial fusion protein 2 (Mfn 2) is a marker protein that can reflect myocardial injury. The mitochondria of the myocardial cells show obvious fragmentation in myocardial ischemia, and the process mainly depends on the regulation of Mfn2 by a mitochondrial membrane fusion protein. Thus, regulation of mitochondrial membrane fusion proteins is essential for maintaining normal physiological activities and metabolism of cardiomyocytes, as the lack of Mfn2 results in insufficient mitochondrial membrane fusion, causing cardiomyocytes to be more susceptible to various death stimuli. During the process of in vitro circulating myocardial reperfusion, the change of Mfn2 protein expression can reflect the change of central function during the process of in vitro circulating myocardial reperfusion injury. The bone marrow mesenchymal stem cells achieve the treatment of myocardial ischemia reperfusion injury through the regulation of Mfn 2.
The LC3 family protein is involved in myocardial autophagy regulation:
bone marrow mesenchymal stem cells may be involved in the regulation of autophagy and apoptosis. Bone marrow mesenchymal stem cells have been shown to be an ideal source of stem cells for myocardial infarction cell therapy. However, the viability of donor stem cells after transplantation is low, limiting their therapeutic efficiency, and the underlying mechanisms are still poorly understood. Autophagy and apoptosis processes also exist in myocardial ischemia reperfusion injury, and the autophagy is inhibited in addition to the anti-apoptosis effect on myocardial cells; the results of the study show that autophagy is not equally effective in different phases, beneficial during ischemia, but opposite during reperfusion. Autophagy also plays an important role in myocardial ischemia-reperfusion injury, and studies have shown that LC3 family protein is involved in myocardial autophagy. The fat-derived mesenchymal stem cell exosome injected into the body of the rat with the myocardial ischemia reperfusion injury can obviously reduce the apoptosis of the myocardial cells and the myocardial infarction area, and improve the cardiac function by the expression of the upper self-aligning muscle LC 3B. The expressions of LC 3-I and LC 3-II in the non-reflow area of myocardial infarction are increased, which shows that the local autophagy expression of myocardial tissue in the non-reflow area of myocardial infarction is enhanced.
AMPK/mTOR signaling pathway regulation:
in the myocardial ischemia and hypoxia stage, the bone marrow mesenchymal stem cells participate in the AMPK/mTOR signal pathway regulation. Researches show that hypoxia increases the activity of AMPK/mTOR signaling pathway, and bone marrow mesenchymal stem cells have potential effect in inhibiting myocardial apoptosis by inducing myocardial autophagy under hypoxia stress. Upon myocardial ischemia and hypoxia, intracellular ATP levels are significantly decreased, and thus AMP/ATP is increased, whereas AMPK, a receptor, receives a signal to be activated, which inhibits the activity of mTOR by phosphorylating nodule-associated protein 2(TSC 2). This process primarily inhibits protein synthesis and induces autophagy to help the myocardium adapt to hypoxic environments. A transgenic mouse myocardial ischemia-reperfusion model was constructed using AMPK inhibitor on cardiomyocytes, and a decrease in the ratio LC 3-II/LC 3-I in cardiomyocytes was observed, and autophagy was inhibited. In myocardial ischemia, the myocardial cells mainly activate AMPK to inhibit an mTOR pathway, induce autophagy, inhibit apoptosis and play a protective role in ischemic myocardial cells.
The AMPK/mTOR signal pathway regulation effect is probably the action mechanism of bone marrow mesenchymal stem cells in treating myocardial ischemia reperfusion injury.
The Bcl-2 protein and Beclin1 protein regulate:
in the myocardial reperfusion stage, the bone marrow mesenchymal stem cells are probably involved in the regulation of the expression of Bcl-2 protein and Beclin1 protein. It was found that, upon ischemia-hypoxia of cardiomyocytes, Bcl-2 was phosphorylated, but was unable to bind to Beclin1 and induce autophagy. During the reperfusion phase, autophagy is dependent on increased Beclin1 expression, which is detrimental to cell survival and aggravates cardiomyocyte injury. The interaction of Beclin1 and Bcl-2 is the major mechanism of autophagy. Research evidence suggests that knockout of the mouse Beclin1 gene decreased both autophagy and apoptosis in the myocardial ischemia-reperfusion model, and thus decreased myocardial infarction area. Increased expression of Beclin1 during the myocardial reperfusion phase can induce autophagy. The expression of Beclin1 was inhibited, as was autophagy by cardiomyocytes. The interaction between Beclin1 and Bcl-2 is one of the important mechanisms of action during the reperfusion phase.
In summary, the mechanism of occurrence of myocardial ischemia-reperfusion injury has many effects, and after occurrence of myocardial ischemia-reperfusion injury, irreversible necrosis and death of myocardial cells occur, and despite the progress in optimal drug therapy and interventional therapy, the prognosis of patients with myocardial ischemia-reperfusion injury and the resulting chronic heart failure are still very serious. Animal experiments and clinical trials using treatment with mesenchymal stem cells showed a general improvement in myocardial function after myocardial ischemia reperfusion injury occurred. The bone marrow mesenchymal stem cells have the potential of multi-system, self-renewal and proliferation, and are expected to repair the heart after myocardial ischemia reperfusion injury. In addition, the mesenchymal stem cells of the bone marrow are easy to separate and amplify, and have immune function to the host. The bone marrow mesenchymal stem cells can be differentiated into myocardial cells and vascular cells, can secrete a large amount of growth factors and cytokines, mediate endogenous regeneration by activating resident cardiac stem cells and other stem cells, and induce neovascularization, inflammation resistance, apoptosis resistance, reconstruction resistance and the like in a paracrine mode. At present, Mfn2, LC3 family, AMPK-mTOR signal path, Bcl-2 and Beclin1 related action mechanisms and the like are possibly involved in the treatment of the mesenchymal stem cells of the bone marrow. The bone marrow mesenchymal stem cells and the exosomes secreted by the bone marrow mesenchymal stem cells form a clinically feasible treatment scheme, so that the infarction area is reduced, the scar formation is reduced, the neovascularization is promoted, and the cardiac function is improved.
The clinical application of the mesenchymal stem cells also has the limitationThe survival rate of transplanted cells can be improved by the methods of pretreatment before stem cell transplantation, stem cell transplantation treatment and the like, firstly, the pretreatment of stem cells is to maintain a standby state so that the stem cells can resist the severe environment and the massive apoptosis of the transplanted cells can be prevented, secondly, the combined application of the stem cells and extracellular matrix molecules, nano fibers, hydrogel or fibrin glue and the combined treatment using two stem cells can improve the survival rate of the transplanted cells, and at present, the transplantation approach of the mesenchymal stem cells mainly comprises 3 methods, namely, the transplantation of the mesenchymal stem cells under the condition of myocardium is performed by injection (①, extracardiac injection, ②, peripheral arterial injection, and ③, the study of the treatment of the mesenchymal stem cells under the condition of heart is performed by intravenous injection, and the study of the coronary arterial injection is performed by coronary artery injection, and the study of the coronary artery injection shows that the treatment effect of the mesenchymal stem cells under the condition of myocardium is performed by injection, the intracardiac injection is performed by the intracardiac injection, and the coronary arterial injection is performed by the coronary artery injection, and the study of the coronary artery injection is performed by the coronary artery injection of the coronary stem cells under the heart is performed by the heart, and the coronary artery injection is performed by the coronary artery injection7And 1X 109The marrow mesenchymal stem cells of the dosage are followed for 12 months by intracardiac injection, the curative effect is observed and the safety is evaluated, and the result shows that the two cell dosages can reduce the scar of the patient, but only 1 multiplied by 109The ejection fraction of patients in the dose-treated group was significantly increased.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (8)

1. The preparation method of the mesenchymal stem cell membrane is characterized by comprising the following steps:
s1: sucking 10ml of bone marrow liquid by using a 20ml syringe, and slowly injecting the cell suspension into a test tube which is preset with human lymphocyte separation liquid with the equal volume density of 1.077kg/L to form a clear interface between the two;
s2: centrifuging at 2500r/min for 20min, sucking the middle milky cloudy mononuclear cell layer, rinsing with PBS, centrifuging at 1500r/min for 10min, discarding supernatant, and adding 10ml of culture solution containing 15% FBSDMEM-LG to obtain single cell suspension;
s3: inoculating the cells into a 50ml culture dish at the density of 3 multiplied by 105/cm2, culturing the cells under the conditions of 37 ℃ and 5% CO2 saturated humidity, changing the liquid for the first half after 4 days, discarding all cells which are not attached to the wall, and changing the liquid for the full amount every 2-3 days later;
s4: after the cells are confluent into a monolayer, digesting with 0.25% trypsin (containing 0.02% EDTA) to obtain primary MSCs suspension, and adding 1 × 104Density of/cm 2 in a ratio of 1: 2 subculturing to form a cell patch;
s5: observing cell growth by an inverted microscope;
s6: drawing a cell growth curve;
s7: the anchorage rate is determined and an anchorage rate curve is plotted.
2. The method for preparing a mesenchymal stem cell membrane sheet according to claim 1, wherein in the S1, the syringe contains about 0.5ml of diluted heparin sodium 2500U/ml.
3. The method for preparing a mesenchymal stem cell membrane of claim 1, wherein in step S1, the bone marrow fluid is extracted by selecting a posterior superior iliac spine puncture site, pulling out the core after the successful puncture of the bone marrow puncture needle after local anesthesia under aseptic conditions, and puncturing 10ml bone marrow with a 10ml syringe containing 5ml physiological saline and heparin (100U/ml).
4. The method for preparing a mesenchymal stem cell sheet according to claim 1, wherein in S2, the centrifuge for centrifugation is a table-top high-speed centrifuge.
5. The method for preparing a mesenchymal stem cell membrane of claim 1, wherein in S5, the growth and morphological change characteristics of the cells are observed day by using an inverted phase contrast microscope and photographed.
6. The method for preparing a mesenchymal stem cell sheet according to claim 1, wherein the 3 rd to 5 th generation of cells are selected at 5X 10 cells per well in S64One of the cells was inoculated into a 24-well plate, cultured under the above conditions, and the number of cells in each well was counted by a hemocytometer counting method in 3 wells per day for 7 days continuously.
7. The method of claim 1, wherein the 3 rd generation of cells is selected at 5 x 10 cells per bottle in S74Inoculating the cells into a 50ml culture bottle for culture, taking 2 bottles every 2h, pouring out the culture solution (containing non-adherent cells), digesting the adherent cells by 0.25% trypsin, counting, and calculating the adherence rate.
8. The use of a mesenchymal stem cell sheet according to claim 1 for the treatment of myocardial ischemia reperfusion injury.
CN201911080070.1A 2019-11-07 2019-11-07 Preparation method and application of mesenchymal stem cell membrane Pending CN110669727A (en)

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