阅读说明：本技术 一种使骨髓间充质干细胞靶向于心肌梗死灶的试剂及制备方法 (Reagent for targeting bone marrow mesenchymal stem cells to myocardial infarction focus and preparation method ) 是由 孙铃 王庆捷 纪元 于 2020-11-18 设计创作，主要内容包括：本发明提供一种使骨髓间充质干细胞靶向于心肌梗死灶的试剂,所述使骨髓间充质干细胞靶向于心肌梗死灶的培养基,由溶质和溶媒组成,所述溶质包括100％血浆、低分子量肝素钠400-700U/mL、磷酸氢二钾溶液40-70mmol/L、腺苷0.4-10μmol/L、人参皂苷Rb1为0.5-10mg/mL、葡萄糖4.5g/L-5.5g/L、碳酸钠0.15g/L-0.25g/L、L-谷氨酰胺0.590g/L-0.710g/L、转铁蛋白50g/L-65g/L、胰岛素5g/L-7g/L、碱性成纤维细胞生长因子0.01g/L-0.05g/L,所述溶媒为复方电解质注射液、葡萄糖注射液或生理盐水,可使通过静脉注射的骨髓间充质干细胞靶向于z心肌梗死灶,解决了目前通过冠状动脉内注射所留下的细胞量较少的问题。(The invention provides a reagent for targeting mesenchymal stem cells to a myocardial infarction focus, which consists of a solute and a solvent, wherein the solute comprises 100 percent of plasma, 400U/mL of low molecular weight heparin sodium, 40-70mmol/L of dipotassium hydrogen phosphate solution, 0.4-10 mu mol/L of adenosine, 0.5-10mg/mL of ginsenoside Rb1, 4.5-5.5 g/L of glucose, 0.15g/L-0.25g/L, L-glutamine, 0.590g/L-0.710g/L of sodium carbonate, 50g/L-65g/L of transferrin, 5g/L-7g/L of insulin, 0.01g/L-0.05g/L of basic fibroblast growth factor, the solvent is compound electrolyte injection, glucose injection or normal saline, can target the mesenchymal stem cells injected by vein to the z myocardial infarction focus, and solves the problem that the amount of the cells left by the current intracoronary injection is less.)

1. An agent for targeting a mesenchymal stem cell to a myocardial infarction focus, which consists of a culture medium for targeting the mesenchymal stem cell to the myocardial infarction focus and an isolated mesenchymal stem cell, and is characterized in that: the culture medium for targeting the mesenchymal stem cells to the myocardial infarction focus consists of solute and solvent;

the solute comprises 100% blood plasma, 700U/mL of low molecular weight heparin sodium, 40-70mmol/L of dipotassium phosphate solution, 0.4-10 mu mol/L of adenosine, 0.5-10mg/mL of ginsenoside Rb1, 4.5-5.5 g/L of glucose, 0.15-0.25 g/L, L-glutamine, 0.590-0.710 g/L of sodium carbonate, 50-65 g/L of transferrin, 5-7 g/L of insulin and 0.01-0.05 g/L of alkaline fibroblast growth factor, and the solvent is compound electrolyte injection, glucose injection or physiological saline.

2. The agent for targeting bone marrow mesenchymal stem cells to a myocardial infarction focus according to claim 1, wherein: the ratio of the culture medium to the in vitro bone marrow mesenchymal stem cells is 1.0 multiplied by 10 per milliliter of the culture medium⁶-4×10⁶And the isolated bone marrow mesenchymal stem cells.

3. The agent for targeting bone marrow mesenchymal stem cells to a myocardial infarction focus according to claim 1, wherein: the in vitro human mesenchymal stem cells are in vitro human allogeneic mesenchymal stem cells.

4. A preparation method of a reagent for targeting bone mesenchymal stem cells to a myocardial infarction focus is characterized by comprising the following steps: the method comprises the following steps:

step (1): preparing a complete culture solution, wherein the complete culture solution is L-DMEM, FBS with the mass concentration of 20%, penicillin 100U/ml and streptomycin 100 mu g/ml;

step (2): taking bone marrow, placing in a sterile heparin tube, adding 100U heparin per ml bone marrow for anticoagulation, mixing well, and usingPBS liquid regulates the concentration of bone marrow cells to 10⁶-10⁷Adding Ficoll separating medium and bone marrow cell suspension at volume ratio of 1:1 into the mixture without damaging interface, centrifuging at 20 deg.C for 25-30 min with 500g horizontal centrifuge, naturally settling, taking out mononuclear cells from interface, and washing with PBS for 2-3 times;

and (3): adding the cells obtained in the step (2) into the complete culture solution prepared in the step (1) for cell counting until 10⁶-10⁷After the primary culture, the cells were cultured at (1-5). times.10⁶cells/ml are inoculated into T25 or T75 cell culture bottles, the volume of complete culture solution in each cell culture bottle is 5-25ml, and the cells are placed in CO₂Culturing at 37 deg.C culture box with volume ratio concentration of 5%, changing liquid after 3d to remove non-adherent cells, changing liquid for 1 time every 3-5d half, digesting with pancreatin with mass concentration of 0.25% at 37 deg.C when cells reach 80% fusion, and culturing at 1 × 10⁴Passage amplification is carried out on cells/ml, and mesenchymal stem cells of P2-P5 generation are taken;

and (4): placing the P2-P5 generation bone marrow mesenchymal stem cells obtained in the step (3) into O₂Culturing the cells in the complete culture solution prepared in the step (1) in a 37 ℃ culture box with the volume ratio concentration of 21% until the cells are fused to 60-70%, changing the serum-free starvation culture solution into the complete culture solution prepared in the step (1) containing 10ng/ml of epidermal growth factor, and culturing for 12-15h until the cells are in a logarithmic growth phase;

and (5): and (3) uniformly mixing the mesenchymal stem cells obtained in the step (4), blood plasma, low molecular weight heparin sodium, dipotassium hydrogen phosphate solution, adenosine, ginsenoside Rb1, glucose, sodium carbonate, L-glutamine, transferrin, insulin, basic fibroblast growth factor and a solvent to obtain the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus.

Technical Field

The invention discloses a reagent for targeting bone marrow mesenchymal stem cells to a myocardial infarction focus and a preparation method thereof, belonging to the technical field of stem cells.

Background

The bone marrow mesenchymal stem cells are another type of stem cells in the bone marrow except the hematopoietic stem cells, are important components of the bone marrow hematopoietic microenvironment, and have the functions of supporting and regulating hematopoiesis in vivo and in vitro. It is also called a fibroblast colony-forming unit because of its relative ease of attachment and formation of fiber-like clones. Under certain induction conditions, BMSCs have the capacity to differentiate into mesodermal cells such as osteoblasts, adipocytes, skeletal muscle, chondrocyte smooth muscle, tenocytes, hematopoietic support matrix and the like; and can be differentiated to astrocytes, neurons, vascular endothelial cells and cardiac muscle cells of the ectoderm. The plasticity of BMSCs to differentiate transversely across lineages, even across germ layers, and its strong differentiation potential make it possible to provide tissue repair. BMSCs have the advantages of multidirectional differentiation potential and high proliferation property, easily obtained materials, small damage to organisms, easy gene operation, good histocompatibility, weak immune rejection reaction of autograft or allograft and the like, can secrete various growth factors such as IL-6, IL-11, LIF, M-CSF, SCF and the like, is considered as an ideal target cell for tissue engineering and genetic engineering, and has wide application prospect in clinic.

Based on the above characteristics of mesenchymal stem cells, most of the studies reported so far have been made on the injection of stem cells into coronary arteries and muscles, in order to achieve the purpose of specifically repairing and improving damaged skin and fundamentally retarding the aging of skin cells. While the need for target revascularization in patients treated with cells for acute myocardial infarction with stem cell intracoronary injection has not increased to achieve relevant clinical results in restoration of cardiac function and cardiac remodeling, studies have shown that treatment is safe, while intracoronary injection is, at first glance, one way of cellular application, but radiolabeling bone marrow cells has been shown: compared with intramuscular injection, the amount of cells left by the intracoronary injection is small, but intramuscular injection only can play a role in local areas, and a reagent for targeting the mesenchymal stem cells to a myocardial infarction focus and a preparation method are urgently needed to solve the problems.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a reagent for targeting bone marrow mesenchymal stem cells to a myocardial infarction focus and a preparation method thereof, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention is realized by the following technical scheme: an agent for targeting a mesenchymal stem cell to a myocardial infarction focus, which consists of a culture medium for targeting a mesenchymal stem cell to a myocardial infarction focus and an isolated mesenchymal stem cell, wherein the culture medium for targeting a mesenchymal stem cell to a myocardial infarction focus consists of a solute and a solvent;

further, the solute comprises 100% blood plasma, 700U/mL of low molecular weight heparin sodium, 40-70mmol/L of dipotassium phosphate solution, 0.4-10 mu mol/L of adenosine, 0.5-10mg/mL of ginsenoside Rb1, 4.5g/L-5.5g/L of glucose, 0.15g/L-0.25g/L, L-glutamine, 0.590g/L-0.710g/L of sodium carbonate, 50g/L-65g/L of transferrin, 5g/L-7g/L of insulin and 0.01g/L-0.05g/L of alkaline fibroblast growth factor, and the solvent is compound electrolyte injection, glucose injection or physiological saline.

Further, the ratio of the culture medium to the isolated bone marrow mesenchymal stem cells is 1.0 × 106 to 4 × 106 isolated bone marrow mesenchymal stem cells per milliliter of the culture medium.

Further, the in vitro human mesenchymal stem cells are in vitro human allogeneic mesenchymal stem cells.

Further, a preparation method of the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus comprises the following steps:

step (1): preparing a complete culture solution, wherein the complete culture solution is L-DMEM, FBS with the mass concentration of 20%, penicillin 100U/ml and streptomycin 100 mu g/ml;

step (2): taking bone marrow, placing in a sterile heparin tube, adding 100U heparin per ml bone marrow for anticoagulation, mixing well, and adjusting bone marrow cell concentration to 10 with PBS liquid⁶-10⁷Per ml, added in a volume ratio of 1:1Separating Ficoll solution and bone marrow cell suspension, centrifuging at 20 deg.C for 25-30 min at 500g in horizontal centrifuge without damaging interface, naturally settling, taking out mononuclear cell from interface, and washing with PBS for 2-3 times;

and (3): adding the cells obtained in the step (2) into the complete culture solution prepared in the step (1) for cell counting until 10⁶-10⁷After the primary culture, the cells were cultured at (1-5). times.10⁶cells/ml are inoculated into T25 or T75 cell culture bottles, the volume of complete culture solution in each cell culture bottle is 5-25ml, and the cells are placed in CO₂Culturing in a 37 ℃ incubator with the volume ratio concentration of 5%, changing the culture solution after 3d to remove non-adherent cells, changing the culture solution for 1 time every 3-5d, digesting the cells at 37 ℃ by pancreatin with the mass concentration of 0.25% when the cells reach 80% fusion, carrying out passage expansion by 1 × 104cells/ml, and taking the mesenchymal stem cells of P2-P5 generation;

and (4): placing the P2-P5 generation bone marrow mesenchymal stem cells obtained in the step (3) into O₂Culturing the cells in the complete culture solution prepared in the step (1) in a 37 ℃ culture box with the volume ratio concentration of 21% until the cells are fused to 60-70%, changing the serum-free starvation culture solution into the complete culture solution prepared in the step (1) containing 10ng/ml of epidermal growth factor, and culturing for 12-15h until the cells are in a logarithmic growth phase;

and (5): and (3) uniformly mixing the mesenchymal stem cells obtained in the step (4), blood plasma, low molecular weight heparin sodium, dipotassium hydrogen phosphate solution, adenosine, ginsenoside Rb1, glucose, sodium carbonate, L-glutamine, transferrin, insulin, basic fibroblast growth factor and a solvent to obtain the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus.

The invention has the following beneficial effects:

(1) the invention takes P2-P5 generation bone marrow mesenchymal stem cells with strong reproductive capacity, adopts serum-free culture to lead the cells to be in a hungry state, then adds epidermal cell growth factor for induction, and cultures until the cells are in a logarithmic phase, the obtained bone marrow mesenchymal stem cells have good differentiation capacity and proliferation capacity, have better differentiation potential under specific induction conditions,

(2) the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus can target the mesenchymal stem cells injected by vein to the myocardial infarction focus, solves the problem that the cell amount left by the current intracoronary injection is less, can make the part of the myocardial infarction obtain enough stem cells to be differentiated into new myocardial cells by the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus, and has the advantages of effective skin tissue repairing effect, multidirectional differentiation potential and high proliferation property.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

As an embodiment of the present invention: an agent for targeting a mesenchymal stem cell to a myocardial infarction focus, which consists of a culture medium for targeting a mesenchymal stem cell to a myocardial infarction focus and an isolated mesenchymal stem cell, wherein the culture medium for targeting a mesenchymal stem cell to a myocardial infarction focus consists of a solute and a solvent;

in this embodiment: the solute comprises 100% plasma, 400/mL of low molecular weight heparin sodium, 40mmol/L of dipotassium phosphate solution, 0.4 mu mol/L of adenosine, 0.5mg/mL of ginsenoside Rb1, 4.5g/L of glucose, 0.15g/L, L g/L of sodium carbonate, 0.590g/L of transferrin, 5g/L of insulin and 0.01g/L of basic fibroblast growth factor, and the solvent is compound electrolyte injection.

In this embodiment: the ratio of the culture medium to the in vitro bone marrow mesenchymal stem cells is 1.0 x 10 per milliliter of the culture medium⁶And the isolated bone marrow mesenchymal stem cells.

In this embodiment: the in vitro human mesenchymal stem cells are in vitro human allogeneic mesenchymal stem cells.

In this embodiment: a preparation method of a reagent for targeting bone mesenchymal stem cells to a myocardial infarction focus comprises the following steps:

step (1): preparing a complete culture solution, wherein the complete culture solution is L-DMEM, FBS with the mass concentration of 20%, penicillin 100U/ml and streptomycin 100 mu g/ml;

step (2): taking bone marrow, placing in a sterile heparin tube, adding 100U heparin per ml bone marrow for anticoagulation, mixing well, and adjusting bone marrow cell concentration to 10 with PBS liquid⁶Adding Ficoll separating medium and bone marrow cell suspension at volume ratio of 1:1 into the mixture without damaging interface, centrifuging at 20 deg.C for 25 min with horizontal centrifuge, naturally settling, taking out mononuclear cell from interface, and washing with PBS for 2 times;

and (3): adding the cells obtained in the step (2) into the complete culture solution prepared in the step (1) for cell counting until 10⁶After the primary culture, 1X 10⁶cells/ml were inoculated into T25 cell culture flasks, each containing 5ml of complete medium, placed in CO₂Culturing in a 37 ℃ incubator with the volume ratio concentration of 5%, changing the culture solution after 3d to remove non-adherent cells, changing the culture solution for 1 time every 3-5d, digesting the cells at 37 ℃ by pancreatin with the mass concentration of 0.25% when the cells reach 80% fusion, carrying out passage amplification by 1 × 104cells/ml, and taking the mesenchymal stem cells of P2;

and (4): placing the P2 generation bone marrow mesenchymal stem cells obtained in the step (3) into O₂Culturing the cells in the complete culture solution prepared in the step (1) in a 37 ℃ culture box with the volume ratio concentration of 21% until the cells are fused to 60%, culturing for 12h by using a serum-free starvation culture solution, and replacing the serum-free starvation culture solution with the complete culture solution prepared in the step (1) containing 10ng/ml of epidermal growth factor to culture the cells until the cells are in a logarithmic growth phase;

and (5): and (3) uniformly mixing the mesenchymal stem cells obtained in the step (4), blood plasma, low molecular weight heparin sodium, dipotassium hydrogen phosphate solution, adenosine, ginsenoside Rb1, glucose, sodium carbonate, L-glutamine, transferrin, insulin, basic fibroblast growth factor and a solvent to obtain the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus.

As an embodiment of the present invention: an agent for targeting a mesenchymal stem cell to a myocardial infarction focus, which consists of a culture medium for targeting the mesenchymal stem cell to the myocardial infarction focus and an isolated mesenchymal stem cell, and is characterized in that: the culture medium for targeting the mesenchymal stem cells to the myocardial infarction focus consists of solute and solvent;

in this embodiment: the solute comprises 100% plasma, 700U/mL of low molecular weight heparin sodium, 70mmol/L of dipotassium phosphate solution, 10 mu mol/L of adenosine, 10mg/mL of ginsenoside Rb1, 5.5g/L of glucose, 0.25g/L, L g/L of sodium carbonate-0.710 g/L of glutamine, 65g/L of transferrin, 7g/L of insulin and 0.05g/L of basic fibroblast growth factor, and the solvent is compound electrolyte injection, glucose injection or physiological saline.

In this embodiment: the ratio of the culture medium to the isolated bone marrow mesenchymal stem cells is 4 multiplied by 10 per milliliter of the culture medium⁶And the isolated bone marrow mesenchymal stem cells.

In this embodiment: the in vitro human mesenchymal stem cells are in vitro human allogeneic mesenchymal stem cells.

In this embodiment: a preparation method of a reagent for targeting bone mesenchymal stem cells to a myocardial infarction focus comprises the following steps:

step (1): preparing a complete culture solution, wherein the complete culture solution is L-DMEM, FBS with the mass concentration of 20%, penicillin 100U/ml and streptomycin 100 mu g/ml;

step (2): taking bone marrow, placing in a sterile heparin tube, adding 100U heparin per ml bone marrow for anticoagulation, mixing well, and adjusting bone marrow cell concentration to 10 with PBS liquid⁷Adding Ficoll separating medium and bone marrow cell suspension at volume ratio of 1:1 into the mixture without damaging interface, centrifuging at 20 deg.C for 30 min with horizontal centrifuge, naturally settling, taking out mononuclear cells from the interface, and washing with PBS for 3 times;

and (3): adding the cells obtained in the step (2) into the complete culture solution prepared in the step (1) for cell counting until 10⁷After the primary culture, 5X 10⁶cells/ml were inoculated into T75 cell culture flasks, each containing 25ml of complete medium, placed in CO₂5% volume ratio concentration 37 ℃ incubatorCulturing for 3 days, removing non-adherent cells by changing the solution, changing the solution for 1 time every 3-5 days, digesting with pancreatin with mass concentration of 0.25% at 37 deg.C when the cells are 80% fused, and concentrating at 1 × 10⁴Passage amplification of cells/ml, taking the mesenchymal stem cells of P5 generation;

and (4): placing the P5 generation bone marrow mesenchymal stem cells obtained in the step (3) into O₂Culturing the cells in the complete culture solution prepared in the step (1) in a 37 ℃ culture box with the volume ratio concentration of 21% until the cells are 70% fused, using a serum-free starvation culture solution for culturing for 15h, replacing the serum-free starvation culture solution with the complete culture solution prepared in the step (1) containing 10ng/ml of epidermal growth factor, and culturing until the cells are in a logarithmic growth phase;

and (5): and (3) uniformly mixing the mesenchymal stem cells obtained in the step (4), blood plasma, low molecular weight heparin sodium, dipotassium hydrogen phosphate solution, adenosine, ginsenoside Rb1, glucose, sodium carbonate, L-glutamine, transferrin, insulin, basic fibroblast growth factor and a solvent to obtain the reagent for targeting the mesenchymal stem cells to the myocardial infarction focus.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.