CN111973632A - Stem cell preparation for treating diabetes and preparation method thereof - Google Patents
Stem cell preparation for treating diabetes and preparation method thereof Download PDFInfo
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- 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
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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Abstract
The invention discloses a stem cell preparation for treating diabetes and a preparation method thereof. The apoptotic bodies can participate in the links of immune regulation, angiogenesis, cell proliferation, apoptosis and the like of organisms, so that the tissue repair function is exerted, and the curative effect of the stem cell preparation can be enhanced by adding the apoptotic bodies. Compared with mesenchymal stem cells, the apoptotic bodies have more stable properties, convenient storage and transportation, and no immunological rejection and tumor formation risks caused by transplanted cells. Substances such as human serum albumin, compound amino acid, vitamin C, low molecular heparin and the like are added to provide nutrition and survival environment for the mesenchymal stem cells, and the treatment effect of the mesenchymal stem cells is promoted. The solution medium is compound electrolyte solution, can maintain the osmotic pressure of cells, and is beneficial to the survival of the cells.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a stem cell preparation for treating diabetes and a preparation method thereof.
Background
With the change of modern life style, the incidence rate of diabetes is higher and higher, and a recent survey shows that the incidence rate of diabetes of people over 20 years old in China is nearly 10%, namely, more than 90% of patients are in the vicinity of one hundred million, and the patients suffer from type 2 diabetes, and the quality of life of the patients is seriously reduced due to the pain of the patients and a large number of cardiovascular and cerebrovascular diseases and unpredictable complications, and the patients are afflicted by the pain. The promotion of islet function recovery and improvement of blood sugar and insulin resistance are key to the treatment of diabetes, but in reality, traditional methods of blood sugar-reducing drugs, insulin and the like are not obvious, a large number of patients still face the concomitant symptoms of blood sugar deterioration, beta cell function reduction, weight increase, cardiovascular diseases and a large number of concurrent diseases, and the search for a more effective treatment method is expected by a large number of patients and is the direction of attack of a plurality of medical scientific researchers.
Mesenchymal stem cells are multifunctional stem cells, are derived from mesoderm and ectoderm in early development, can be separated from various tissues such as bone marrow, fat, synovium, skeleton, muscle, umbilical cord and the like, can be differentiated into various tissues such as fat, bone, cartilage and the like after induction, have an immunoregulation function, and have great potential for regenerative medicine and autoimmune diseases. In the past, it was often thought that mesenchymal stem cells could function by homing to the damaged site and then directly committed to differentiate into target cells. However, studies have shown that the rate of homing of mesenchymal stem cells to the site of loss is very limited in the number of target cells differentiated, and that this process of differentiation into the desired target cells is very short, and contradicts the observed therapeutic effect over time. At present, researchers think that the realization of the repair effect through the secretion of certain substances is an important way for the stem cells to play a biological role, and how to enhance the curative effect of the mesenchymal stem cells through tissue repair so as to effectively improve the blood sugar and the insulin resistance still needs to be researched.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a stem cell preparation for treating diabetes and a preparation method thereof.
In order to realize the purpose of the invention, the invention is realized by adopting the following technical scheme:
a stem cell preparation for use in the treatment of diabetes, the stem cell preparation comprising stem cells, apoptotic bodies and a dispersion medium.
Apoptotic bodies are bodies containing cytoplasm, organelles and nuclear fragments which appear at the end of the process of apoptosis, are mainly involved in the transfer of substances among cells, and play an important role in the processes of antigen presentation and immunosuppression. The organelles in the apoptotic bodies are surrounded by cell membranes, and the surrounding inflammatory reaction can not be caused because the cell contents are not leaked out.
Preferably, the stem cell is a mesenchymal stem cell having a final cell concentration of 106～108one/mL.
Preferably, the mesenchymal stem cell is selected from umbilical cord mesenchymal stem cell, bone marrow mesenchymal stem cell, dental pulp mesenchymal stem cell, periodontal ligament mesenchymal stem cell or adipose mesenchymal stem cell.
Preferably, the concentration of apoptotic bodies is 0.01-0.02 mg/mL. The apoptosis corpuscle is derived from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, periodontal ligament mesenchymal stem cells or adipose mesenchymal stem cells.
Preferably, the dispersion medium is selected from one or more of human serum albumin, low molecular heparin, compound amino acid, vitamin C or compound electrolyte.
The human serum albumin and the compound amino acid adopted by the invention are all components of clinical injection, can provide nutrition for cells and are beneficial to metabolism of the cells. The addition of vitamin C can maintain the activity of various peroxidases and is also beneficial to the maintenance of cell metabolism and activity. The addition of the low-molecular heparin ensures that cells maintain a good cell dispersion state in the preservation process, reduces the phenomena of intercellular adhesion agglomeration and cell wall adhesion, reduces the risk of intravascular cell agglomeration embolism possibly occurring during clinical cell infusion, and simultaneously reduces cell loss caused by cell aggregation and filtration by an infusion filter, and the addition of trace heparin does not cause adverse reactions such as clinical bleeding. The compound electrolyte solution can maintain the osmotic pressure of cells and is beneficial to the survival of the cells.
Preferably, the mass-to-volume ratio of the human serum albumin is 1-5%, the mass-to-volume ratio of the low molecular heparin is 0.5%, the mass-to-volume ratio of the compound amino acid is 1-10%, the mass-to-volume ratio of the vitamin C is 0.3-0.7%, and the balance is the compound electrolyte injection. The mass-volume ratio is the ratio of the mass of human serum albumin, low molecular heparin, compound amino acid and vitamin C to the total volume of the dispersion medium.
The invention provides a preparation method of a stem cell preparation for treating diabetes, which comprises the following steps:
(1) after mesenchymal stem cells were isolated, the cells were inoculated into an α -MEM medium containing 10% fetal bovine serum and placed in CO2Culturing in an incubator, and digesting and passing by adopting 0.2 percent of pancreatin when the fusion degree of the mesenchymal stem cells is 80 to 85 percent to obtain the fifth generation mesenchymal stem cells;
(2) when the adherent fusion rate of the fifth generation mesenchymal stem cells reaches about 80%, replacing the culture medium with a complete alpha-MEM culture medium with the concentration of staurosporine of 300nM, and collecting the cell culture solution supernatant after incubation; centrifuging the supernatant at a temperature of 4 ℃ in a centrifuge of 16000 Xg for 30 minutes, discarding the supernatant, adding phosphate buffer solution for cleaning, centrifuging at 16000 Xg for 30 minutes again, discarding the supernatant to obtain an apoptotic body;
(3) weighing human serum albumin, low molecular heparin, compound amino acid and vitamin C, dissolving in compound electrolyte to prepare a dispersion medium, and precooling at 4 ℃ for later use;
(4) after the mesenchymal stem cells are digested and counted, the mesenchymal stem cells and the apoptosis corpuscles are suspended by a dispersion medium, and the final cell concentration is adjusted to 106～108The concentration of the apoptotic bodies is 0.01-0.02 mg/mL.
Preferably, the incubation temperature in the step (2) is 37 ℃, and the incubation time is 12-24 h.
The invention provides an application of a stem cell preparation in preparing a medicament for treating diabetes.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a stem cell preparation for treating diabetes and a preparation method thereof. The apoptotic bodies can participate in the links of immune regulation, angiogenesis, cell proliferation, apoptosis and the like of organisms, so that the tissue repair function is exerted, and the curative effect of the stem cell preparation can be enhanced by adding the apoptotic bodies. After the mesenchymal stem cells enter the body, the influence of the microenvironment in the receptor can be quickly eliminated, the micro-environment cannot play a role for a long time, the influence of the microenvironment in the receptor is avoided when the apoptosis corpuscle plays a role in the body, and the combined use of the mesenchymal stem cells and the apoptosis corpuscle can prolong the treatment time. Compared with mesenchymal stem cells, the apoptotic bodies have more stable properties, convenient storage and transportation, and no immunological rejection and tumor formation risks caused by transplanted cells. Substances such as human serum albumin, compound amino acid, vitamin C, low molecular heparin and the like are added to provide nutrition and survival environment for the mesenchymal stem cells, and the treatment effect of the mesenchymal stem cells is promoted. The solution medium is compound electrolyte solution, can maintain the osmotic pressure of cells, and is beneficial to the survival of the cells.
Drawings
Fig. 1A is a flow detection result of a positive surface marker of umbilical cord mesenchymal stem cells CD90 in an embodiment of the present invention;
fig. 1B is a flow detection result of an umbilical cord mesenchymal stem cell CD105 positive surface marker in an embodiment of the present invention;
fig. 1C is a flow detection result of a positive surface marker of umbilical cord mesenchymal stem cells CD73 in an embodiment of the present invention;
fig. 1D is a flow detection result of an umbilical cord mesenchymal stem cell CD34 negative surface marker in an embodiment of the present invention;
fig. 1E is a flow detection result of an umbilical cord mesenchymal stem cell CD45 negative surface marker in an embodiment of the present invention;
fig. 1F is a flow detection result of an umbilical cord mesenchymal stem cell CD14 negative surface marker in an embodiment of the present invention;
fig. 1G is a flow detection result of an umbilical cord mesenchymal stem cell CD19 negative surface marker in an embodiment of the present invention;
FIG. 1H shows the result of flow detection of HLA-DR negative surface marker of umbilical cord mesenchymal stem cells according to an embodiment of the present invention;
FIG. 2 is an electron micrograph of umbilical cord mesenchymal stem cell-derived apoptotic bodies in an embodiment of the present invention;
FIG. 3 is a graph showing the effect of the treatment group, the model group and the control group on the blood sugar of diabetic mice in the example of the present invention;
FIG. 4 is a graph showing a comparison of the effects of the treatment group, the model group and the control group on glycated hemoglobin (HbA 1 c) in diabetic mice in examples of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Description of the reagents:
α -MEM Medium: purchased from Gibco.
Human serum albumin: purchased from jertbelin biologicals ltd, switzerland, under production lot number P10009968.
Low molecular weight heparin calcium injection: purchased from Shenzhen Saurur Biopharmaceutical Limited, Production lot 1809103.
Compound amino acid: purchased from Tokyo pharmaceutical Co., Ltd, Jiangxi, pharmaceutical Standard H19993208.
Vitamin C: procurement was from sienlijun pharmaceutical llc, production lot A8L 708.
Compound electrolyte: purchased from Shanghai Baite medical supplies, Inc., drug Standard H2000475.
Example 1
The present embodiment provides a stem cell preparation for treating diabetes, which includes stem cells, apoptotic bodies, and a dispersion medium.
In some embodiments, the stem cell is mesenchymalStem cells, said mesenchymal stem cells having a final cell concentration of 106～108Per mL, e.g. 106one/mL, 107one/mL, 108one/mL.
In some embodiments, the mesenchymal stem cell is selected from an umbilical cord mesenchymal stem cell, a bone marrow mesenchymal stem cell, a dental pulp mesenchymal stem cell, a periodontal ligament mesenchymal stem cell, or an adipose mesenchymal stem cell.
In some embodiments, the concentration of apoptotic bodies is 0.01-0.02 mg/mL, such as 0.01 mg/mL, 0.015 mg/mL, 0.02 mg/mL.
In some embodiments, the dispersion medium comprises human serum albumin, low molecular heparin, complexed amino acids, vitamin C, and complexed electrolytes.
In some embodiments, the mass-to-volume ratio of the human serum albumin is 1-5%, the mass-to-volume ratio of the low molecular heparin is 0.5%, the mass-to-volume ratio of the compound amino acid is 1-10%, the mass-to-volume ratio of the vitamin C is 0.3-0.7%, and the balance is the compound electrolyte.
Example 2
The embodiment provides a preparation method of a stem cell preparation for treating diabetes, which comprises the following steps:
isolated culture of mesenchymal stem cells
The umbilical cord tissue is taken out from the collection bottle, is placed in alcohol for cleaning for 2 times, is transferred into sterile PBS to remove connective tissue, blood stasis and the like, and is fully cleaned to remove blood stains. Transferring the umbilical cord into a large-size culture dish, cutting to a length of 3-4cm, and longitudinally separating to convert the umbilical cord tissue into a sheet shape. Further separating the sheet umbilical cord tissue, and cutting into pieces of 2-3mm3The tissue blocks of the same size were planted one by one in a petri dish previously coated with an alpha-MEM medium containing 10% fetal bovine serum, and then the petri dish was placed at 37 ℃ with a volume fraction of 5% CO2And (3) in a saturated humidity incubator, supplementing 0.5 mL of culture medium every 24 hours, fully exchanging the culture medium after 72 hours, exchanging the culture medium 2 times every week, digesting and passaging by using 0.20% trypsin when the cells grow and fuse to about 80%, and obtaining the umbilical cord mesenchymal stem cells of P5 generation.
Identification of mesenchymal stem cells
The P5 generation umbilical cord mesenchymal stem cells are digested by 0.25% trypsin, and the stable proliferation cells are collected. Will 106The human umbilical cord mesenchymal stem cells obtained by collection are suspended in 100mL PBS buffer solution containing 1% bovine serum albumin, and the expression conditions of positive antibodies (CD 90, CD105 and CD 73) and negative antibodies (CD 45, CD34, CD11b, CD19 and HLA-DR) are detected by flow cytometry, and the results are shown in FIGS. 1A-H, wherein the positive surface markers CD90, CD105 and CD73 are all greater than or equal to 95%, and the negative surface markers CD45, CD34, CD11b, CD19 and HLA-DR are all less than 2%. Therefore, the umbilical cord mesenchymal stem cells obtained in the embodiment meet the stem cell standard.
Extraction of apoptotic bodies
When the adherent fusion rate of the mesenchymal stem cells of the generation P5 reaches about 80 percent, replacing complete alpha-MEM with 300nM STS concentration, incubating for 15 hours at 37 ℃, collecting cell culture solution supernatant, and temporarily storing at 4 ℃; centrifuging the supernatant at 16000 Xg for 30 min at 4 deg.C, discarding the supernatant, washing with PBS once, centrifuging at 16000 Xg for 30 min, discarding the supernatant, and collecting apoptotic body precipitate.
Fourth, identification of apoptotic bodies
The apoptotic bodies obtained in the above were resuspended in PBS, diluted at 1:50 times, 10uL of the resuspension was taken, added to a carbon-containing sample-carrying copper mesh with a pore size of 2nm, left to stand at room temperature for 5 minutes, then the excess liquid was gently blotted with filter paper, negatively stained with 2% phosphotungstic acid (PH = 7.0) at room temperature for 5 minutes, dried at room temperature, placed in a sample chamber of a transmission electron microscope, and the morphology of the apoptotic bodies was observed and photographed, as shown in fig. 2, which is an apoptotic body electron microscope image derived from umbilical mesenchymal stem cells, and it can be seen from the image that the apoptotic bodies obtained in this example are circular, have a diameter of about 100nm, have a complete membrane structure, and contain low-density substances.
Preparation of mesenchymal stem cell preparation
A mesenchymal stem cell preparation comprising the following components: mesenchymal stem cells, apoptotic bodies and dispersion medium.
In this example, 5% human serum albumin stock solution and 0.5% human serum albumin stock solution are weighed according to the mass percentage% low molecular weight heparin, 10% compound amino acid and 0.5% vitamin C are dissolved in compound electrolyte to prepare dispersion medium, and precooled at 4 ℃ for later use. After the umbilical cord mesenchymal stem cells are digested and counted, the mesenchymal stem cells and the apoptotic bodies are suspended in the solution to prepare suspension, and the number of the umbilical cord mesenchymal stem cells in each milliliter of preparation is 106～108The concentration of the apoptotic bodies is 0.01-0.02 mg/mL.
Example 3
Effect verification experiment:
40 db/db mice with the age of 8 weeks are selected in the experiment, the sex is half of each, the mice are randomly divided into 4 groups, each group comprises 10 mice, and the groups are respectively a model group, a mesenchymal stem cell group, an apoptotic body group and a mesenchymal stem cell + apoptotic body group, and in addition, normal C57 mice are selected as a control group. The mice in the model group are subjected to tail vein injection by 200uL of dispersion medium; the tail vein of the mesenchymal stem cell group is administered with 200uL of 1.0X 106(ii) a dispersion medium of mesenchymal stem cells of the individual; the apoptotic body group was given 200uL of dispersion medium containing 2ug apoptotic bodies; mesenchymal stem cell + apoptotic body group mice were administered with 200uL of mesenchymal stem cell preparation (containing 1.0X 10 mesenchymal stem cells)6Individual, apoptotic bodies 2 ug) tail vein injection, mice were monitored weekly for blood glucose changes. At the 6 th week of treatment, the mice are fasted for 6h, the blood sugar of each group of mice is measured, and the blood is recorded, the eyeball is removed for blood sampling, and the content of the glycosylated hemoglobin and the hemoglobin are measured by ELISA.
Fig. 3 is a graph showing the effect of the model group, the mesenchymal stem cell group, the apoptotic body group, the mesenchymal stem cell + apoptotic body group and the control group on blood sugar of the diabetic mouse in the embodiment of the present invention. The mesenchymal stem cell preparation does not cause a car toxicological reaction and a rejection reaction by intravenous injection. Compared with the model group, in the first 6 weeks, the mesenchymal stem cells and the apoptotic bodies can obviously reduce the blood sugar, the mesenchymal stem cells can not control the blood sugar from the 6 th week, and the apoptotic bodies can not control the blood sugar from the 8 th week, however, the mesenchymal stem cells and the apoptotic bodies can be continuously controlled to the 10 th week and are basically consistent with the blood sugar content of the control group.
Fig. 4 is a graph comparing the effects of the model group, the mesenchymal stem cell group, the apoptotic body group and the mesenchymal stem cell + apoptotic body group in the example of the present invention on glycated hemoglobin (HbA 1 c) in diabetic mice. The content of the glycosylated hemoglobin of the mice with the mesenchymal stem cells and the apoptotic body group is basically consistent with that of the model group, and the content of the glycosylated hemoglobin of the mice with the mesenchymal stem cells and the apoptotic body group is controlled compared with that of the model group and is basically consistent with that of the control group. Therefore, the stem cell preparation can effectively improve the treatment effect of diabetes.
The above description is only an embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions within the technical scope of the present disclosure should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (9)
1. A stem cell preparation for use in the treatment of diabetes, said stem cell preparation comprising stem cells, apoptotic bodies and a dispersion medium.
2. The stem cell preparation of claim 1, wherein the stem cells are mesenchymal stem cells having a final cell concentration of 106～108one/mL.
3. The stem cell preparation of claim 2, wherein the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, periodontal ligament mesenchymal stem cells, or adipose mesenchymal stem cells.
4. The stem cell preparation of claim 1, wherein the concentration of apoptotic bodies is 0.01-0.02 mg/mL.
5. The stem cell preparation of claim 1, wherein the dispersion medium comprises human serum albumin, low molecular heparin, complexed amino acids, vitamin C, and complexed electrolytes.
6. The stem cell preparation according to claim 5, wherein the human serum albumin comprises 1-5% by mass/volume, 0.5% by mass/volume of low molecular heparin, 1-10% by mass/volume of the compound amino acid, 0.3-0.7% by mass/volume of the vitamin C, and the balance of the compound electrolyte.
7. A method of preparing a stem cell preparation according to any one of claims 1 to 6, comprising the steps of:
(1) after mesenchymal stem cells were isolated, the cells were inoculated into an α -MEM medium containing 10% fetal bovine serum and placed in CO2Culturing in an incubator, and digesting and passing by adopting 0.2 percent of pancreatin when the fusion degree of the mesenchymal stem cells is 80 to 85 percent to obtain the fifth generation mesenchymal stem cells;
(2) when the adherent fusion rate of the fifth generation mesenchymal stem cells reaches about 80%, replacing the culture medium with a complete alpha-MEM culture medium with the concentration of staurosporine of 300nM, and collecting cell culture solution supernatant after incubation for 12 h; centrifuging the supernatant at a temperature of 4 ℃ in a centrifuge of 16000 Xg for 30 minutes, discarding the supernatant, adding phosphate buffer solution for cleaning, centrifuging at 16000 Xg for 30 minutes again, discarding the supernatant to obtain an apoptotic body;
(3) weighing human serum albumin, low molecular heparin, compound amino acid and vitamin C, dissolving in compound electrolyte to prepare a dispersion medium, and precooling at 4 ℃ for later use;
(4) after the fifth generation mesenchymal stem cells are digested and counted, the mesenchymal stem cells and the apoptotic bodies are suspended by a dispersion medium, and the final cell concentration is adjusted to 106～108The concentration of the apoptotic bodies is 0.01-0.02 mg/mL.
8. The preparation method according to claim 7, wherein the incubation temperature in the step (2) is 37 ℃ and the incubation time is 12-24 h.
9. Use of a stem cell preparation according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of diabetes.
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