CN113101302A - Research method for treating diabetes by in-vivo islet homing differentiation of autologous MSCs - Google Patents
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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
Abstract
The invention discloses a research method for treating diabetes by in vivo islet homing differentiation of autologous MSCs (mesenchymal stem cells), which comprises the steps of in vitro induction of human MSCs into islet-like cell masses, dithizone staining, preparation of a rat diabetes model, transplantation of the islet-like cell masses, immunohistochemical detection of pancreatic tissues and analysis by a statistical method. The invention has the beneficial effects that: the research leads us to realize that autologous MSCs can be converted into islet-like cell masses after induced differentiation, and the islet-like cell masses have a certain insulin secretion function after transplantation, so that the purpose of treating diabetes can be achieved. These all provide experimental basis for the application of mesenchymal stem cell transplantation in clinical diabetes treatment, and are expected to become a more convenient, economic and effective treatment means.
Description
Technical Field
The invention relates to a research method for treating diabetes by in-vivo islet homing differentiation of autologous MSCs, in particular to a research method for treating diabetes by in-vivo islet homing differentiation of autologous MSCs, belonging to the technical field of diabetes research.
Background
Diabetes mellitus is a metabolic disease seriously threatening human health and life, and is the third largest disease after cardiovascular diseases and tumors at present. Although the etiology and pathogenesis of diabetes are not well understood at present, progressive failure of islet beta cell function is a currently recognized critical link and determinant in pathogenesis. Therefore, replacing and restoring the beta cell function of the pancreatic islet becomes an inevitable choice for treating diabetes, and in recent years, although dosage forms and administration routes of insulin have been remarkably developed, clinical practice proves that exogenous insulin treatment cannot accurately control blood sugar like self-secretion of insulin, and chronic complications such as diabetic nephropathy and retinopathy gradually become main causes of death and disability of diabetic patients. Therefore, islet cell transplantation is becoming a growing concern for researchers.
According to literature data, theories and experimental techniques of stem cell research are further developed, two problems of donor deficiency and immune rejection are avoided through autotransplantation, and reports show that transplantation of islet cells in-vitro experiments can effectively reduce blood sugar and reduce occurrence of complications, induced differentiation of MSCs to islet-like cells in vitro and treatment effect of the islet-like cells after transplantation on diabetic rats provide basis for clinical treatment of diabetes.
Disclosure of Invention
The invention aims to solve the problems and provide a research method for treating diabetes by islet homing differentiation in vivo of autologous MSCs.
The invention realizes the purpose through the following technical scheme: a research method for treating diabetes by in-vivo islet homing differentiation of autologous MSCs comprises the following steps:
step one, inducing human MSCs into islet-like cell masses in vitro, taking human MSCs which are passaged to the 3 rd generation, and after the cells are fused to 70-80%, using a DMEM high-sugar medium containing 10ug/L basic fibroblast growth factor and nicotinamide for induced differentiation;
step two, carrying out dithizone dyeing, dissolving 10mg of dithizone powder in 1mL of dimethyl sulfoxide to prepare a stock solution, and storing at-20 ℃ for later use;
step three, preparing a rat diabetes model, which comprises the following processes:
(1) preparing 2% injection from 0.1mol/L sodium citrate buffer solution (pH4.5), and injecting 70mg/Kg streptozotocin and 0.1mol/L sodium citrate buffer solution into the abdominal cavity after the rat fasts for 12 h;
(2) and respectively collecting blood of rats with fasting and broken tail before injection and 48h, 5d and 8d after injection, and determining the blood glucose concentration of whole blood;
(3) the normal value of the blood sugar concentration of the rat is 2.80-7.56mmol/L, and the rat with the blood sugar concentration higher than 16.7mmol/L for 2 times continuously is selected as a diabetes model.
Step four, transplanting the islet-like cell mass, after modeling, transplanting 2 x 106 islet-like cells into rats in the islet-like cell group under the kidney capsule, transplanting 2 x 106 umbilical cord mesenchymal stem cells into rats in the mesenchymal stem cell group under the kidney capsule, transplanting 1mL culture solution without any cells into rats in the model control group under the kidney capsule, measuring the concentration of fasting blood glucose in each group of rats before and 48 hours after transplantation, and measuring the fasting blood glucose and the body mass 1 time per week;
step five, carrying out immunohistochemical detection on pancreatic tissues, taking out pancreatic glands, quickly putting the pancreatic glands into a formaldehyde solution, fixing for 3 hours, then dyeing by a conventional immunohistochemical method, carrying out enzyme repair for 30min, adding goat anti-mouse primary antibody overnight, and carrying out second-day rehabilitation repair next day;
and step six, analyzing by a statistical method.
As a still further scheme of the invention: in the first step, before induction, the MSCs are in a long fusiform shape and grow adherent. After induction for 4d, cells were concentrated towards the center with islet-like cell mass. After 7d of islet-like cell clusters are induced to be stained for 15min by dithizone, most cells of the cell clusters can be seen to be stained under an inverted microscope, the cells present a brownish red positive expression, and a few cells at the periphery are not stained.
As a still further scheme of the invention: in the second step, the dithizone is temporarily mixed with PBS buffer solution 1: diluting with 100 percent, and filtering by a filter membrane with the aperture of 0.22 mu m. Adding the randomly selected induced islet-like cell mass under an inverted microscope, incubating in an incubator at 37 ℃ for 15min, washing with PBS for 3 times, and performing microscopic examination.
As a still further scheme of the invention: in the third step, the fasting blood sugar concentration of each group of rats before molding is in a normal range. After streptozotocin is injected into the abdominal cavity for 48 hours, the blood sugar concentration of the rat is rapidly increased. On both day 5 and day 8 post-injection, the blood glucose concentration in rats was greater than 16.7mmol/L, demonstrating successful modeling.
As a still further scheme of the invention: in the fourth step, 2 weeks after the islet cell group transplantation, the blood glucose concentration of the rat in the islet-like cell group is obviously reduced, and the insulin secretion is increased until the 6 th week. The blood glucose concentration of rats in the mesenchymal stem cell group was decreased after transplantation, insulin secretion was increased, and insulin secretion was decreased by 4 weeks after transplantation, and then maintained at a lower level. The normal control group was always in a diabetic glycemic state with no insulin secretion.
As a still further scheme of the invention: in the fifth step, hematoxylin counterstaining and SABC kit color development are utilized during pancreas tissue immunohistochemical detection.
As a still further scheme of the invention: and in the sixth step, SPSS software is adopted for statistical analysis.
The invention has the beneficial effects that: the research method for treating diabetes by in-vivo islet homing differentiation of the autologous MSCs is reasonable in design, and the research leads us to realize that the autologous MSCs can be converted into islet-like cell masses after induced differentiation, and the islet-like cell masses have a certain insulin secretion function after transplantation, so that the purpose of treating diabetes can be achieved. These all provide experimental basis for the application of mesenchymal stem cell transplantation in clinical diabetes treatment, and are expected to become a more convenient, economic and effective treatment means.
Drawings
FIG. 1 is a schematic flow chart of the present invention.
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.
Referring to fig. 1, a method for studying islet homing differentiation in vivo of autologous MSCs for diabetes treatment includes the following steps:
step one, inducing human MSCs into islet-like cell masses in vitro, taking human MSCs which are passaged to the 3 rd generation, and after the cells are fused to 70-80%, using a DMEM high-sugar medium containing 10ug/L basic fibroblast growth factor and nicotinamide for induced differentiation;
step two, carrying out dithizone dyeing, dissolving 10mg of dithizone powder in 1mL of dimethyl sulfoxide to prepare a stock solution, and storing at-20 ℃ for later use;
step three, preparing a rat diabetes model, which comprises the following processes:
(1) preparing 2% injection from 0.1mol/L sodium citrate buffer solution (pH4.5), and injecting 70mg/Kg streptozotocin and 0.1mol/L sodium citrate buffer solution into the abdominal cavity after the rat fasts for 12 h;
(2) and respectively collecting blood of rats with fasting and broken tail before injection and 48h, 5d and 8d after injection, and determining the blood glucose concentration of whole blood;
(3) the normal value of the blood sugar concentration of the rat is 2.80-7.56mmol/L, and the rat with the blood sugar concentration higher than 16.7mmol/L for 2 times continuously is selected as a diabetes model;
step four, transplanting the islet-like cell mass, after modeling, transplanting 2 x 106 islet-like cells into rats in the islet-like cell group under the kidney capsule, transplanting 2 x 106 umbilical cord mesenchymal stem cells into rats in the mesenchymal stem cell group under the kidney capsule, transplanting 1mL culture solution without any cells into rats in the model control group under the kidney capsule, measuring the concentration of fasting blood glucose in each group of rats before and 48 hours after transplantation, and measuring the fasting blood glucose and the body mass 1 time per week;
step five, carrying out immunohistochemical detection on pancreatic tissues, taking out pancreatic glands, quickly putting the pancreatic glands into a formaldehyde solution, fixing for 3 hours, then dyeing by a conventional immunohistochemical method, carrying out enzyme repair for 30min, adding goat anti-mouse primary antibody overnight, and carrying out second-day rehabilitation repair next day;
and step six, analyzing by a statistical method.
Further, in the embodiment of the present invention, in the first step, before the induction, the MSCs grow in a long fusiform shape and adhere to the wall. After induction for 4d, cells were concentrated towards the center with islet-like cell mass. After 7d of islet-like cell clusters are induced to be stained for 15min by dithizone, most cells of the cell clusters can be seen to be stained under an inverted microscope, the cells present a brownish red positive expression, and a few cells at the periphery are not stained.
Further, in the present embodiment, in the second step, the dithizone is temporarily mixed with PBS buffer 1: diluting with 100 percent, and filtering by a filter membrane with the aperture of 0.22 mu m. Adding the randomly selected induced islet-like cell mass under an inverted microscope, incubating in an incubator at 37 ℃ for 15min, washing with PBS for 3 times, and performing microscopic examination.
Further, in the present embodiment, in step three, the fasting blood glucose concentration of each group of rats before molding is within the normal range. After streptozotocin is injected into the abdominal cavity for 48 hours, the blood sugar concentration of the rat is rapidly increased. On both day 5 and day 8 post-injection, the blood glucose concentration in rats was greater than 16.7mmol/L, demonstrating successful modeling.
Further, in the fourth step of the present invention, the blood glucose level of the rat in the islet-like cell group is significantly decreased and the insulin secretion is increased up to the 6 th week after 2 weeks of islet mass cell transplantation. The blood glucose concentration of rats in the mesenchymal stem cell group was decreased after transplantation, insulin secretion was increased, and insulin secretion was decreased by 4 weeks after transplantation, and then maintained at a lower level. The normal control group was always in a diabetic glycemic state with no insulin secretion.
Further, in the present embodiment, in the fifth step, hematoxylin counterstaining and SABC kit color development are used for performing immunohistochemical detection on pancreatic tissues.
Further, in the embodiment of the present invention, in the sixth step, statistical analysis is performed by using SPSS software.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes 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. Any reference sign in a claim should not be construed as limiting the claim concerned.
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.
Claims (7)
1. A research method for treating diabetes by islet homing differentiation in vivo of autologous MSCs is characterized in that: the method comprises the following steps:
step one, inducing human MSCs into islet-like cell masses in vitro, taking human MSCs which are passaged to the 3 rd generation, and after the cells are fused to 70-80%, using a DMEM high-sugar medium containing 10ug/L basic fibroblast growth factor and nicotinamide for induced differentiation;
step two, carrying out dithizone dyeing, dissolving 10mg of dithizone powder in 1mL of dimethyl sulfoxide to prepare a stock solution, and storing at-20 ℃ for later use;
step three, preparing a rat diabetes model, which comprises the following processes:
(1) preparing streptozotocin into 2% injection by using 0.1mol/L sodium citrate buffer solution, and injecting 70mg/Kg streptozotocin and 0.1mol/L sodium citrate buffer solution into the abdominal cavity after the rat fasts for 12 hours;
(2) and respectively collecting blood of rats with fasting and broken tail before injection and 48h, 5d and 8d after injection, and determining the blood glucose concentration of whole blood;
(3) the normal value of the blood sugar concentration of the rat is 2.80-7.56mmol/L, and the rat with the blood sugar concentration higher than 16.7mmol/L for 2 times continuously is selected as a diabetes model.
Step four, transplanting the islet-like cell mass, after modeling, transplanting 2 x 106 islet-like cells into rats in the islet-like cell group under the kidney capsule, transplanting 2 x 106 umbilical cord mesenchymal stem cells into rats in the mesenchymal stem cell group under the kidney capsule, transplanting 1mL culture solution without any cells into rats in the model control group under the kidney capsule, measuring the concentration of fasting blood glucose in each group of rats before and 48 hours after transplantation, and measuring the fasting blood glucose and the body mass 1 time per week;
step five, carrying out immunohistochemical detection on pancreatic tissues, taking out pancreatic glands, quickly putting the pancreatic glands into a formaldehyde solution, fixing for 3 hours, then dyeing by a conventional immunohistochemical method, carrying out enzyme repair for 30min, adding goat anti-mouse primary antibody overnight, and carrying out second-day rehabilitation repair next day;
and step six, analyzing by a statistical method.
2. The method of claim 1, wherein the method comprises the following steps: in the first step, before induction, the MSCs are in a long fusiform shape and grow adherent. After induction for 4d, cells were concentrated towards the center with islet-like cell mass. After 7d of islet-like cell clusters are induced to be stained for 15min by dithizone, most cells of the cell clusters can be seen to be stained under an inverted microscope, the cells present a brownish red positive expression, and a few cells at the periphery are not stained.
3. The method of claim 1, wherein the method comprises the following steps: in the second step, the dithizone is temporarily mixed with PBS buffer solution 1: diluting with 100 percent, and filtering by a filter membrane with the aperture of 0.22 mu m. Adding the randomly selected induced islet-like cell mass under an inverted microscope, incubating in an incubator at 37 ℃ for 15min, washing with PBS for 3 times, and performing microscopic examination.
4. The method of claim 1, wherein the method comprises the following steps: in the third step, the fasting blood sugar concentration of each group of rats before molding is in a normal range. After streptozotocin is injected into the abdominal cavity for 48 hours, the blood sugar concentration of the rat is rapidly increased. On both day 5 and day 8 post-injection, the blood glucose concentration in rats was greater than 16.7mmol/L, demonstrating successful modeling.
5. The method of claim 1, wherein the method comprises the following steps: in the fourth step, 2 weeks after the islet cell group transplantation, the blood glucose concentration of the rat in the islet-like cell group is obviously reduced, and the insulin secretion is increased until the 6 th week. The blood glucose concentration of rats in the mesenchymal stem cell group was decreased after transplantation, insulin secretion was increased, and insulin secretion was decreased by 4 weeks after transplantation, and then maintained at a lower level. The normal control group was always in a diabetic glycemic state with no insulin secretion.
6. The method of claim 1, wherein the method comprises the following steps: in the fifth step, hematoxylin counterstaining and SABC kit color development are utilized during pancreas tissue immunohistochemical detection.
7. The method of claim 1, wherein the method comprises the following steps: and in the sixth step, SPSS software is adopted for statistical analysis.
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| CN107460158A (en) * | 2017-08-10 | 2017-12-12 | 河南省银丰生物工程技术有限公司 | A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin |
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| 李俊林等: "人脐带间充质干细胞体外向胰岛样细胞诱导分化及其治疗糖尿病效果", 《中国组织工程研究与临床康复》, vol. 13, no. 14, pages 2637 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115850486A (en) * | 2022-11-16 | 2023-03-28 | 艾可泰科(浙江)控股有限公司 | Use of islet stem cells in the treatment of diabetes |
| CN115850486B (en) * | 2022-11-16 | 2023-05-30 | 艾可泰科(浙江)控股有限公司 | Use of islet stem cells in the treatment of diabetes |
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