CN112546070A - Mesenchymal stem cell preparation and preparation method and application thereof - Google Patents
Mesenchymal stem cell preparation and preparation method and application thereof Download PDFInfo
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- 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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Abstract
The invention provides a mesenchymal stem cell preparation, which comprises the following components: mesenchymal stem cells and a suspending agent; the mesenchymal stem cells are processed by mannose. The umbilical cord mesenchymal stem cells with the capacity of differentiating into pancreatic beta cells and the potential of treating diabetes and mannose with the immunoregulation function are combined to prepare the medicine for treating diabetes and complications thereof, and on the premise of controlling the inflammatory environment in a host, a good action environment is created for the umbilical cord mesenchymal stem cells, so that the continuous treatment effect of the umbilical cord mesenchymal stem cells is promoted.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a mesenchymal stem cell preparation, a preparation method and application thereof in preparing medicines for treating diabetes and complications thereof.
Background
Diabetes mellitus, which is the clinically major endocrine-metabolic disease, rapidly increases in prevalence. Statistically, it is predicted that by 2025, diabetics will reach 3 billion worldwide, 75% of which are in developing countries of china, india, etc. Diabetes is largely classified into type 1 and type 2 diabetes. Among them, diabetes and vascular complications are the most common chronic complications of diabetes, and are directly related to prognosis and quality of life of the diabetic, and also the main cause of death and disability of the diabetic. The diabetic vascular complications are characterized by systemic vascular damage and mainly comprise diabetic microangiopathy and macroangiopathy, wherein the diabetic microangiopathy is characteristic pathological change of diabetes and is a common risk factor of chronic complications of diabetes. The pathological changes are very extensive, especially the microvasculature of the parts such as glomerulus, retina, nerve, cardiac muscle and the like, which cause nephropathy, fundus oculi pathological changes, neuropathy, cardiac muscle pathological changes and the like, and become main factors for determining the prognosis of patients, wherein the latter mainly comprises accelerated atherosclerosis, ischemic heart disease, increased risk of cerebrovascular disease, serious peripheral angiopathy and the like. Diabetic nephropathy is one of the major microvascular pathological complications of diabetic patients. And is also the main cause of end-stage renal failure.
At present, the method for treating diabetes mainly adopts a traditional blood sugar control method, wherein insulin injection is the most commonly used blood sugar reduction measure, but the insulin injection can not stabilize the blood sugar level in vivo for a long time and can not prevent the occurrence of diabetic complications, insulin dependence and resistance of patients can be caused finally, and short-term rapid blood sugar reduction can also generate related symptoms such as dizziness caused by hypoglycemia. The current research also proposes the strategies of islet beta cell repair and regeneration, such as cell infiltration repair therapy, beta cell recombination activation technology, islet transplantation surgery therapy and the like, but the treatment methods are limited by technical feasibility or lack of pancreas donors, and the application effect is not ideal.
In recent years, the treatment effect of the mesenchymal stem cell transplantation on diabetes is receiving attention, and the preparation developed based on the mesenchymal stem cell can obviously improve the hyperglycemia condition. The mesenchymal stem cells have wide sources, convenient material taking, strong proliferation and differentiation potentials, low immunogenicity, unique immunoregulation function, no limitation of moral ethical problems, easy industrial preparation and wide clinical application prospects.
However, there are some problems with the current mesenchymal stem cell-based diabetes therapy, the biggest disadvantages of which are unstable therapeutic effect on diabetic hyperglycemia and insignificant relief from diabetic complications. A series of researches show that the curative effect of the mesenchymal stem cells is probably influenced by hyperglycemia in a patient body on one hand and the inflammatory environment in the body on the other hand, but whether and how to promote the curative effect of the mesenchymal stem cells by controlling the in-vivo environment of a host have no related methods and research reports at present.
Disclosure of Invention
The invention aims to: the umbilical cord mesenchymal stem cells with the capacity of differentiating into pancreatic beta cells and the potential of treating diabetes and mannose with the immunoregulation function are combined to prepare the medicine for treating diabetes and complications thereof, and on the premise of controlling the inflammatory environment in a host, a good action environment is created for the umbilical cord mesenchymal stem cells, and the continuous treatment effect of the umbilical cord mesenchymal stem cells is promoted.
In order to realize the purpose of the invention, the invention provides the following technical scheme:
in a first aspect of the invention, a mesenchymal stem cell preparation is provided, which comprises the following components: mesenchymal stem cells and a suspending agent; the mesenchymal stem cells are processed by mannose.
Preferably, the suspending agent is selected from PBS buffer or physiological saline.
Preferably, the concentration of mesenchymal stem cells in the mesenchymal stem cell preparation is 1 × 106Per mL-5X 106one/mL.
Preferably, the mesenchymal stem cell is processed by the following steps: the mesenchymal stem cells passaged to P4 generation were inoculated into alpha-MEM medium containing 10% fetal bovine serum and 10-50mM mannose at 37 deg.C with 5% CO2Culturing in an incubator for 24-48 h.
Preferably, the source of mesenchymal stem cells comprises umbilical cord, umbilical cord blood, bone marrow, teeth or adipose tissue of a human or animal.
In a second aspect of the present invention, a method for preparing the above mesenchymal stem cell preparation is provided, which comprises the following steps:
(1) the mesenchymal stem cells passaged to P4 generation were inoculated into alpha-MEM medium containing 10% fetal bovine serum and 10-50mM mannose at 37 deg.C with 5% CO2Culturing in an incubator for 24-48 h.
(2) After the mesenchymal stem cells treated by mannose are digested and counted, the mesenchymal stem cells are evenly suspended in normal saline or PBS buffer solution until the concentration is 1 × 106Per mL-5X 106And (4) per mL, obtaining the mesenchymal stem cell preparation.
In a third aspect of the invention, an application of the mesenchymal stem cell preparation in preparing a medicament for treating diabetes or diabetic complications is provided.
Preferably, the diabetes mellitus comprises diabetic islet damage, decreased insulin secretion levels, increased blood glucose concentration, or an increased proportion of glycated hemoglobin.
Preferably, the diabetic complication comprises diabetes-induced nephropathy, osteoporosis, renal failure, retinal degeneration, heart disease, stroke, nerve damage, non-healing infection, or ketoacidosis.
Compared with the prior art, the invention has the beneficial effects that:
the invention breaks the fence between the drug therapy of diabetes and the stem cell therapy method, skillfully improves the immunoregulation capability and stability of the mesenchymal stem cell by applying mannose, so that the mesenchymal stem cell can regenerate damaged islet tissues and recover the secretion level of insulin after transplantation, slow and long-acting blood sugar reduction is realized, the long-term use amount of the insulin is controlled, and hypoglycemia or insulin resistance caused by insulin injection is avoided. Can also improve the treatment effect on the diabetic complications, obviously improve the pathological changes of target organs of the diabetes, and fundamentally achieve the aim of treating the diabetes and the diabetic complications.
Drawings
FIG. 1 is a statistical result of blood glucose concentrations of 4 groups of mice in example 3 of the present invention;
FIG. 2 is a graph showing glucose tolerance comparison among 4 groups of mice in example 3 of the present invention;
FIG. 3A is a graph showing MASSON staining comparison of kidney of mice in UCMSC group in example 3 of the present invention;
FIG. 3B is a graph comparing MASSON staining of the kidney of mice in the experimental group of example 3 according to the present invention;
FIG. 3C is a graph comparing MASSON staining of kidney of mice in model group according to example 3 of the present invention;
FIG. 3D is a graph comparing MASSON staining of kidney in normal group mice in example 3 of the present invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following description is given by way of example only with reference to the accompanying drawings and examples and should not be construed as limiting the scope of the invention.
Reagent and formulation (the following ingredients were purchased from Invitrogen, USA)
1. PBS for washing
0.8 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate and 0.2 g of potassium dihydrogen phosphate, and deionized water is added to the mixture to reach the constant volume of 1000 mL. Filtering, sterilizing, and storing in refrigerator at 4 deg.C.
2. Pancreatin solution for digestion
Weighing 0.25 g of trypsin, adding the trypsin into 100 mL of serum-free alpha-MEM culture medium, uniformly stirring, filtering and sterilizing, and storing in a refrigerator at 4 ℃ to prevent repeated freeze thawing.
3. PBS for injection (containing 3% fetal bovine serum to maintain cell viability)
0.8 g of sodium chloride, 0.2 g of potassium chloride, 2.9 g of disodium hydrogen phosphate dodecahydrate and 0.2 g of potassium dihydrogen phosphate, adding deionized water to a constant volume of 1000 mL, and adding 3% fetal calf serum. Filtering, sterilizing, and storing in refrigerator at 4 deg.C.
Example 1
The embodiment provides a mesenchymal stem cell preparation, which comprises the following components: mesenchymal stem cells and a suspending agent; the mesenchymal stem cells are processed by mannose.
In this embodiment, the suspending agent is selected from PBS buffer or physiological saline.
In this embodiment, the concentration of mesenchymal stem cells in the mesenchymal stem cell preparation is 1 × 106one/mL.
In some embodiments, the mesenchymal stem cellThe concentration of mesenchymal stem cells in the stem cell preparation was 2X 106 3X 10 pieces/mL64X 10 units/mL6one/mL or 5X 106one/mL.
In this embodiment, the processing process of the mesenchymal stem cells is as follows: the mesenchymal stem cells passaged to P4 generation were inoculated into alpha-MEM medium containing 10% fetal bovine serum and 10-50mM mannose at 37 deg.C with 5% CO2Culturing in an incubator for 24-48 h.
In some embodiments, the concentration of mannose is 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM.
In this embodiment, the mesenchymal stem cell source is mouse bone marrow.
In some embodiments, the source of mesenchymal stem cells comprises umbilical cord, umbilical cord blood, teeth, or adipose tissue of a human or animal.
Example 2
This example provides a method of preparing the mesenchymal stem cell preparation of example 1, comprising the steps of:
(1) the mesenchymal stem cells passaged to P4 generation were inoculated into alpha-MEM medium containing 10% fetal bovine serum and 10mM mannose at 37 ℃ with 5% CO2Culturing in an incubator for 24-48 h.
(2) After the mesenchymal stem cells treated by mannose are digested and counted, the mesenchymal stem cells are evenly suspended in normal saline or PBS buffer solution until the concentration is 1 × 106And (4) per mL, obtaining the mesenchymal stem cell preparation.
In some embodiments, the concentration of mannose is 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM.
In some embodiments, the mesenchymal stem cell preparation has a concentration of mesenchymal stem cells of 2 x 106 3X 10 pieces/mL64X 10 units/mL6one/mL or 5X 106one/mL.
Example 3: treatment experiment of combination of mesenchymal stem cells and mannose on diabetic mice
1. 18 mice were selected for inclusion in the experiment and were completely randomized into 3 groups (6 per group): model group, UCMSC group, experimental group, and normal group. The model group is a diabetes model mouse, the UCMSC group is a diabetes mouse injected with common mesenchymal stem cells, the experimental group is a diabetes mouse injected with the mesenchymal stem cell preparation in the embodiment 1 of the invention, and the normal group is a normal mouse.
2. On the 25 th day of the experiment, glucose tolerance test was performed, each mouse was subcutaneously injected with 1 g/kg of glucose, and the mice received blood glucose concentration measurement by sampling the tail vein blood collection needles before injection (0 th min) and 15 th, 30 th, 60 th, 90 th, and 120 th min after injection, respectively, and recorded.
3. Sample drawing detection
(1) On day 54 of the experiment (i.e., about 1 month after injection, after the end of the last blood glucose measurement), mice were decapitated and sacrificed, the peritoneal skin and peritoneum were quickly cut open with an ophthalmic scissors, the kidneys were found, and they were fixed in 4% paraformaldehyde overnight.
(2) The following day, the fixed kidney tissue was washed, dehydrated, paraffin-embedded the next day, sectioned on selected days and MASSON stained.
4. Analysis of Experimental results
(1) Statistical analysis was performed using Graphpad Prism software, all data were expressed as mean ± standard deviation, the statistical analysis method used one-way anova, the group-by-group comparison used Newman-Keuls test, α was set to 0.05, P < 0.05 considered the difference to be statistically significant (marked as x), and P > 0.05 considered the difference to be not statistically significant (marked as NS).
(2) And (3) blood sugar measurement results:
1) referring to fig. 1, fig. 1 is a statistical result of blood glucose concentrations of 4 mice in this example, and it can be seen that fasting blood glucose of the mice in the model group is higher than 11.1, blood glucose of the experimental group and UCMSC group is significantly reduced compared to the blood glucose of the model group, blood glucose is lower than 11.1, there is a significant difference, and blood glucose control of the experimental group is more stable.
2) Glucose tolerance assay results: referring to fig. 2, fig. 2 is a graph comparing glucose tolerance of 4 groups of mice in example 3 of the present invention, and it can be seen that the glucose tolerance of the mice in the model group is severe, the glucose tolerance of the experimental group and the UCMSC group is alleviated to some extent compared with the glucose tolerance of the model group, and the alleviating effect of the experimental group is more significant.
3) Renal histology test results: referring to fig. 3A to 3D, fig. 3A is a graph comparing MASSON staining of kidney of mice in UCMSC group according to example 3 of the present invention; FIG. 3B is a graph comparing MASSON staining of the kidney of mice in the experimental group of example 3 according to the present invention; FIG. 3C is a graph comparing MASSON staining of kidney of mice in model group according to example 3 of the present invention; FIG. 3D is a graph comparing MASSON staining of kidney in normal group mice in example 3 of the present invention. It can be seen that the fibrosis of the model group mice is severe, the fibrosis of the experimental group and UCMSC is relieved, and the relieving effect of the experiment is more obvious.
This summary merely illustrates some embodiments which are claimed, wherein one or more of the features recited in the claims can be combined with any one or more of the embodiments, and such combined embodiments are also within the scope of the present disclosure as if they were specifically recited in the disclosure.
Claims (9)
1. A mesenchymal stem cell preparation, comprising the following components: mesenchymal stem cells and a suspending agent; the mesenchymal stem cells are processed by mannose.
2. Mesenchymal stem cell preparation according to claim 1, wherein the suspending agent is selected from PBS buffer or physiological saline.
3. The mesenchymal stem cell preparation of claim 1, wherein the concentration of mesenchymal stem cells in the mesenchymal stem cell preparation is 1 x 106Per mL-5X 106one/mL.
4. The mesenchymal stem cell preparation according to claim 1, wherein the mesenchymal stem cells are processed by: collecting mesenchymal stem cells passaged to P4 generation, inoculating to 10% fetal calf serum and 10-50mM glycerolSugar-exposed alpha-MEM Medium with 5% CO at 37 deg.C2Culturing in an incubator for 24-48 h.
5. The mesenchymal stem cell preparation of claim 1, wherein the source of mesenchymal stem cells comprises human or animal umbilical cord, umbilical cord blood, bone marrow, teeth or adipose tissue.
6. A method of preparing a mesenchymal stem cell preparation according to any of claims 1 to 5, comprising the steps of:
(1) the mesenchymal stem cells passaged to P4 generation were inoculated into alpha-MEM medium containing 10% fetal bovine serum and 10-50mM mannose at 37 deg.C with 5% CO2Culturing in an incubator for 24-48 h;
(2) after the mesenchymal stem cells treated by mannose are digested and counted, the mesenchymal stem cells are evenly suspended in normal saline or PBS buffer solution until the density is 1 × 106Per mL-5X 106And (4) per mL, obtaining the mesenchymal stem cell preparation.
7. Use of a mesenchymal stem cell preparation according to any one of claims 1 to 5 in the manufacture of a medicament for the treatment of diabetes or diabetic complications.
8. The use of claim 7, wherein the diabetes mellitus comprises diabetic islet damage, decreased insulin secretion levels, increased blood glucose concentration, or an increased proportion of glycated hemoglobin.
9. The use of claim 7, wherein the diabetic complication comprises diabetes-induced nephropathy, osteoporosis, renal failure, retinal degeneration, heart disease, stroke, nerve damage, non-healing infection, or ketoacidosis.
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