CN111956784A

CN111956784A - Pharmaceutical preparation, method for the production thereof and use thereof

Info

Publication number: CN111956784A
Application number: CN201910417771.3A
Authority: CN
Inventors: 安欢笑; 高迎凤; 胡成虎
Original assignee: Guangdong Fujin Stem Cell Regeneration Medicine Co ltd
Current assignee: Guangdong Fujin Stem Cell Regeneration Medicine Co ltd
Priority date: 2019-05-20
Filing date: 2019-05-20
Publication date: 2020-11-20

Abstract

The invention provides a pharmaceutical preparation, a preparation method and application thereof, wherein the pharmaceutical preparation comprises the following components: mesenchymal stem cell injection and insulin injection. Aiming at the defects and the defects of the prior art in the treatment of the type 1 diabetes, the invention provides a medicinal preparation, which promotes the continuous treatment effect of mesenchymal stem cells by quickly controlling the blood sugar environment of a host, improves the method for treating the type 1 diabetes and the complications thereof, solves the dilemma of long-term insulin injection of patients, realizes the tissue regeneration of target organs of the pancreatic islets and the complications, and can fundamentally achieve the aim of treating the type 1 diabetes and the complications thereof.

Description

Pharmaceutical preparation, method for the production thereof and use thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a medicinal preparation for treating diabetes and complications thereof, and a preparation method and application 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, type 1 diabetes is a critical complication such as severe insulin secretion disorder, persistent rise of blood sugar, unhealed infection, renal failure, retinal degeneration, heart disease, stroke, nerve damage, osteoporosis, ketoacidosis, etc. due to the destruction of islet beta cells caused by autoimmunity under the interaction of genetic susceptibility factors and environmental factors.

At present, the method for treating type 1 diabetes is mainly a traditional blood sugar control method, wherein insulin injection is the most common blood sugar reduction measure, but insulin injection can not stabilize the blood sugar level in vivo for a long time and can not prevent 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 mesenchymal stem cell transplantation on type 1 diabetes is receiving attention, and a preparation developed based on mesenchymal stem cells 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 type 1 diabetes therapy, the biggest disadvantages of which are unstable therapeutic effect on type 1 diabetes 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, 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 provide a medicinal preparation for treating diabetes and complications thereof and a preparation method thereof, aiming at the defects and shortcomings of the treatment of the type 1 diabetes, the medicinal preparation promotes the continuous treatment effect of mesenchymal stem cells by rapidly controlling the blood sugar environment of a host, improves the type 1 diabetes and complications thereof, solves the dilemma of long-term insulin injection of patients, realizes tissue regeneration of target organs of pancreatic islets and complications, and can fundamentally achieve the aim of treating the type 1 diabetes and the complications thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, there is provided a pharmaceutical formulation comprising the following components: mesenchymal stem cell injection and insulin injection. The medicinal preparation can rapidly control the blood sugar of a type 1 diabetes patient after injection.

Preferably, the mesenchymal stem cell injection is 1 part by volume, and the insulin injection is 1 part by volume.

Further, the components of the mesenchymal stem cell injection comprise mesenchymal stem cells and normal saline, and the concentration of the mesenchymal stem cells in the mesenchymal stem cell injection is 1 x 10⁶ 1X 10 pieces/ml⁷Per ml; the concentration of insulin in the insulin injection is 0.1 IU/ml-1 IU/ml.

Further, the pharmaceutical preparation also contains heparin sodium, and the concentration of the heparin sodium is 40IU/ml-80 IU/ml.

Preferably, the tissue source of the mesenchymal stem cell comprises umbilical cord, umbilical cord blood, bone marrow, tooth, adipose tissue of human or animal.

Preferably, the mesenchymal stem cells are prepared by a primary isolation culture method.

In a second aspect of the present invention, there is provided a process for preparing said pharmaceutical formulation, comprising the steps of:

(1) taking the inactivated mammal, disinfecting the mammal with 75% ethanol, and separating the bone of the mammal;

(2) placing the skeleton in a culture dish, cleaning by using a physiological saline cleaning solution, and removing periosseous muscles and connective tissues to obtain the cleaned skeleton;

(3) immersing the cleaned bone into a cell culture solution, shearing joint heads (sockets) at two ends of the bone to expose a marrow cavity, and flushing the marrow cavity by using the cell culture solution to obtain dark red marrow;

(4) sucking the dark red bone marrow into a centrifuge tube, gently blowing and beating the dark red bone marrow to uniformly mix the bone marrow, and counting cells in the centrifuge tube;

(5) adjusting the concentration of cells to 2 × 10⁵Per cm²Bottom area, inoculating in a culture dish, culturing in a constant-temperature incubator, after culturing for 48 h, absorbing the supernatant, cleaning the cell surface with a physiological saline cleaning solution for 3 times, removing the non-adherent cells, injecting a fresh cell culture solution, putting back into the incubator for culturing, changing the solution for 1 time every 48 h, and culturing for 7-9 days to obtain primary culture cells;

(6) after the culture process in the step (5) is finished, pouring out the culture solution, washing the primary culture cells for 2-3 times by using a normal saline cleaning solution, sucking trypsin, injecting the trypsin into a culture dish, rubbing and vibrating the trypsin, blowing the trypsin by using a suction pipe until the primary culture cells are dispersed, and allowing the cells to suspend under a mirror, wherein the total digestion time is not more than 2 min;

(7) adding cell culture solution to terminate digestion, transferring the liquid into a centrifuge tube, centrifuging for 5 min at 800 r/min with a horizontal centrifuge, discarding the upper layer liquid, suspending cell mass with the cell culture solution, filtering with 200 mesh filter screen, removing the cell mass which can not be blown off, counting cells, centrifuging for 5 min at 800 r/min with a horizontal centrifuge, discarding the upper layer liquid, suspending the cells with physiological saline injection to density of 2 × 10⁷Per mL, obtaining the mesenchymal stem cellsAnd (4) refrigerating the injection for injection.

(8) Preparing insulin injection, and mixing the mesenchymal stem cell injection and the insulin injection according to the volume ratio of 1:1 to obtain the medicinal preparation. The pharmaceutical preparation needs to be injected within half an hour after preparation.

Preferably, the step (8) further comprises the step of adding heparin sodium into the pharmaceutical preparation, wherein the concentration of the heparin sodium is 40IU/ml-80 IU/ml.

Preferably, the mammal is selected from the group consisting of 8 week old C57 female mice; the bone is selected from femur, tibia or humerus.

Preferably, the cell culture fluid comprises the following components: 20% fetal bovine serum, 2 mmol/L-glutamine, 100. mu.g/mL penicillin sodium and 100U/mL streptomycin sulfate.

In a third aspect of the invention, the pharmaceutical preparation is provided for use in preparing a medicament for treating diabetes and complications, wherein the diabetes comprises type 1 diabetes, and the type 1 diabetes comprises islet injury, decreased insulin secretion level, increased blood glucose concentration and proportion of glycosylated hemoglobin; the diabetic complications include osteoporosis, renal failure, retinal degeneration, heart disease, stroke, nerve damage, non-healing infections, or ketoacidosis.

Compared with the prior art, the invention has the advantages that:

the invention breaks the fence between the drug treatment of type 1 diabetes and the stem cell treatment method, skillfully creates good action environment and opportunity for mesenchymal stem cells by applying insulin, so that the mesenchymal stem cells 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 consumption of the insulin is controlled, and hypoglycemia or insulin resistance caused by insulin injection is avoided. More importantly, the method can promote the treatment effect of the mesenchymal stem cells on the type 1 diabetes complications by controlling the host environment, obviously improve the target organ pathological changes of the type 1 diabetes, and can fundamentally achieve the purpose of treating the type 1 diabetes and the complications thereof.

Other features and advantages of the present invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

Drawings

FIGS. 1A and 1B are graphs comparing the blood glucose concentration of 4 groups of mice according to the present invention with time;

FIG. 1C is a graph comparing hemoglobin ratios of 4 groups of mice in example of the present invention;

FIG. 2A is a micrograph of a pancreatic tissue section of a type 1 diabetes control group according to an embodiment of the present invention;

FIG. 2B is a micrograph of a pancreatic tissue section of an insulin injection group according to an embodiment of the present invention;

fig. 2C is a micrograph of a pancreatic tissue section of a mesenchymal stem cell injection group according to an embodiment of the present invention;

FIG. 2D is a micrograph of a section of pancreatic tissue from an insulin + mesenchymal stem cell-injected group according to an embodiment of the present invention;

FIG. 3A is a graph comparing the change in islet area of 4 groups of mice in accordance with the present invention;

FIG. 3B is a graph showing a comparison of the changes in serum insulin concentrations in 4 groups of mice in the example of the present invention;

FIGS. 4A-4D are microscopic CT images of femurs of 4 groups of mice in the example of the present invention;

FIG. 5A is a graph showing a comparison of bone mass in 4 groups of mice in example of the present invention;

FIG. 5B is a graph comparing bone density of 4 groups of mice in example of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

The present embodiment provides a pharmaceutical formulation comprising the following components: mesenchymal stem cell injection and insulin injection. The mesenchymal stem cell injection is 1 part by volume, and the insulin injection is 1 part by volume. The components of the mesenchymal stem cell injection compriseMesenchymal stem cells and physiological saline, wherein the concentration of the mesenchymal stem cells in the mesenchymal stem cell injection is 1 multiplied by 10⁶Per ml or 1X 10⁷Per ml; the concentration of the insulin in the insulin injection is 0.1 IU/ml, 0.2 IU/ml, 0.3 IU/ml, 0.4 IU/ml, 0.5 IU/ml, 0.6 IU/ml, 0.7 IU/ml, 0.8 IU/ml, 0.9 IU/ml or 1 IU/ml.

Further, the present embodiment provides a method for preparing the pharmaceutical preparation, comprising the following steps:

(1) taking an 8-week-old C57 female mouse, inactivating, disinfecting with 75% ethanol for 3-5 minutes, placing a primary dissecting box on an ice block under an aseptic condition in an ultra-clean bench, cutting the skin of limbs of the mouse with an ophthalmologic scissors, carefully separating the muscle of the limbs with the ophthalmologic forceps and the ophthalmologic scissors, and separating the femur, the tibia and the humerus at the joint;

(2) placing the femur, the tibia and the humerus in a culture dish, cleaning by using 10ml of physiological saline cleaning solution, and removing periosseous muscles and connective tissues to obtain cleaned bones;

(3) immersing the cleaned bone into 10mL of cell culture solution, shearing joint heads (sockets) at two ends of the bone to expose a marrow cavity, and sucking the cell culture solution by using a 1 mL syringe to flush the marrow cavity to obtain dark red marrow;

(5) adjusting the concentration of cells to 2 × 10⁵Per cm²Bottom area, inoculating in culture dish, 37 deg.C, 5% CO₂Culturing in a constant-temperature incubator at saturated humidity for 48 h, absorbing the supernatant, cleaning the cell surface with PBS cleaning solution for 3 times, discarding the non-adherent cells, injecting fresh cell culture solution, culturing in the incubator, changing the solution for 1 time every 48 h, and culturing for 7-9 days to obtain primary culture cells;

(6) after the culture process in the step (5) is finished, pouring out the culture solution, washing the primary culture cells for 2-3 times by using a normal saline cleaning solution, sucking trypsin at 37 ℃, injecting the trypsin into a culture dish, then rubbing and vibrating the trypsin, blowing the trypsin by using a suction pipe until the primary culture cell colony is changed in a falling sand sample, and suspending the cells under a microscope, wherein the total digestion time is not more than 2 min;

(7) adding cell culture solution to terminate digestion, transferring the liquid into a centrifuge tube, centrifuging for 5 min at 800 r/min with a horizontal centrifuge, discarding the upper layer liquid, suspending cell mass with the cell culture solution, filtering with 200 mesh filter screen, removing the cell mass which can not be blown off, counting cells, centrifuging for 5 min at 800 r/min with a horizontal centrifuge, discarding the upper layer liquid, suspending the cells with physiological saline injection to density of 2 × 10⁷And (4) obtaining the mesenchymal stem cell injection per mL, and refrigerating for injection.

(8) Preparing insulin injection, and mixing the prepared mesenchymal stem cell injection and the insulin injection according to the volume ratio of 1:1 to obtain the pharmaceutical preparation.

The concentration of the prepared insulin injection is 0.1 IU/ml, 0.2 IU/ml, 0.3 IU/ml, 0.4 IU/ml, 0.5 IU/ml, 0.6 IU/ml, 0.7 IU/ml, 0.8 IU/ml, 0.9 IU/ml or 1 IU/ml.

The insulin injection described in this example was purchased from Novo Nordisk Producao clinical do music Ltd, Inc.: noohalin R (biosynthetic human insulin injection) with a specification of 3ML and a concentration of 1 IU/ML.

The cell culture fluid described in this example contained the following components: the alpha-MEM culture medium contains 20% fetal bovine serum (obtained by screening serum special for culturing human bone marrow mesenchymal stem cells), 2 mmol/L-glutamine, 100 mu g/mL penicillin sodium and 100U/mL streptomycin sulfate.

Example 2

The present embodiment provides a pharmaceutical formulation comprising the following components: mesenchymal stem cell injection, insulin injection and heparin sodium.

In this embodiment, the mesenchymal stem cell injection is 1 part by volume, and the insulin injection is 1 part by volume. The components of the mesenchymal stem cell injection of the embodiment comprise mesenchymal stem cells and normal saline, and the concentration of the mesenchymal stem cells in the mesenchymal stem cell injection of the embodiment is 1×10⁶Per ml or 1X 10⁷Per ml; the concentration of insulin in the insulin injection is 0.1 IU/ml, 0.2 IU/ml, 0.3 IU/ml, 0.4 IU/ml, 0.5 IU/ml, 0.6 IU/ml, 0.7 IU/ml, 0.8 IU/ml, 0.9 IU/ml or 1 IU/ml. The concentration of heparin sodium in the pharmaceutical preparation is 40IU/ml, 50 IU/ml, 60 IU/ml, 70 IU/ml or 80 IU/ml.

referring to the steps (1) to (7) in the preparation method of the pharmaceutical preparation in embodiment 1 of the present invention, the mesenchymal stem cell injection is prepared, and the pharmaceutical preparation is obtained after mixing the mesenchymal stem cell injection, the insulin injection and the heparin sodium according to the formula ratio.

The concentration of the insulin injection in the embodiment is 0.1 IU/ml, 0.2 IU/ml, 0.3 IU/ml or 0.4 IU/ml.

EXAMPLE 3 therapeutic experiments on type 1 diabetic mice with the pharmaceutical formulations of the present invention

First, model mouse type 1 diabetes (the following drugs were purchased from Sigma, USA)

1. Preparing a sodium citrate buffer solution: 2.1 g of citric acid and 2.94 g of sodium citrate, adding deionized water to a constant volume of 200 mL, and adjusting the pH value to 4.2-4.5. Filtering, sterilizing, and storing in refrigerator at 4 deg.C.

2. Preparing streptozotocin injection: the injection is prepared freshly before injection, streptozotocin is weighed under the condition of keeping out of the sun and dissolved in sodium citrate buffer solution by 5 mg/mL, and the mixture is placed on ice to be stored in the sun and is injected within 30 min.

3. C57 female mice with the age of 8 weeks are taken, and tail vein blood collection needles are sampled 24 h before molding (experiment day 0) to detect random blood sugar concentration.

4. The injection is administered by intraperitoneal injection of 50 mg/kg/d streptozotocin for 5 days (figure 1A). When the mouse is injected into the abdominal cavity, the head of the mouse is kept low, the needle is inserted into the lower part of the right side of the abdomen and is about 1 cm away from the midline of the abdomen, and the needle insertion depth is not more than 1/2 of a 1 mL syringe needle.

5. From the 3 rd day of the experiment, sampling a tail vein blood taking needle at the same time every 3 days to detect random blood sugar concentration, and defining mice with blood sugar concentration above 250 mg/dL for 3 times of continuous measurement as type 1 diabetes mice to be included in subsequent experiments.

Injection of mesenchymal stem cell injection

1. 24 type 1 diabetic mice were selected for inclusion in the experiment and were completely randomized into 4 groups (6 per group): a type 1 diabetes control group, an insulin injection group, a mesenchymal stem cell injection group and a medicinal preparation (insulin + mesenchymal stem cell) injection group. Mice were injected with tail vein every 3 days at the same time, blood sampling needles were sampled to detect random blood glucose concentration and recorded.

2. On the 25 th day of experiment, different groups of mice received blood glucose concentration detection and recording by sampling tail vein blood taking needles respectively before injection (0 th min) and at 15 th, 30 th, 60 th, 90 th and 120 th min after injection.

Third, sample drawing detection

1. On day 54 of the experiment (i.e., about 1 month after injection and after the last blood glucose measurement), one eyeball was removed from the mouse under anesthesia, whole blood was removed from the retrobulbar sinus without anticoagulation, and the mouse was allowed to stand at room temperature for 30 min, and then placed in a centrifuge at 3000 r/min and centrifuged for 15 min to separate serum. The supernatant was collected and placed in a centrifuge at 12000 r/min for 15 min to completely remove the cellular components. The serum sample obtained at last is light yellow and clear liquid, and is subpackaged and frozen at-80 ℃ to avoid repeated freezing and thawing.

2. Pancreatic tissue from mice was taken and fixed overnight in 4% paraformaldehyde.

3. After pancreatic tissue was removed, the mouse femurs were harvested, stripped of surrounding muscle and connective tissue, and fixed in 4% paraformaldehyde overnight.

4. Washing and dehydrating the fixed pancreatic tissues, embedding paraffin, selecting days to carry out slicing and HE staining, photographing and observing, and quantitatively analyzing the area proportion of the pancreatic tissues by adopting ImageJ software.

5. And (3) flushing the fixed pancreatic tissues, flushing the fixed thighbone together, cutting off the proximal end of the thighbone by a slow cutting machine, and reserving a tissue block with the length of about 1 cm from the distal end of the thighbone. The sample was then subjected to micro CT examination with a resolution set at 8 μm, a voltage of 80 kV and a current of 80 μ A. And performing three-dimensional reconstruction on the obtained scanning image, and quantitatively analyzing the bone mass and the bone density value of the cancellous bone area by adopting Micview V2.1.2 software.

6. Serum glycated hemoglobin and insulin levels were measured on the selected day. Serum samples were thawed on ice, glycated hemoglobin, total protein, insulin detection experiments were performed according to the ELISA related kits (all purchased from RB, USA), absorbance was measured at 450 nm with a spectrophotometer, a standard curve was calculated, and results were quantitatively analyzed.

Fourth, 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. Blood glucose assay results (see fig. 1A, fig. 1B): the baseline blood glucose levels in all groups of mice were about 120 mg/dL, with blood glucose rising gradually since type 1 diabetes molding, reaching the 250 mg/dL limit of diabetes on day 9 of the experiment, and reaching and stabilizing at a high blood glucose level of about 400 mg/dL on day 18. Thereafter, the blood glucose concentration of the type 1 diabetes control mice fluctuated around 400 mg/dL until the end of the experiment; the blood sugar concentration of the mice in the insulin injection group is instantly reduced to hypoglycemia after the insulin injection, wherein the blood sugar concentration reaches a valley value of about 120 mg/dL at 60 min after the injection, and the hyperglycemia is recovered at 120 min after the injection until the experiment is finished; the blood glucose concentration of the mice in the mesenchymal stem cell injection group slowly declined after the mesenchymal stem cell injection, declined to the diabetes limit of 250 mg/dL on the 30 th day of the experiment (5 th day after the injection), declined to a baseline level of about 120 mg/dL on the 39 th day (14 th day after the injection) and fluctuated to the end of the experiment; the blood glucose concentration of the mice in the insulin + mesenchymal stem cell injection group is instantaneously reduced along with the insulin injection, and is slowly reduced along with the mesenchymal stem cell injection for a long time, and finally is reduced to a baseline level of about 120 mg/dL and fluctuates to the end of the experiment.

3. Glycated hemoglobin assay results (fig. 1C): the proportion of glycated hemoglobin in the mice of the type 1 diabetes control group is higher than about 6% of the critical line, reaching about 9%. The glycated hemoglobin level of the insulin injection group mice was not decreased, while the glycated hemoglobin level of the mesenchymal stem cell injection group and the insulin + mesenchymal stem cell injection group mice was decreased to less than 6% compared to the type 1 diabetes control group mice.

4. Results of islet histology (fig. 2A-2D and fig. 3A): the islet area proportion of the type 1 diabetes control group mice is less than 10%, and only small islet tissues are scattered and distributed. Compared with the mice of the type 1 diabetes control group, the islets of langerhans of the mice of the insulin injection group are hardly recovered, while the islet areas of the mice of the mesenchymal stem cell injection group and the insulin + mesenchymal stem cell injection group are recovered to about 20 percent, and rope-like and large-block islet tissues appear.

5. Serum insulin assay results (fig. 3B): the insulin level of the mice in the type 1 diabetes control group is lower than 40 mIU/L. Compared with the mice of the type 1 diabetes control group, the serum insulin level of the mice of the insulin injection group is not improved, while the serum insulin level of the mice of the mesenchymal stem cell injection group and the insulin + mesenchymal stem cell injection group is increased by about 10 mIU/L.

6. Microscopic CT analysis results (fig. 4A-5B): the type 1 diabetes control group mice have severe osteoporosis, the bone mass proportion is less than 5 percent, and the bone density is less than 100 mg/cm³. Compared with the mice of the type 1 diabetes control group, the bone mass and the bone density of the mice of the insulin injection group and the mesenchymal stem cell injection group are hardly improved, and the bone loss of the mice of the insulin + mesenchymal stem cell injection group can be recovered to reach about 13 percent of bone mass proportion and 180 mg/cm³The bone density of (a).

Fifth, summarize

1. The animal experiments prove that the mesenchymal stem cells and the insulin can be used for treating type 1 diabetes and complications thereof. In the invention, the mesenchymal stem cell system can restore normal blood sugar level by regenerating islet tissues and promoting insulin secretion, but the effect is slow, and the bone complications cannot be treated. However, after the mesenchymal stem cells are injected in combination with insulin, the treatment effect of the mesenchymal stem cells is enhanced, and the mesenchymal stem cells not only can treat type 1 diabetes, but also can play a treatment effect on target organs of complications.

2. The invention firstly proposes the treatment application effect of promoting the mesenchymal stem cells under the condition of rapidly controlling the blood sugar environment in the body of a patient, and can solve the problem of synchronous treatment of diabetes and complications thereof. The mesenchymal stem cells used in the invention have wide sources, are not limited by ethics and have low immunogenicity, and the insulin is also a first-line medicament for controlling the blood sugar of clinical type 1 diabetes. Therefore, the insulin + mesenchymal stem cell combined treatment method has wide prospect in future clinical application.

3. In this specification, the invention has been described with reference to specific embodiments, but it will be apparent that various modifications and changes can be made without departing from the spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.

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

1. A pharmaceutical formulation, comprising the following components: mesenchymal stem cell injection and insulin injection.

2. The pharmaceutical preparation of claim 1, wherein the mesenchymal stem cell injection is 1 part by volume and the insulin injection is 1 part by volume.

3. The pharmaceutical preparation of claim 1, wherein the components of the mesenchymal stem cell injection comprise mesenchymal stem cells and physiological saline, and the concentration of the mesenchymal stem cells in the mesenchymal stem cell injection is 1 x 10⁶1X 10 pieces/ml⁷Per ml; the concentration of insulin in the insulin injection is 0.1 IU/ml-1 IU/ml.

4. The pharmaceutical formulation of claim 1, further comprising heparin sodium at a concentration of 40IU/ml to 80 IU/ml.

5. The pharmaceutical preparation of claim 3, wherein the tissue source of the mesenchymal stem cells comprises human or animal umbilical cord, umbilical cord blood, bone marrow, teeth, adipose tissue.

6. The pharmaceutical preparation of claim 3, wherein the mesenchymal stem cells are prepared by a primary isolation culture method.

7. A process for the preparation of a pharmaceutical formulation according to any one of claims 1 to 6, characterized in that it comprises the following steps:

(7) adding cell culture solution to terminate digestion, transferring the liquid into a centrifuge tube, centrifuging for 5 min at 800 r/min with a horizontal centrifuge, discarding the upper layer liquid, suspending cell mass with the cell culture solution, filtering with 200 mesh filter screen, removing the cell mass which can not be blown off, counting cells, centrifuging for 5 min at 800 r/min with a horizontal centrifuge, discarding the upper layer liquid, suspending the cells with physiological saline injection to density of 2 × 10⁷Obtaining the mesenchymal stem cell injection per mL, and refrigerating for injection;

(8) preparing an insulin injection, and mixing the mesenchymal stem cell injection and the insulin injection according to the volume ratio of 1:1 to obtain the pharmaceutical preparation.

8. The method according to claim 7, wherein the step (8) further comprises a step of adding heparin sodium to the pharmaceutical preparation, and the concentration of the heparin sodium is 40IU/ml to 80 IU/ml.

9. The method according to claim 7, wherein in the step (1), the mammal is selected from the group consisting of 8-week-old C57 female mice; the bone is selected from femur, tibia or humerus.

10. The method according to claim 7, wherein the cell culture solution comprises the following components: 20% fetal bovine serum, 2 mmol/L-glutamine, 100. mu.g/mL penicillin sodium and 100U/mL streptomycin sulfate.

11. The use of the pharmaceutical formulation of claim 1 for the manufacture of a medicament for the treatment of diabetes and complications thereof, wherein said diabetes comprises type 1 diabetes, and wherein said type 1 diabetes comprises damage to the islets of langerhans, decreased levels of insulin secretion, increased blood glucose concentration and proportion of glycated hemoglobin; the diabetic complications include osteoporosis, renal failure, retinal degeneration, heart disease, stroke, nerve damage, non-healing infections, or ketoacidosis.