CN110585242A - Application of multisystem differentiation continuous stress cells, medicine for treating diabetes and preparation method of medicine - Google Patents

Abstract

The invention provides the application of multi-line differentiated continuous stress cells in the preparation of medicines for treating diabetes. The present invention also provides a medicine for treating diabetes, which is characterized in that it at least includes cell suspension of multilineage differentiated continuous stress cells. A medicine for treating diabetes of the present invention may also contain pharmaceutically acceptable auxiliary materials. The present invention also provides a method for preparing a drug for treating diabetes, which is characterized by comprising the following steps: (1) preparing and culturing multilineage differentiated persistent stress cells; (2) preparing multilineage differentiated persistent stress cells Suspension. The application of the multi-line differentiated persistent stress cell in the preparation of the medicine for treating diabetes provides a new direction for the treatment of diabetes.

Description

Application of multilineage differentiated persistent stress cells, drug for treating diabetes and its preparation method

technical field

The invention belongs to the technical field of biomedicine, and specifically relates to the application of multilineage differentiated persistent stress cells in the preparation of drugs for treating diabetes, the drug for treating diabetes and a preparation method thereof.

Background technique

Diabetes is caused by genetic factors, immune dysfunction, microbial infection and its toxins, free radical toxins, mental factors and other pathogenic factors acting on the body to cause hypofunction of pancreatic islets and insulin resistance. Clinically, hyperglycemia is the main feature. Typical cases may show polyuria, polydipsia, polyphagia, weight loss, etc., that is, "three more and one less" symptoms. Once diabetes (blood sugar) Poor control will lead to complications, leading to failure of the kidneys, eyes, feet and other parts of the disease, and can not be cured. Diabetes is divided into type 1 diabetes, type 2 diabetes, gestational diabetes and other special types of diabetes. Type 2 diabetes accounts for approximately 95% of diabetic patients.

The number and incidence of diabetes in China rank first in the world. According to a survey in JAMA magazine in 2013, nearly 12% of Chinese adults (approximately 113.9 million people) suffer from diabetes. The number of diabetic patients in China accounts for 1/3 of the global diabetes patients (in 2012, there were more than 371 million diabetic patients in the world). In October 2018, the well-known foreign journal "Diabetes and Metabolism" published a research report, the data of which came from the National Center for Disease Control and Prevention (CDC). The study pointed out that from 2000 to 2016, the prevalence of diabetes and the risk of death in China increased by 60%. In 2016 alone, 140,000 people died from diabetes. At present, the treatment of diabetes is mainly symptomatic treatment, and patients need to take medicine for life. At present, there is no medicine that can treat diabetes thoroughly, long-actingly, and without side effects. Patients urgently need more effective treatments. Therefore, finding and developing new drugs for treating diabetes has important medical significance and far-reaching social significance.

Contents of the invention

The present invention provides the application of multi-lineage differentiated persistent stress cells in the preparation of the medicine for treating diabetes, the medicine for treating diabetes and its preparation method, so as to solve the problems raised in the background technology.

In order to solve the above technical problems, the embodiments of the present invention firstly provide the application of multi-lineage differentiated persistent stress cells in the preparation of drugs for treating diabetes.

Further, the multilineage differentiated persistent stress cells are derived from mesenchymal tissues of bone marrow, fat, umbilical cord and dermis. The multilineage differentiated sustained stress cells to be protected in the present invention are not limited to mesenchymal tissues derived from bone marrow, fat, umbilical cord or dermis, and the multilineage differentiated sustained stress cells used in the embodiments of the present invention are derived from bone marrow.

Further, the multilineage differentiated persistent stress cells grow in suspension into spheres or adhere to the wall. The multiline differentiated continuous stress cells of the present invention express SSEA-3, Oct3/4, Sox2, CD105 and CD90, but do not express CD34, CD45 and CD11b.

Another medicament for treating diabetes according to the embodiment of the present invention is characterized in that it at least includes a cell suspension of multilineage differentiated persistent stress cells. A medicine for treating diabetes of the present invention may also contain pharmaceutically acceptable auxiliary materials.

Further, the drug dosage of the multilineage differentiated persistent stress cell suspension is 5~10×106 cells/kg body weight based on the number of multilineage differentiated persistent stress cells, and local injection (intrathecal injection, subcutaneous injection, intramuscular injection, etc.) Injection, etc.) 1~5 times; or 5~10×107/kg body weight, intravenous infusion 1~5 times.

The embodiment of the present invention also provides a preparation method of a drug for treating diabetes, which is characterized in that it includes the following steps: (1) preparing and culturing multi-lineage differentiated persistent stress cells; (2) preparing multi-lineage differentiated persistent stress cells cell suspension of cells.

Specifically, the method for preparing multilineage differentiated sustained stress cells in step (1) of the present invention is as follows:

The method disclosed in the reference (Kuroda, et al., PNAS, 2010, 107:8639-8643.) prepared bone marrow cells, using the characteristics of multilineage differentiated persistent stress cells expressing SSEA-3+/CD105+/CD45- Bone marrow cells were sorted by the advanced cell sorting technology, and the sorted multi-lineage differentiated continuous stress cells were cultured in suspension using a-MEM + 0.9% MethoCult H4100 + 20% FBS medium. Cells began to aggregate after 1 day of culture. A single cell aggregates into a cluster to form a cell colony, and then the cell colony divides and grows to form a stem cell sphere. The number of cells gradually increases in 3-5 days, and the cell sphere gradually increases, showing a large and transparent shape. The sphere formation time of multilineage differentiated continuous stress cells after subculture is significantly shorter than that of the first generation cells. From the second generation, cell spheres can generally be formed within 2 days. The pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox2 were used to identify multilineage differentiated persistent stress cells.

The multi-lineage differentiated continuous stress cells prepared by the method of the present invention have good passage stability, and studies have shown that the cell properties remain stable after 60 generations of passage, which is reflected in: 1. Cell spheres can still grow in suspension; 2. MUSE cells within 5 passages had the same analgesic effect; 3. Protein profile analysis showed that compared with MUSE cells passaged 5 passages, there were no statistical differences in the protein expression of cytokines, chemokines and receptors.

Further, the cell suspension of the multilineage differentiated persistent stress cells in the step (2) is prepared according to the following process: the multilineage differentiated persistent stress cells cultured in the step (1) are digested with 0.25% trypsin-EDTA A single cell suspension was prepared, washed with normal saline, and then suspended with normal saline to obtain a multilineage differentiated continuous stress cell suspension.

Further, the cell concentration of the multilineage differentiated persistent stress cells in the multilineage differentiated persistent stress cell suspension is 1-10×10 ⁷ cells/ml.

The beneficial effects of above-mentioned technical scheme of the present invention are as follows:

(1) The application of the multilineage differentiated persistent stress cells of the present invention in the preparation of drugs for treating diabetes provides a new option for clinically treating diabetes.

(2) The multilineage differentiated persistent stress cells in the present invention are a kind of adult stem cells newly discovered in adult bone marrow and dermal tissue in 2010, and have the following characteristics: 1) Specifically express stage specific embryonic antigen 3 (stage specificembryonic Antigen-3, SSEA3) and CD105; 2) Stress tolerance; 3) Single cells can form cell spheres and have strong self-renewal ability; 4) Can differentiate into cells of inner, middle and outer germ layers , but no teratomas were formed in nude mice. The present invention finds that multilineage differentiated persistent stress cells can effectively treat diabetes; injection of multilineage differentiated persistent stress cells can promote the recovery of diabetic islet cells and lower blood sugar, which has great clinical application value.

(3) In the examples of the present invention, the streptozotocin-induced diabetic mouse model was used to prove for the first time that transplantation of multi-lineage differentiated persistent stress cells by local injection or intravenous injection can significantly inhibit the diabetic model mice for a long time. It can protect damaged islet cells, lower blood sugar, and inhibit early hyperalgesia and late stocking-like sensory disturbance caused by diabetes.

(4) The stable passage characteristics of the multilineage differentiated persistent stress cells described in the present invention are extremely beneficial to the requirement of providing a large number of standardized cell products for the preparation of drugs for treating diabetes.

Description of drawings

Figure 1 is a diagram of the identification results of human multilineage differentiated sustained stress cells (MUSE cells) in an example of the present invention; among them, Figure 1A is a collection of human bone marrow cells cultured to the third generation, using multilineage differentiated continuous stress The characteristics of the SSEA-3+/CD105+ cells are sorted by flow cytometry; Figure 1B shows that the sorted multilineage differentiated continuous stress cells can form cells from sorted single cells in suspension culture Figure 1C is a diagram of cell morphology when the suspension cell spheres of multi-lineage differentiated continuous stress cells were digested into single cells and cultured on the wall.

Fig. 2 is the immunofluorescence staining picture of the pancreatic tissue frozen section in the embodiment of the present invention; Wherein, what Anti-Insulin marks is the beta cell of pancreatic islet, and what Anti-Glucagon marks is the alpha cell of pancreatic islet; STZ-induced mouse diabetes model It mainly damages the beta cells of pancreatic islets, and the intravenous injection of Muse cells can protect the damaged beta cells of pancreatic islets.

Fig. 3 is a diagram showing the results of blood glucose detection in mice in the embodiment of the present invention.

Detailed ways

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will describe in detail in conjunction with specific embodiments.

Example 1

Preparation of multilineage differentiated persistent stress cells

Professional doctors performed bone marrow aspiration on the donor's anterior superior iliac spine, and extracted 1ml of bone marrow. Mix the bone marrow and phosphate buffered saline solution at a ratio of 1:1 and add it to the upper layer of the lymphocyte separation medium. After centrifugation, the liquid is divided into four layers. The uppermost layer accounts for about 1/2 of the liquid column and is a light red plasma layer; Accounting for 1/4 of the liquid column, it is a clear lymphocyte separation liquid layer; at the junction of the two layers is a milky white cloud-like layer with a height of about 5 mm, which is the mononuclear cell layer (this layer is the cell layer to be extracted); The bottom layer is tightly attached to the wall of the test tube and is dark red, mainly composed of denser red blood cell layer. Take the cells in the second layer, use DMEM/F12 complete medium containing 15% FBS, and culture them in a constant temperature incubator at 37°C and 5% CO ₂ for 24 hours. Scattered single cells can be seen attached to the wall; 2-3 days later The parietal cells began to divide and proliferate, and gradually stretched from the initial round shape, with irregular orientation of the two poles of the cells, irregular shape, and mixed cells around; after 4-7 days, the cells grew in a colony-like manner, most of which were spindle-shaped; after 1 week, the cells were flat, Multiple protrusions, cell bodies fused with each other. After the third passage, the cells were basically single in shape and showed a spindle shape, and about 95% of the cells were CD90+/CD45-/CD11b- detected by flow cytometry. The cells cultured to the third generation were collected, and the characteristics of Muse cells SSEA-3+/CD105+ were sorted by flow cytometry technology, and the sorted Muse cells were cultured in suspension, using a-MEM +0.9%MethoCult In H4100 + 20% FBS medium, cells began to aggregate after 1 day, and single cells aggregated into clusters to form cell colonies, and then the cell colonies divided and grew to form stem cell spheres. The number of cells gradually increased in 3-5 days, and the cell spheres gradually increased, showing a large And transparent shape. The sphere formation time of Muse cells after subculture is significantly shorter than that of the first generation cells. From the second generation, cell spheres can generally be formed within 2 days. Muse cells were identified by pluripotent stem cell markers SSEA-3, Nanog, Oct4 and Sox2.

The identification results are shown in Figure 1, in which Figure 1A is the result of collecting and culturing human bone marrow cells up to the third passage, and using the characteristics of multi-lineage differentiated continuous stress cells SSEA-3+/CD105+ to sort by flow cytometry. The results indicated that about 0.89% of the cultured bone marrow cells were multilineage differentiated persistent stress cells. Figure 1B shows that the sorted multilineage differentiated continuous stress cells can form cell spheres from the sorted single cells in suspension culture. This result indicates that the multilineage differentiated persistently stressed cells can be massively expanded in vitro. Figure 1C is the cell morphology when the suspension cell spheres of multi-lineage differentiated persistent stress cells were digested into single cells and cultured on the wall. The results indicated that multilineage differentiated continuous stress cells could also be cultured adherently, and the cell morphology was consistent. available for further experiments.

Example 2

Multilineage differentiated continuous stress cells for the treatment of diabetic model mice

1) Streptozotocin-induced mouse diabetes model

Streptozotocin (STZ) injection modeling: STZ selectively destroys certain species of pancreatic islet β cells, which can cause diabetes in many animals. Because of its low tissue toxicity and high animal survival rate, it is an ideal animal model that is widely used at home and abroad to study diabetes and its chronic complications. The preparation of animal models of type 1 diabetes and type 2 diabetes is related to the dose of STZ injection: when a large dose of STZ is injected, it will directly cause extensive damage to the islet β cells, resulting in a type 1 diabetes model; while injecting a small amount of STZ, due to It only destroys the function of a part of the islet β cells, making the peripheral tissue insensitive to insulin. At the same time, it is fed with high-calorie feed. The combination of the two induces an animal model with pathological and physiological changes close to human type 2 diabetes.

Streptozotocin preparation method: take 2.1g of citric acid (FW:210.14) and add it to 100mL of double distilled water to make solution A; take 2.94g of sodium citrate (FW:294.10) and add it to 100mL of double distilled water to make solution B . Prepare citric acid buffer solution when injecting: mix A and B solutions in a certain ratio (1:1.32), measure the pH value with a pH meter, and adjust ph=4.2-4.5, which is the citric acid buffer solution required to configure STZ.

Streptozocin (STZ)-induced diabetes model preparation: STZ was dissolved according to the final concentration (fasting body weight 80 mg/kg), the mice were continuously anesthetized with isoflurane gas on the operating platform, and were treated within 1 minute. The intraperitoneal injection was completed. A total of 3 injections of STZ were given with an interval of 24 hours.

2) Muse cell transplantation

Two weeks after injection of STZ, the blood glucose level of the mice was detected. Modeling was successful if the blood glucose level was above 25mmol/L after fasting for 12 hours (the blood glucose level of normal mice was less than 10mmol/L after fasting for 12 hours). After the Muse cells were resuspended in PBS, 5~10×107 cells/kg body weight were infused intravenously within 10 minutes.

3) Histopathological detection of mouse pancreas

The mice were perfused with 4% formaldehyde solution and the pancreas tissues were collected. Placed in 4% paraformaldehyde and fixed overnight at 4°C. Then the pancreas tissues were put into 10%, 20% and 30% sucrose solutions prepared by 0.1 M PB for dehydration, then frozen sections, immunohistochemical staining, and observation under a fluorescent microscope. The results are shown in Figure 2. Compared with the model group injected with normal saline, the number of pancreatic cells in the model group injected intravenously with Muse cells increased, suggesting that the transplantation of Muse cells can protect damaged pancreatic cells.

4) Blood glucose detection in mice

After the mice were fasted for 12 hours, 75% ethanol was used to disinfect the back of the tail, and after drying, the tip of the tail was cut off, and a drop of naturally flowing blood was taken to measure the glucose concentration in the whole blood with a rapid blood glucose meter. The results are shown in Figure 3. Compared with the model group injected with normal saline, the blood sugar content in the model group injected intravenously with Muse cells was significantly lower, suggesting that the transplantation of Muse cells can lower blood sugar. It can be seen from Figure 3 that after intraperitoneal injection of Muse cells, the blood sugar level of diabetic model mice can be significantly reduced for a long time.

To sum up, transplantation of multilineage differentiated persistent stress cells by intravenous injection can effectively treat diabetes, promote the recovery of damaged pancreatic tissue, and reduce blood sugar for a long time.

The above description is a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (8)

1. Application of multi-differentiated sustained stress cells in preparation of medicines for treating diabetes is provided.

2. The use of the multi-lineage differentiation continuously stressed cell according to claim 1, wherein the multi-lineage differentiation continuously stressed cell is derived from mesenchymal tissue of bone marrow, fat, umbilical cord, and dermis.

3. The use of the multi-lineage differentiated continuously stressed cell according to claim 1, wherein the multi-lineage differentiated continuously stressed cell is suspended in spherulitic or adherent growth for the treatment of diabetes.

4. A medicament for the treatment of diabetes comprising at least a cell suspension of a plurality of differentiated continuously stressed cells.

5. The agent for treating diabetes mellitus as claimed in claim 4, wherein the amount of said agent for suspension of multisystem differentiation-sustaining stress cells is 5 ~ 10X 10 in terms of the number of multisystem differentiation-sustaining stress cells⁶Injecting the mixture 1 ~ 5 times or 5 ~ 10X 10 times per kg body weight⁷One/kg body weight, 1 ~ 5 times by intravenous infusion.

6. A method for preparing a medicament for the treatment of diabetes according to claim 4, comprising the steps of: (1) preparing and culturing multisystem differentiation sustained stress cells; (2) preparing cell suspension of multi-series differentiation sustained stress cells.

7. The method for preparing a medicine for treating diabetes according to claim 6, wherein the cell suspension of the multi-lineage differentiation stress-sustaining cells in the step (2) is prepared by the following process: digesting the multisystem differentiation continuous stress cells cultured in the step (1) by using 0.25% trypsin-EDTA to prepare single cell suspension, washing by using normal saline, and suspending by using the normal saline to obtain multisystem differentiation continuous stress cell suspension.

8. The method of claim 6, wherein the cell concentration of the multi-lineage differentiation stress-sustaining cells in the multi-lineage differentiation stress-sustaining cell suspension is 1 ~ 10X 10⁷ One per ml.