CN112280742A

CN112280742A - Anti-aging pharmaceutical composition or health product prepared from stem cells

Info

Publication number: CN112280742A
Application number: CN202011219730.2A
Authority: CN
Inventors: 张海涛; 陈帅
Original assignee: Beijing Xinsong Biotechnology Co ltd
Current assignee: Beijing Xinsong Biotechnology Co ltd
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-01-29

Abstract

The invention relates to an anti-aging pharmaceutical composition or health product prepared from stem cells. According to the invention, miRNA-16 is transfected into umbilical cord mesenchymal stem cells, so that the transfected umbilical cord mesenchymal stem cells can improve the anti-aging function of the stem cells on one hand, improve the activity of the stem cells on the other hand, and improve the anti-aging function of human, and the anti-aging characteristic is realized in a high-sugar environment, so that the anti-aging stem cells are suitable for anti-aging treatment of diabetes patients, and have great market value.

Description

Anti-aging pharmaceutical composition or health product prepared from stem cells

Technical Field

The invention relates to the field of medicines and health-care products, in particular to an anti-aging medicine composition or health-care product prepared from stem cells.

Background

Aging is a process and phenomenon in which the body exhibits progressive structural degeneration and hypofunction with age. Population aging has become a serious political, social, economic and health problem, and it is an urgent problem to find a technology for effectively improving the quality of life of the elderly and reversing or delaying aging. The stem cells have the potential of self-renewal and multidirectional differentiation, and theoretically have the feasibility of participating in and promoting tissue cell regeneration, injury repair and reversing human body aging.

Mesenchymal Stem Cells (MSCs) are pluripotent stem cells derived from early-developing mesoderm and ectoderm, and MSCs are initially found in bone marrow and are increasingly receiving attention due to their characteristics such as multipotentiality, hematopoietic support, and immune regulation. The mesenchymal stem cells can differentiate various tissue cells under in vivo or in vitro specific induction conditions, and can be used as ideal seed cells for repairing tissue and organ damage caused by aging and pathological changes. Research shows that bone marrow mesenchymal stem cells (BM-MSCs) have anti-aging effect on D-galactose aging model mice. With the progress of research on MSCs, scientists found that umbilical cord mesenchymal stem cells (UC-MSCs) have many advantages over BM-MSCs such as no tumor contamination, strong proliferation capacity, convenient collection, low immunocompetence, no need of formulation, etc., and umbilical cord as medical waste has no adverse effect on donors and is not limited by ethical issues, so that UC-MSCs are very likely to become an ideal substitute for BM-MSCs.

However, stem cells themselves also present aging problems. Cellular senescence is a cellular state in which cells respond to a variety of stressors, including genotoxic factors, nutrient deficiency, hypoxia, mitochondrial dysfunction, and oncogene activation, among others. Research shows that the aging affects the functions of the MSCs, including proliferation capacity, clonogenic capacity, differentiation potential, immunological characteristics, telomerase activity, cell migration capacity, adhesion and the like, and is reflected in the attenuation of the cell proliferation capacity and the differentiation potential. The literature indicates that the expressions of MSCs surface specific markers CD44, CD90, CD105, Stro-1 and the like are reduced after aging; the cell morphology is gradually flattened and enlarged, the cell membrane components are changed, and the content of the integral protein caveolin-1 in the membrane is up-regulated; cytosolic pH changes, granule shrinkage; increased lysosomal spacing and increased activity, increased β -galactosidase (β -gal) production; lipofuscin is accumulated in the lysosome, and the autofluorescence level of cells is increased; mitochondria are large but dysfunctional, leading to excessive Reactive Oxygen Species (ROS) production; meanwhile, due to the deletion of lamin B1, the integrity of the nucleus is damaged, chromatin fragments appear in cytoplasm, telomerase is obviously shortened, and the like. In conclusion, the ultimate outcome of senescent MSCs is to stop proliferation, resulting in the cessation of the role of stem cells for the treatment of senescence.

The aging-prone characteristic of in vitro culture of the MSCs limits the amplification quantity of the MSCs, thereby limiting the clinical application of the MSCs. How to make aged MSCs reproduce a state of rejuvenation to prolong its utility value has become one of research hotspots in regenerative medicine. Romanov et al pioneered the use of human mammary epithelial cells to explore whether the state of cellular senescence could be reversed. Since then, more and more scholars have tried to delay the aging process of MSCs by adding bioactive substances or drugs, etc. Lysophosphatidic acid (LPA) is a metabolite in membrane phospholipid synthesis, and studies have shown that LPA participates in and maintains normal physiological functions of the body. The MSCs preferentially express LPA receptor subtype 1, and by adding the receptor antagonist Ki16425, the expression of cell senescence-associated p16Ink4a, Rb, p53 and p21Cip1 in hBMSCs is inhibited, so that the senescence state of the hBMSCs cultured in vitro continuously is obviously slowed down. The combination of the bioactive molecular carrier silk protein and curcumin can delay the aging process of BMSCs, and the delay degree has a certain relation with curcumin dosage and stimulation time, but the specific mechanism needs to be further researched. Rapamycin (rapamycin, RAPA) is an mTOR inhibitor and has been shown to have the effect of delaying cell senescence, probably through the inhibition of AKT/mTOR to regulate intracellular ROS production, expression of pluripotent genes Nanog and Oct-4 and accumulation of DNA damage. Researches of Cao Yulin and the like find that the cell volume of the UCMSCs with replicative senescence is reduced after being treated by RAPA, beta-gal staining positive cells are reduced, the expression of p53 is reduced, and the expression of blood vessel related formation factors is increased; in vivo experiments of mice show that the RAPA treatment group can enhance angiogenesis in ischemic areas compared with a control group, and the RAPA can be used as a new means for delaying the aging state of MSCs. Zeng-Ying et al also obtained the same results by using Ku0063794 and RAPA to act on aging BMSCs. RAPA can also obviously improve the aging phenotype and the immunoregulation function of BMSCs from systemic lupus erythematosus patients by inhibiting mTOR signaling pathway, and brings new hope for curing the diseases. However, these drugs are all the effects of drugs, and the drugs themselves have uncertain side effects or liver damage to human bodies, so that there are many obstacles to popularization and application.

Disclosure of Invention

The invention overcomes the defects of the prior art, on one hand, the anti-aging function of the stem cells is improved by improving the anti-aging activity of the stem cells, and on the other hand, the anti-aging function of people is improved while the activity of the stem cells is improved.

The method comprises the steps of continuously culturing the umbilical cord mesenchymal stem cells in a high-sugar microenvironment high-sugar culture medium for 16 generations to simulate the cell aging state of a diabetic patient, detecting the miRNA expression of the cells, carrying out statistical analysis on the nuclear miRNA expression conditions before and after aging, screening the miRNA with the difference multiple of more than 2 times and P of less than 0.001 as the miRNA with the obvious difference in expression, and further carrying out differential expression analysis on miRNA expression profile data. Furthermore, 2 miRNAs with the most obvious difference are screened out as a research basis for anti-aging research.

In one aspect of the invention, the application of the umbilical cord mesenchymal stem cells transformed with miR-16 in the preparation of a pharmaceutical composition is claimed; the pharmaceutical composition functions as follows:

(a) delaying aging;

(b) delaying cell aging;

(c) and (3) delaying the aging of the mesenchymal stem cells.

The invention provides a preparation method of miR-16-transformed umbilical cord mesenchymal stem cells, which comprises the following steps:

(1) improving the content of miR-16 in umbilical cord mesenchymal stem cells; or

(2) Reducing the expression of the encoding gene for inhibiting the expression of miR-16 in the umbilical cord mesenchymal stem cells.

The invention further provides application of the miR-16-transformed umbilical cord mesenchymal stem cells in preparation of an anti-aging health-care product.

Furthermore, the medicine or the health care product can also contain other anti-aging medicines.

In another preferred embodiment, the dosage form of the pharmaceutical composition is: oral preparation, injection, infusion solution, tablet, powder, capsule, and pill; preferably oral.

The invention also provides an anti-aging freeze-drying agent which consists of the anti-aging composition, a freeze-drying protective agent and a stabilizing agent.

In some embodiments, the stabilizing agent is one or more of a lyoprotectant, an acid-base modifier, an emulsifier.

Wherein the freeze-drying protective agent is one or more of trehalose, glycine, mannitol, dextran and lactose;

the acid-base regulator is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, amino acid and potassium citrate.

The emulsifier is one or more of polysorbate-20, polysorbate-60 and polysorbate-80.

Advantageous effects

According to the invention, miRNA with significant difference in differential expression is screened out as a research basis for anti-aging research by analyzing miRNA differential expression and functions before and after aging of umbilical cord mesenchymal stem cells in a high-sugar environment. The miRNA is transfected into the umbilical cord mesenchymal stem cells, so that the transfected umbilical cord mesenchymal stem cells can improve the anti-aging function of the stem cells on one hand, improve the activity of the stem cells on the other hand, improve the anti-aging function of human bodies, realize the anti-aging characteristic in a high-sugar environment, are suitable for anti-aging treatment of diabetes patients, and have great market value.

Drawings

FIG. 1miR-16 expression level result chart

FIG. 2 is a diagram showing the results of the activity of transgenic stem cells detected by the CCK-8 method

FIG. 3 is a graph showing the results of anti-aging cell assay

Detailed Description

To further illustrate the objects, aspects and advantages of the present invention, we shall now describe the invention with reference to the following specific examples, which are only for better illustrating the patent of the present invention and are not intended to limit the scope of the present invention. All other embodiments that can be obtained by a person skilled in the art without making any inventive step based on the examples of the present invention belong to the protection scope of the present invention.

Example 1 isolation and identification of umbilical cord mesenchymal Stem cells

Washing umbilical cord 5cm or so with PBS, separating and removing umbilical cord adventitia and blood vessel tissue, and cutting into pieces of 1mm³Washing with PBS after the small pieces, centrifuging at 3000r/min for 5min, discarding the supernatant, and retaining the precipitate; after 3 washes with PBS, 1X 10⁶The cells were seeded in a T-75 flask at a cell density of/mL and cultured in DMEM with a volume fraction of 10% FBS at 37 ℃ in a saturated humidity environment of 5% carbon dioxide. After 5 days, the liquid is changed for the first time, the cells which are not attached to the wall are discarded, and the liquid is changed for 1 time every 3 to 4 days later. Observing that the cells reach 85% fusion, digesting with 0.25% pancreatin for 5min, carrying out passage according to the ratio of 1:3, and continuously culturing. During the culture process, flow cytometry was used to identify cell surface markers. Collecting umbilical cord mesenchymal stem cells, collecting 5 generations of cells in logarithmic phase, removing culture medium, washing with PBS for 2 times, digesting with 0.25% trypsin digestion solution, removing digestion solution, adding PBS, and making into 1 × 10⁶Single cell suspension in ml, 100. mu.L of cell suspension per Eppendorf tube. 20 mul of antihuman FITC 90, FITC-CD34, PE-CD45, PE-HLA-DR, FITC 105 and PE-CD73 are respectively added into the cell suspension, the control tube is respectively added with antihuman FITC-IgG1 and PE-IgG1 with the same amount, the cell suspension is prepared again after being incubated for 30min at the temperature of 25 ℃ in a dark place, washed for 2 times by PBS and centrifuged for 5min at 1500r/min, and the cell suspension is detected by a flow cytometer. The results of the flow cytometry measurements are shown in Table 1.

TABLE 1 expression results of human umbilical cord MSCs markers

Marker substance	Amount of expression
		CD73	98.34％
CD90	99.87％
		CD105	97.42％
CD34	0.05％
		CD45	0.15％

From the results in table 1, it can be seen that human umbilical cord MSCs highly express CD73 (98.34%), CD90 (99.87%) and CD105 (97.42%), and lowly express hematopoietic stem cell markers CD34 (0.05%) and CD45 (0.15%), which are consistent with the international identification of mesenchymal stem cells, indicating that stem cells are successfully isolated.

Example 2 differential expression and functional analysis of miRNA before and after aging of umbilical cord mesenchymal stem cells in high sugar environment

Dividing the stem cells obtained by the identification into 3 groups, and continuously culturing a group of cells in a DMEM/NG (medium containing 5.5mmol/L of sugar) as a normal sugar control group (NG group) for 16 generations; another group of cells was cultured in DMEM/HG (25 mmol/L saccharide-containing) medium as the HG group (to simulate a high-glucose microenvironment in diabetic patients) for 16 serial passages. The third group of cells was cultured in DMEM/HG (25 mmol/L saccharide-containing) medium as a control (to simulate a high-glucose microenvironment in diabetic patients) for 2 consecutive passages. And respectively extracting RNA from the three cells by adopting an RNA extraction kit.

Detection of miRNA expression profiling Using Megaplex Pools miRNA expression detection System (Applied Biosystems, USA) and operating according to the instructions, the main steps include reverse transcription of RNA samples into cDNA using Megaplex RT Primers specific Primers; miRNA expression was detected by ABI7900HT fluorescent quantitative PCR system (Applied Biosystems) using taqman microrna Arrays kit (Applied Biosystems); the final results were normalized with U6 as an internal control. And (3) carrying out statistical analysis on the expression conditions of the nuclear miRNA before and after aging by using a DEGseq tool, and screening the miRNA with the difference multiple of more than 2 times and P of less than 0.001 as the miRNA with the significant difference in expression. The results show that RNA is detected by using the miRNA quantitative PCR chip to detect the expression condition of miRNA. The result of differential expression analysis of miRNA expression profile data shows that under the high-sugar culture environment of 16 generations, compared with the high-sugar culture environment of 2 generations and the sugar-free culture environment of 16 generations of MSCs, the expression of 243 miRNAs in the high-sugar culture environment is different (the difference multiple is more than 2 times and P is less than 0.001), wherein 187 miRNAs are obviously up-regulated, and 56 miRNAs are obviously down-regulated. The results for the 5 mirnas with the most significant differences are shown in table 2 below.

TABLE 2 high-sugar differential expression of miRNA in umbilical cord mesenchymal stem cells

ID	Fold change (Log2)
		miR-375	-10.986201
miR-16	-9.855023
		miR-330-5p	9.221345
miR-362-3p	8.521436
		miR-609	-7.253691

The most significant of these mirnas were associated with senescence of stem cells, as initially determined from the results in table 2. The inventors expect that the anti-aging properties of the cells could be improved by up-regulating two mirnas, which are clearly down-regulated.

Example 3 establishment of miR-16 transfected umbilical cord mesenchymal Stem cells

miR-16 and miR-16 inhibitor are purchased from Sharp Biotech, Guangzhou.

Respectively transfecting miR-16 and miR-16 inhibitor to umbilical cord mesenchymal stem cell culture cells: specifically, when the density of the umbilical cord mesenchymal stem cells separated in example 1 is 80%, 2ug of miR-16 and 2ug of miR-16 inhibitor are respectively mixed with 10uL of lipofectamine2000 in a serum-free culture medium according to the specification of a transfection reagent, the mixture is stood at room temperature for 20min, and then the mixture is slowly added into the culture medium at 37 ℃ and 5% CO₂Culturing, and harvesting cells after 24 h.

Detecting the expression level of miR-16: after the cells are transfected for 24 hours, the cells are harvested, mRNA is extracted by using a mirVanaTMqRT-PCR miRNA Detection Kit of Ambion company for reverse transcription, and then RT-PCR Detection is carried out, and U6snRNA is used as an internal reference gene; the reaction system is as follows: 10 μ L SYBR Green supermix; 0.5. mu.L of mir VanaPCR primers; 0.5 μ L of product from TR reaction; 9 μ L of nucleic-free Water; reaction conditions are as follows: 95 ℃ for 3 min; data were analyzed at 95 ℃ for 15sec, 60 ℃ for 30sec (40cycles) using the LightCycler 480 system. The detection primer of miR-16 is an upstream primer: 5'-acactccagctgggtagcagcacgtaaat-3', respectively; the downstream primer is 5'-ctcaactggtgtcgtggagtc ggcaattcagttgagcgccaata-3'; the results are shown in FIG. 1.

As can be seen from FIG. 1, the expression level of miR-16 in the umbilical cord mesenchymal stem cells transfected with miR-16 is remarkably improved, while the expression of the corresponding miR-16 in the stem cells transfected with miR-16 inhibitor is remarkably reduced, which indicates that the corresponding transgenic stem cells are successfully constructed.

Example 4CCK-8 method for determining the viability of transgenic Stem cells

The transgenic stem cells are inoculated in a 96-well plate according to 3000 cells per well, and the change of the activity of 48h cells under the high-sugar environment after miR-16, miR-16 inhibitor and blank control are transfected respectively is detected by a CCK-8 method. The specific operation refers to the CCK-8 kit specification, and the absorbance (A) value of the sample at the wavelength of 450nm is measured by an enzyme-labeling instrument. The results are shown in FIG. 2.

The relation between miR-16 and umbilical cord mesenchymal stem cell activity under the high-sugar condition is detected by a CCK-8 method in the experiment, the activity of the stem cell transfected with miR-16 and miR-16 inhibitor for 48h is detected by the CCK-8 method, and the result shows that the activity A value of the miR-16 transfected cell reaches 1.42 in the high-sugar environment on the corresponding 4 th day of high-sugar culture, which is obviously stronger than the activities of 0.45 of miR-16-inhibitor transfected cell and 0.55 of non-transgenic control. It is fully demonstrated that miR-16 can improve the cell activity of umbilical cord mesenchymal stem cells under high sugar.

Example 5 cellular anti-aging assay

And (3) detecting the senescence condition of the cells by adopting a senescence-associated beta-galactosidase staining method. After each group of cells are transfected, continuously culturing in a high-sugar culture medium for 48 hours, washing by PBS, adding beta-galactosidase staining fixing solution, fixing for 15min at room temperature, washing again by PBS, adding staining working solution, and keeping the temperature at 37 ℃ without CO₂Incubating overnight under conditions; under the observation of a common optical microscope, each sample counts 800 cells, the cytoplasmic blue stained cells are aged cells, and the counted positive cells account for the percentage of the total number of the observed cells. The results are shown in FIG. 3.

As can be seen from the results of FIG. 3, we detected the senescence level of the stem cells after 48h transfection of miR-16 and miR-16 inhibitor, and the results showed that the number of beta-galactosidase positive staining cells of the HG + miR-16 group was significantly reduced by 35, and the number of beta-galactosidase positive staining cells of the HG + miR-16 inhibitor group was significantly increased by 683. This indicates that miR-16 can enhance the anti-aging property of umbilical cord mesenchymal stem cells in a high-sugar environment.

Example 6 diabetic aging mouse model test

Female Wistar rats, provided by Shanghai laboratory animal center of Chinese academy of sciences, weigh about 140 g.

After the Wistar rat is adaptively fed for 1 week, an animal model is prepared according to the following mode, and the specific operation method is as follows:

group A (normal control group) is prepared by injecting 0.01mL/100 (g.d-1) normal saline into abdominal cavity of rat for 40d, then feeding normal saline into stomach (1mL/100 g.d-1) for 2 weeks, and simultaneously feeding free drinking water and feed;

group B (aging diabetes model control group) rats were given D-galactose intraperitoneal injection (50mg/kg, 1 time per day) for 40 days; meanwhile, filling high-fat high-sugar emulsion (1mL/100g of body weight, 1 time per day, containing 100g/L of cane sugar, 100g/L of lard and 10g/L of cholesterol) from the 11 th day, injecting STZ (30mg/kg, the STZ is prepared immediately, and the STZ is prepared into 0.25% solution by using citric acid buffer solution with pH4.2 under the ice bath condition) into the abdominal cavity after continuously 4 weeks, and screening the aged diabetic adult mice with the blood sugar of more than or equal to 11.1mmol/L after fasting for 12 hours without water prohibition after 4 days. Continuously administering physiological saline for 2 weeks, wherein the concentration of the physiological saline is 0.01mL/100 (g.d);

the treatment administration mode comprises the following steps: each time of continuous 3d injection of the transgenic umbilical cord mesenchymal stem cell suspension of example 3 into the tail vein of the model group mice is 0.3 mL/mouse, and the cell concentration is 1X 10⁶mL, interval time 7d, total 9 injections. The model control group and the normal control group were administered with the same amount of cell culture solution to the tail vein. The other feeding conditions of each group were the same as those of the normal control group. After the experiment, the patient was anesthetized with ether, and the abdominal aorta was bled to examine various indices. Blood sugar, MDA, SOD and CAT are respectively detected by a glucose oxidase method, a TBA method, a xanthine oxidase method and Catalase (CAT) activity: and detecting by an ammonium molybdate colorimetric method, and strictly operating according to the requirements of the kit. The results are shown in table 3 below.

TABLE 3 influence of transgenic umbilical cord mesenchymal Stem cells on blood glucose, CAT, SOD, MDA content

P <0.05 compared to the corresponding control group, P < 0.01.

As can be seen from the results in Table 3, through detection, the activity values of SOD and CAT in mice are obviously reduced after the model group is injected with D-galactose and fed with high sugar, the MDA content is obviously increased, and the blood sugar content is also obviously increased compared with the normal control group, which indicates that the model construction is more successful. After the transgenic stem cells are treated, the result of blood sugar shows that the transgenic stem cell treatment has certain effect of reducing blood sugar concentration. Comparing CAT and SOD activity values and MDA content of the model group and the treatment group, the CAT and SOD activities of the mice injected with the transgenic umbilical cord mesenchymal stem cells are obviously improved compared with the model group, and the MDA content is obviously reduced, which indicates that the anti-aging effect is obvious.

Claims

1. A preparation method of miR-16-transferred umbilical cord mesenchymal stem cells comprises the following steps:

2. The application of the umbilical cord mesenchymal stem cells of trans-miR-16 in the preparation of a pharmaceutical composition; the pharmaceutical composition functions as follows:

(a) delaying the aging of the human body in a high-sugar environment;

(b) delaying the aging of cells in a high-sugar environment;

(c) delaying the aging of umbilical cord mesenchymal stem cells in a high-sugar environment.

3. The application of the miR-16-transferred umbilical cord mesenchymal stem cells in the preparation of health products; the health product has the following functions:

(a) delaying the aging of the human body in a high-sugar environment;

(b) delaying the aging of cells in a high-sugar environment;

4. Use according to claim 2 or 3, characterized in that: the medicine or health product may also contain other antiaging medicine.

5. The use according to claim 2, wherein said pharmaceutical composition is in the form of: oral preparation, injection, infusion solution, tablet, powder, capsule, and pill; preferably oral.

6. Use according to claim 2, characterized in that the pharmaceutical composition is a lyophilizate consisting of 16 transferred umbilical cord stem cells together with lyoprotectants and stabilizers and acid-base regulators.

7. Use according to claim 6, characterized in that: the stabilizer is one or more of a freeze-drying protective agent, an acid-base regulator and an emulsifier.

8. Use according to claim 6, characterized in that: the freeze-drying protective agent is one or more of trehalose, glycine, mannitol, dextran and lactose.

9. Use according to claim 6, characterized in that: the acid-base regulator is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, amino acid and potassium citrate.

10. Use according to claim 7, characterized in that the emulsifier is one or more of polysorbate-20, polysorbate-60, polysorbate-80.