CN111718897A

CN111718897A - Application of melatonin in maintaining dryness of mesenchymal stem cells for in vitro continuous passage

Info

Publication number: CN111718897A
Application number: CN202010630308.XA
Authority: CN
Inventors: 刘文佳; 帅逸; 廖立
Original assignee: Air Force Medical University of PLA
Current assignee: Air Force Medical University of PLA
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2020-09-29

Abstract

The invention discloses an application of melatonin in maintaining the dryness of mesenchymal stem cells subjected to in vitro continuous passage, wherein the mesenchymal stem cells subjected to in vitro long-time passage are continuously treated by the melatonin with physiological concentration, and the high expression of dryness-related genes and the low expression of senescence-related genes are maintained through the antioxidation effect, so that the physiological function of high-algebraic mesenchymal stem cells is preserved, the aim of collecting fewer primary cells with single source to obtain a large amount of available seed cells is fulfilled, the consistency and the stability of the cell source in treatment can be ensured, and the potential safety hazard caused by the mixed application of cells with multiple sources can be avoided. The melatonin pretreatment concentration in the invention is screened out by gradient concentration, and has higher species conservation through double verification of in vitro experiments and in vivo experiments, so that the problem of dryness loss in the in vitro passage process of mesenchymal stem cells can be solved. Is suitable for maintaining the dryness of the human and mouse derived mesenchymal stem cells in the in vitro culture process.

Description

Application of melatonin in maintaining dryness of mesenchymal stem cells for in vitro continuous passage

Technical Field

The invention relates to the technical field of biology, in particular to application of melatonin in maintaining dryness of mesenchymal stem cells subjected to in vitro continuous passage.

Background

Mesenchymal Stem Cells (MSCs) are a type of seed cell with tissue regeneration and disease treatment potential. At present, mesenchymal stem cell-based therapeutic methods have been widely used in regenerative medicine and disease treatment. By 2015, more than 500 relevant clinical trials of mesenchymal stem cell therapy have been approved worldwide. In most studies, the dose for a single cell therapy is about 1000 ten thousand to 1 million cells. Since the source of bone marrow is less and mesenchymal stem cells only occupy a very small part of tissue cells, in vitro passage expansion is indispensable for the application of mesenchymal stem cells.

However, several studies have reported that mesenchymal stem cells undergo a series of functional degenerations during long-term in vitro passaging. For example, mesenchymal stem cells that have been serially passaged for a long period of time may exhibit abnormal cell morphology, loss of mesenchymal stem cell-specific surface markers, self-renewal, and decreased multipotentiality. Therefore, loss of the sternness of mesenchymal stem cells will directly lead to tissue regeneration or failure of cell therapy. Research shows that after long-time passage, the treatment effect of the mesenchymal stem cells on cardiovascular diseases, pulmonary diseases, nervous system diseases, graft-versus-host diseases, septicemia and bone diseases is reduced. Therefore, the loss of the sternness in the process of passage is a great dilemma faced by the application field of the mesenchymal stem cells.

In vivo, the maintenance of stem cell dryness and function relies on a complex and large set of intracellular and extracellular signaling networks. Loss of mesenchymal stem cell function for in vitro passaging is largely due to loss of the physiological cell niche. Currently, some application strategies have been reported to promote or maintain the function of the mesenchymal stem cells expanded in vitro by regulating the intracellular and extracellular internal environments and signal pathways. For example, the mesenchymal stem cells are cultured under the condition of low oxygen or physiological oxygen concentration, the mesenchymal stem cells are inoculated and cultured on the tissue-specific extracellular matrix, exogenous signal proteins such as FGF, PDGF and EGF are added to the mesenchymal stem cell culture medium, and the properties of the mesenchymal stem cells are changed from the molecular level by using the genetic engineering technology. However, these methods have their application limitations. For example, hypoxic or physiological oxygen culture conditions are generally used to maintain the dryness of cells by slowing the proliferation of stem cells by reducing the cyclic progression of the cell cycle, and thus this method does not meet the clinical requirements for cell mass. Although extracellular matrix can provide a tissue-specific extracellular microenvironment for stem cells, the source and quality control of extracellular matrix is a bottleneck encountered by this approach in vitro large-scale culture applications. Although exogenous signaling proteins have high specificity for maintaining and promoting stem cell function, their short half-life and expensive price severely limit their use in long-term processing. Although the function of stem cells can be controlled in a targeted way by genetic engineering technology, the mutation or tumor formation risk brought by the genetic engineering technology is irremediable.

The natural small molecular compound is an active ingredient separated and extracted from animal or plant tissues, and can specifically and reversibly regulate related signal paths. A large number of studies report that small molecule compounds play an important role in the maintenance of stem cell function and the regulation of stem cell fate. Because small molecule compounds have targeting specificity, convenience in application and storage and low cost, the small molecule compounds have good application prospects in promotion of stem cell therapy as active substances. However, small molecule compounds still face some challenges in applications where mesenchymal stem cell function is maintained.

Melatonin is an active substance mainly secreted from the pineal body of mammals, and plays an important role in the regulation of physiological rhythms. Studies have reported that melatonin can regulate various physiological functions such as sleep, periodic rhythm, neuroendocrine and the like. In recent years, the role of melatonin in regulating mesenchymal stem cell characteristics has been gradually reported. For example, melatonin can promote its therapeutic effects in bone defects, kidney damage, lung damage, and skin injury by modulating the osteogenic differentiation, survival, and migration capabilities of mesenchymal stem cells. Excessive oxidative stress is one of the main factors causing the aging of the mesenchymal stem cells, and research reports show that excessive oxygen radicals cannot induce the aging of the mesenchymal stem cells under the protection of the melatonin, which suggests the application prospect of the melatonin in the protection of the mesenchymal stem cells.

In conclusion, the existing technical method for maintaining the cell dryness cannot be well applied and popularized due to the defects. Therefore, it is one of the subjects of great interest of the applicant to search for a safe, efficient, inexpensive and easily controllable substance for preventing loss of the sternness of high-generation mesenchymal stem cells after in vitro serial passage and preserving the physiological functions of the high-generation mesenchymal stem cells.

Disclosure of Invention

In order to overcome the defects of the prior technical method for maintaining the dryness of the in vitro continuous passage stem cells, the invention aims to provide the application of the melatonin in maintaining the dryness of the in vitro continuous passage mesenchymal stem cells.

In order to achieve the above task, the applicant has unexpectedly discovered a new use of melatonin in research, namely, an application of melatonin in maintaining the dryness of Mesenchymal Stem Cells (MSCs) serially passaged in vitro.

The applicant continuously treats mesenchymal stem cells which are subjected to long-term passage in vitro by using melatonin (melatonin) with physiological concentration, and maintains high expression of dryness-related genes and low expression of senescence-related genes through antioxidation, so that the physiological function of high-generation mesenchymal stem cells is preserved.

According to the invention, the mesenchymal stem cell is a rat-derived mesenchymal stem cell or a human-derived mesenchymal stem cell.

Further, the mesenchymal stem cells subjected to long-time continuous passage are subjected to digestion passage by using a digestion solution, wherein rat-derived mesenchymal stem cells are subjected to continuous passage to the 15 th generation, and human-derived mesenchymal stem cells are subjected to continuous passage to the 25 th generation.

Preferably, the melatonin is configured into a 1mM melatonin storage solution, stored at 4 ℃ and protected from light all the time; from the 1 st generation of cells, rat-derived mesenchymal stem cells or human-derived mesenchymal stem cells are pretreated by melatonin working solution with the final concentration of 10nM, wherein the rat-derived mesenchymal stem cells are continuously treated to the 15 th generation, the human-derived mesenchymal stem cells are continuously treated to the 25 th generation, and the whole process is protected from light.

Further preferably, the melatonin storage liquid is a dimethyl sulfoxide storage liquid containing 1mM of melatonin; the melatonin working solution is an alpha-MEM culture medium containing 10nM melatonin, 10% fetal calf serum, 1% streptomycin and penicillin.

The application of the melatonin in maintaining the dryness of the mesenchymal stem cells subjected to in vitro continuous passage has the advantages that:

1. high efficiency. In vitro experiments and in vivo experiments show that compared with the 1 st generation cells, the dryness, the regeneration capacity and the disease treatment capacity of the high generation cells treated by the melatonin are better maintained.

2. Specificity. In vitro experiments show that the mesenchymal stem cells have better effect on high-generation mesenchymal stem cells after in vitro continuous passage.

3. And (4) safety. Melatonin treatment was at a concentration of 10nM, within the physiological concentration range; and is subjected to melatonin removal treatment before being used for tissue regeneration or disease treatment to prevent melatonin from remaining.

4. Convenience and high cost performance. Only 1 small molecular compound is needed to be added, which is convenient for large-scale development.

Drawings

Fig. 1 is a graph showing the effect of melatonin on the sternness of mesenchymal stem cells after long serial passages.

Fig. 2 is a graph showing the effects of melatonin on the regenerative capacity of mesenchymal stem cells for long-term serial passages and on therapeutic effects (including ectopic osteogenesis, repair of skull defects, treatment of osteoporosis, and treatment of enteritis).

The present invention will be described in further detail with reference to the following drawings and examples.

Detailed Description

In the following embodiments, the applicant uses melatonin in maintaining the dryness of the mesenchymal stem cells subjected to in vitro continuous passage to prevent loss of the dryness of the high-generation mesenchymal stem cells subjected to in vitro continuous passage, so as to preserve the physiological functions of the high-generation mesenchymal stem cells, achieve the purpose of collecting fewer primary cells from a single source to obtain a large amount of available seed cells, ensure the consistency and stability of cell sources in treatment, and avoid potential safety hazards caused by mixed application of cells from multiple sources. And proves that the melatonin can maintain the dryness of the human and mouse derived mesenchymal stem cells in the in vitro culture process.

The embodiment provides the application of melatonin in maintaining the dryness of mesenchymal stem cells subjected to in vitro continuous passage, and the specific implementation process comprises the steps of long-time continuous passage of the mesenchymal stem cells, melatonin pretreatment and cell function evaluation.

In the embodiment, the melatonin pretreatment concentration is screened out through gradient concentration, and double verification of in vitro experiments and in vivo experiments shows that the melatonin conservativeness between species is higher, so that the problem of dryness loss in the in vitro passage process of the mesenchymal stem cells can be solved. Is suitable for maintaining the dryness of the human and mouse derived mesenchymal stem cells in the in vitro culture process.

In this example, reagents not specifically described are commercially available.

The specific implementation steps of the application of the melatonin in maintaining the dryness of the mesenchymal stem cells subjected to in vitro continuous passage are as follows:

the method comprises the following steps: obtaining of Primary mesenchymal Stem cells

Rat-derived mesenchymal stem cells were obtained by isolating bilateral femurs and tibias of 1-week-old rats, removing the soft tissue, cutting off the ends of the stem, flushing out the whole bone marrow with a 1mL syringe, resuspending in α -MEM medium, and culturing in 8 × 10⁷Individual cells were seeded in 10cm disposable plastic culture dishes.

Human-derived mesenchymal stem cells were obtained by cutting iliac tissue pieces taken from orthognathic surgery patients (22 to 25 years old, ethically approved, and informed consent of patients) into pieces, flushing out all bone marrow with a 1mL syringe, resuspending in α -MEM medium, gently pipetting into single cell suspension, and subjecting to 8 × 10⁷Individual cells were seeded in 10cm disposable plastic culture dishes.

Rat-derived mesenchymal stem cells or human-derived mesenchymal stem cells are cultured in an aseptic constant-temperature incubator. The culture medium is replaced once every 2 days, nonadherent cells are removed, when the adherent cells grow and converge to 80% of the bottom area of the culture dish, the culture medium is discarded, the cells are washed for 3 times by phosphate buffer solution, 2mL of digestive juice is added, and the cells are digested for 30s in a constant-temperature incubator at 37 ℃. The digestion was immediately neutralized by adding medium containing 10% fetal bovine serum. Gently blow the bottom wall for 50 times to obtain single cell suspension, and count. Before inoculation, the surface markers SCA-1/STRO-1, CD44, CD90, CD106, CD34 and CD45 of the mesenchymal stem cells are identified with self-renewal capacity and multi-differentiation capacity.

The alpha-MEM culture medium is as follows: alpha-MEM medium containing 10% fetal bovine serum, 1% streptomycin and penicillin;

the sterile constant-temperature incubator is saturated humidity and 5% CO₂The incubator is a 37 ℃ sterile constant-temperature incubator;

the digestive juice is sterile digestive juice containing 0.25% of pancreatin and 1mM of EDTA.

Step two: obtaining mesenchymal stem cells for long-time continuous passage

After counting, the cells were passaged at 1 × 10⁶The individual cells were seeded at 75cm²In a culture flask. When the adherent cells grew and converged to 80% of the bottom area of the culture flask, the medium was discarded, the cells were washed 3 times with phosphate buffer, 2mL of the digestion solution was added, and the cells were digested in a 37 ℃ incubator for 30 seconds. The digestion was immediately neutralized by adding medium containing 10% fetal bovine serum. Gently blow the bottom wall for 50 times to obtain single cell suspension, and count. Rat-derived mesenchymal stem cells were serially passaged to the 15 th passage, and human-derived mesenchymal stem cells were serially passaged to the 25 th passage.

Step three: long-term melatonin pretreatment

The melatonin is prepared into a melatonin storage solution with the concentration of 1mM, stored at 4 ℃ and protected from light all the time. Starting from the 1 st generation of cells, after the cells are inoculated for 6h to adhere to the wall, the original culture medium is discarded, the mesenchymal stem cells are pretreated by melatonin working solution with the final concentration of 10nM, the rat-derived mesenchymal stem cells are continuously treated to the 15 th generation, the human-derived mesenchymal stem cells are continuously treated to the 25 th generation, and the whole process is protected from light. The melatonin pretreatment is limited to a long-time continuous passage process, and melatonin removal treatment is required in the processes of in vitro induction and function detection.

The melatonin storage liquid is dimethyl sulfoxide storage liquid containing 1mM melatonin;

the melatonin working solution is an alpha-MEM culture medium containing 10nM melatonin, 10% fetal calf serum, 1% streptomycin and penicillin.

Step four: functional detection of mesenchymal stem cells

Collecting the 1 st, 4 th and 15 th generation rat-derived mesenchymal stem cells (named as rP1, rP4, rP15) which are not pretreated with melatonin and the 15 th generation rat mesenchymal stem cells (named as rP15+ mel) which are pretreated with melatonin, and performing in vitro experiment and in vivo experiment double verification; also, generation 1, generation 4 and generation 25 human-derived mesenchymal stem cells (designated as hP1, hP4, hP25) which were not pretreated with melatonin, and generation 25 human-derived mesenchymal stem cells (designated as hP25+ mel) which were pretreated with melatonin were collected to perform double validation of in vitro experiments and in vivo experiments.

In the experiment for detecting self-renewal ability and multi-directional differentiation ability, cells were washed 3 times with phosphate buffer solution before being inoculated to prevent melatonin residue; in experiments for testing the ectopic osteogenesis ability, the skull defect repairing ability, the osteoporosis treating ability, and the ulcerative colitis treating ability, the cells were washed 3 times with a phosphate buffer solution before the inoculation or transplantation of the cells to prevent melatonin residual.

Step five: in vitro experiments

The in vitro experiment is mainly used for comprehensively analyzing the self-renewal capacity, the multidirectional differentiation capacity, the dryness and the aging level of the mesenchymal stem cells. The whole course of the in vitro experiment is not participated in melatonin, and the specific operation steps are as follows:

(ii) clone formation experiment

At 5 × 10²Inoculating the cells in a 5cm disposable plastic culture dish containing α -MEM culture medium, uniformly distributing the cells in the culture plate by a cross method, replacing the culture medium once every 3 days, culturing for 10 days, removing the culture medium, washing the cells for 3 times by using a phosphate buffer solution, removing a washing solution, fixing the cells by using a 4% paraformaldehyde solution for 20min, removing the fixing solution, washing the cells for 3 times by using the phosphate buffer solution, removing the washing solution, impregnating the cells by using a 1% toluidine blue staining solution for 30min, removing the toluidine blue staining solution, washing the cells for 3 times by using the phosphate buffer solution, removing the washing solution, observing under a microscopeCounting, the number of cells in a single colony is greater than or equal to 50, i.e., one clone is counted by the formula [ clone formation rate ═ clone number/500 × 100%]And calculating the clone formation rate.

② cell proliferation curve detection

At 1 × 10³The individual cells/well were seeded in 96-well plates and the cells were distributed evenly in the culture plate by a criss-cross technique. After 6h of inoculation, adding the CCK-8 reagent in the wells on day 0 at the concentration of 10 muL of CCK-8 reagent per 100 muL of culture medium, uniformly mixing for 15s by using a micro-oscillator, incubating for 2h at 37 ℃, detecting the absorbance of each well at the wavelength of 450nm, and avoiding light all the time. Proliferation curves were drawn starting on day 0, continuing the assay until day 6.

③ in vitro osteogenic differentiation capacity detection

At 1 × 10⁵Inoculating each cell/well in 12-well plate, uniformly distributing cells in culture plate by cross method, changing common culture medium into osteogenesis inducing liquid when cell growth reaches 80%, changing osteogenesis inducing liquid once every 3 days, and keeping away from light. By day 7 of induction, the medium was discarded, the cells were washed 3 times with phosphate buffer, and the washing solution was discarded. Cells were fixed in 4% paraformaldehyde solution for 20min and the fixative was discarded. The cells were washed 3 times with phosphate buffer and the wash was discarded. Adding alkaline phosphatase activity reaction reagent, reacting at 37 deg.C for 15min, and keeping away from light. The reaction solution was discarded, the cells were washed 3 times with phosphate buffer, and the washing solution was discarded. The cell staining results were observed to evaluate the alkaline phosphatase activity. By day 7 of induction, the medium was discarded, the cells were washed 3 times with phosphate buffer, and the washing solution was discarded. Adding Trizol lysate to fully crack cells, extracting total RNA and quantifying, obtaining cDNA through reverse transcription, and detecting the expression quantity of osteogenic related genes RUNX2 and OCN by a real-time RT-PCR method. When the induction is carried out to 28 days, the culture medium is discarded, the cells are washed 3 times by phosphate buffer, and the washing solution is discarded. Cells were fixed in 4% paraformaldehyde solution for 20min and the fixative was discarded. The cells were washed 3 times with phosphate buffer and the wash was discarded. The cells were stained with alizarin red stain for 1min, discarded, and the cells were washed 3 times with phosphate buffer. The wash was discarded, cell staining was observed and the amount of mineralized nodule formation was evaluated.

(iv) measurement of dryness and aging level

rP1(hP1), rP4(hP4), rP15(hP25) and rP15+ mel (hP25+ mel) groups of cells, the medium was discarded, the cells were washed 3 times with phosphate buffer, and the washing solution was discarded. Adding Trizol lysate to fully crack cells, extracting total RNA and quantifying, obtaining cDNA through reverse transcription, and detecting the expression quantity of the dry, aging and oxidation resistance related genes Nanog, p53, p16 and SOD2 by a real-time RT-PCR method. Adding lysis solution containing protease inhibitor into the other part of cells to fully lyse the cells, and further lysing the cells by low-frequency ultrasonic oscillation. Collecting cell lysis suspension, centrifuging at 4 deg.C and 12000r for 15min, removing precipitate, collecting supernatant, quantifying protein, and detecting expression levels of dry marker Nanog, aging markers p53 and p16, and antioxidant marker SOD2 by SDS-PAGE electrophoresis. The whole process is operated on ice.

Step six: in vivo experiments

The in vivo experiment is mainly used for analyzing the bone regeneration capacity, the bone metabolism regulation capacity and the immunoregulation capacity of the mesenchymal stem cells.

Detection of ectopic osteogenesis Capacity

At 1 × 10⁶The cells were seeded in 6-well plates per well and distributed evenly in the plates by a criss-cross protocol. When the cell growth is converged to 80%, the common culture medium is replaced by the cell membrane inducing liquid, the induction is continued for 10 days, the inducing liquid is replaced every 3 days, and the whole process is protected from light. And (3) connecting cell membranes, forming a sandwich composite structure by the three cell membranes and two layers of HA/TCP particles (total 20mg), wrapping the sandwich composite structure into a transplantation agglomerate, and transplanting the transplantation agglomerate to the back subcutaneous part of the nude mouse of 8 weeks old. At 8 weeks post-transplantation, the grafts were removed, washed with phosphate buffer and fixed in 4% paraformaldehyde for 48 h. After 14 days of decalcification with 17% EDTA, the sections were dehydrated, embedded in paraffin, HE-stained and massson-trichrome-stained, examined and evaluated for new bone formation.

② skull defect repair ability detection

Four cell patches and three-component HA/TCP particles (50 mg in total) form a sandwich composite structure, and the sandwich composite structure is laid and covered on a standard skull defect position of 8mm of a rat. 12 weeks after transplantation, rat calvaria were separated, washed with phosphate buffer and fixed in 4% paraformaldehyde solution for 1 week. After carrying out the micCT scanning, decalcifying the skull in 17 percent EDTA for 30 days, dehydrating, embedding paraffin, carrying out HE staining on the section, and detecting the formation of the new skull.

(iii) detection of osteoporosis treatment ability

24h after ovariectomy in rats, 2 × 10 in tail vein⁷Mesenchymal stem cells were injected at a concentration of cells/kg. 10 weeks after cell transplantation, rat femurs were isolated, the cranium washed with phosphate buffer and fixed in 4% paraformaldehyde solution for 1 week. After micct scanning, decalcification was carried out for 60 days in EDTA with a concentration of 17%, dehydration and paraffin embedding were carried out, and the sections were subjected to histological staining to examine changes in bone mass and osteoblast and osteoclast activities.

(iv) detection of ability to treat ulcerative colitis

Colitis was induced by 3% dextran sulfate solution, and on

day

3, 1 × 10 was added to the tail vein⁷Mesenchymal stem cells were injected at a concentration of cells/kg. The survival rate, weight change and disease progression of the rats were observed and recorded throughout the course. On induction day 10, rat colons were isolated, intestinal lumens were flushed with phosphate buffer, fixed in 4% paraformaldehyde solution for 48h, dehydrated, paraffin embedded, and sectioned for histological analysis.

The osteogenesis inducing liquid comprises: alpha-MEM medium containing 5mM sodium beta-glycerophosphate, 50. mu.g/mL ascorbic acid, 10nM dexamethasone, 10% fetal bovine serum, 1% streptomycin and penicillin;

the cell patch inducing liquid comprises: alpha-MEM medium containing 100mg/mL ascorbic acid, 10% fetal bovine serum, 1% streptomycin and penicillin;

the HA/TCP particles are hydroxyapatite and tricalcium phosphate, and the ratio of the HA/TCP particles to the tricalcium phosphate is 6: 4, the mixed biological material particles are accepted tissue engineering scaffold materials;

the dextran sulfate solution is a well-known drug for inducing experimental ulcerative colitis.

The influence of melatonin on the sternness of mesenchymal stem cells after long-term continuous passage is shown in figure 1, and the results show that the clonality, the proliferation capacity and the bone differentiation capacity of the rP15+ mel (hP25+ mel) group are stronger than those of rP15(hP25), the expression of corresponding sternness molecules Nanong and antioxidant molecules SOD2 is higher than that of rP15(hP25), and the expression of aging molecules p53 and p16 is lower than that of rP15(hP 25). Also, the dryness level of rP15+ mel (hP25+ mel) approaches P1 and P4.

Fig. 2 shows the influence of melatonin on the regeneration capacity and therapeutic effect of mesenchymal stem cells after long-term continuous passage, and the results show that the ectopic osteogenesis capacity, the skull defect repair capacity, the osteoporosis treatment capacity and the ulcerative colitis treatment capacity of the rP15+ mel group are all stronger than those of the rP 15. The hP25+ mel group has stronger capacity for treating ulcerative colitis than hP 25. Also, the regenerative or therapeutic capacity of rP15+ mel (hP25+ mel) was comparable to P1 and P4.

Claims

1. The application of melatonin in maintaining the dryness of mesenchymal stem cells subjected to in vitro continuous passage.

2. The use according to claim 1, wherein melatonin is used for continuously treating mesenchymal stem cells which are passaged in vitro for a long time, and the high expression of the sternness related gene and the low expression of the senescence related gene are maintained through antioxidation, so that the physiological function of high-generation mesenchymal stem cells is preserved.

3. The use of claim 2, wherein the mesenchymal stem cell is a rat-derived mesenchymal stem cell or a human-derived mesenchymal stem cell.

4. The use according to claim 2 or 3, wherein the mesenchymal stem cells subjected to the long-term serial passage are subjected to digestion passage by using a digestion solution, wherein rat-derived mesenchymal stem cells are serially passed to the 15 th passage, and human-derived mesenchymal stem cells are serially passed to the 25 th passage.

5. The use according to claim 2 or 3, wherein melatonin is formulated as a 1mM storage solution of melatonin, stored at 4 ℃ and protected from light throughout; from the 1 st generation of cells, rat-derived mesenchymal stem cells or human-derived mesenchymal stem cells are pretreated by melatonin working solution with the final concentration of 10nM, wherein the rat-derived mesenchymal stem cells are continuously treated to the 15 th generation, the human-derived mesenchymal stem cells are continuously treated to the 25 th generation, and the whole process is protected from light.

6. The use of claim 5, wherein the melatonin storage solution is a dimethyl sulfoxide storage solution containing 1mM melatonin; the melatonin working solution is an alpha-MEM culture medium containing 10nM melatonin, 10% fetal calf serum, 1% streptomycin and penicillin.