CN114854681A - Method for improving activity, proliferation and migration of human umbilical cord mesenchymal stem cells and application thereof - Google Patents

Abstract

The invention relates to a method for improving cell viability, cell proliferation rate and cell migration of human umbilical cord mesenchymal stem cells and application thereof.

Description

Method for improving activity, proliferation and migration of human umbilical cord mesenchymal stem cells and application thereof

Technical Field

The invention belongs to the technical field of stem cell processing, and relates to a method for improving the activity, proliferation and migration of human umbilical cord mesenchymal stem cells and application thereof, in particular to an application method and application of rapamycin in the field of mesenchymal stem cells, which are suitable for increasing the activity, proliferation and migration of human umbilical cord mesenchymal stem cells by using rapamycin.

Background

Mesenchymal Stem Cells (MSCs) have high self-renewal capacity and multi-differentiation capacity, and have a wide prospect in treating neurodegenerative diseases such as alzheimer disease. However, MSCs cultured in vitro may exhibit replicative senescence, mainly manifested by low cell viability, reduced migration and differentiation capacity, directly affecting the efficacy for cell transplantation. There are many methods for inducing differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into neuron-like cells in vitro. However, these differentiation protocols are inefficient. Therefore, improving the cell viability and neural differentiation rate of MSCs is the key to improving the effect of stem cell transplantation.

Autophagy is an intracellular catabolism mechanism negatively regulated by mTOR signaling, and plays an important role in maintaining stem cell homeostasis and fate through multiple mechanisms such as self-renewal, differentiation, etc. Rapamycin (Rap) is an autophagy activator and also inhibits the mTOR signaling pathway. Research shows that Rap can effectively regulate heart differentiation of mouse embryonic stem cells, cartilage differentiation of hepatic progenitor cells, fat differentiation and liver differentiation. In addition, autophagy is activated during neuronal differentiation of MSCs. Previous studies have shown that autophagy promotes differentiation of mesenchymal stem cells into neurons by modulating the Notch1 signaling pathway. However, the role and mechanism of Rap-induced autophagy in the proliferation and neural differentiation of hUC-MSCs is not clear.

The Wnt/β -catenin signaling pathway plays an important role in the regulation of stem cell fate, such as self-renewal and differentiation. Normally, cytoplasmic beta-catenin binds to APC, Axin, GSK-3 beta and CKI epsilon, resulting in degradation of beta-catenin. Once the Wnt/beta-catenin signal channel is activated, cytoplasmic beta-catenin is transferred to the nucleus and interacts with an LEF1/TCF family, so that downstream target genes of CyclinD1 and c-Myc expression are induced, and cell proliferation and differentiation are regulated. Autophagy has been shown to promote cardiac or hepatic differentiation processes by modulating the Wnt/β -catenin signaling pathway. However, whether Rap can promote the neural differentiation of hUC-MSCs through Wnt/beta-catenin signal transduction is still to be studied.

In the invention, the influence of Rap on the proliferation and neural differentiation of hUC-MSCs is researched, and the roles of Wnt/beta-catenin signal transduction and autophagy in Rap-induced neural differentiation are further discussed. We found that Rap promotes the proliferation, migration and neural differentiation of hUC-MSCs in a dose-dependent manner, while inhibiting senescence and apoptosis, by inhibiting Wnt/β -catenin signaling pathway and activating autophagy.

Disclosure of Invention

Based on the problems in the prior art, the method utilizes the rapamycin to treat the human umbilical cord mesenchymal stem cells so as to improve the cell viability and/or the cell proliferation rate and/or the cell migration of the human umbilical cord mesenchymal stem cells.

The method for improving the viability of the human umbilical cord mesenchymal stem cells comprises the following steps: human umbilical cord mesenchymal stem cells at a density of 0.2-0.8 ten thousand cells/well were treated with rapamycin at a concentration of 1-10 nM.

The method for improving the proliferation rate of the human umbilical cord mesenchymal stem cells comprises the following steps: human umbilical cord mesenchymal stem cells at a density of 0.4 to 1 ten thousand cells per well are treated with rapamycin at a concentration of 1 to 10 nM.

The method for improving migration of human umbilical cord mesenchymal stem cells comprises the following steps: human umbilical cord mesenchymal stem cells at a density of 2-5 ten thousand cells per well were treated with rapamycin at a concentration of 1-10 nM.

The invention also discloses application of improving the activity, proliferation and migration of the human umbilical cord mesenchymal stem cells, in particular application of improving the cell activity and/or cell proliferation rate and/or cell migration of the human umbilical cord mesenchymal stem cells by using the rapamycin.

The invention also discloses a neuron-like cell which is obtained by culturing the human umbilical cord mesenchymal stem cell treated by the method.

The invention further discloses preparation of the neuron-like cells and application of the neuron-like cells in treating neurodegenerative diseases.

Compared with the prior art, the method adopts rapamycin (Rap) to treat the human umbilical cord mesenchymal stem cells, can respectively increase the activity, proliferation and migration of the hUC-MSCs, simultaneously inhibit the aging and apoptosis of the hUC-MSCs, and activate the autophagy of the hUC-MSCs and induce the hUC-MSCs to differentiate into neuron-like cells. Furthermore, the invention can realize the control of the hUC-MSCs in different degrees by adopting different doses of rapamycin (Rap) in a dose-dependent manner, activate corresponding signal paths, and increase or inhibit different gene expressions, thereby realizing the improvement of the fate of the hUC-MSCs according to requirements.

Drawings

FIG. 1 shows the effect of Rap on the viability, proliferation and migration of hUC-MSCs cells.

FIG. 2 shows the regulation of senescence and apoptosis of hUC-MSCs by Rap.

FIG. 3 is a graph showing the effect of Rap on neural differentiation of hUC-MSCs.

FIG. 4 shows the Wnt/beta-catenin signaling pathway in hUC-MSCs promoted by Rap.

FIG. 5 shows that XAV-939 reverses rap-induced Wnt/β -catenin signaling activation.

FIG. 6 is a graph showing that XAV-939 attenuates the effect of Rap on hUC-MSCs.

FIG. 7 shows Rap-activated autophagy of hUC-MSCs.

FIG. 8 shows the regulation effect of 3-MA reversed Rap on hUC-MSCs.

Detailed Description

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

Examples

1. Test materials and methods.

1.1 materials

Dulbecco's modified eagle's medium low glucose (DMEM)/F-12, Phosphate Buffered Saline (PBS), fetal bovine serum (FBS, pH 7.4), dimethyl sulfoxide (DMSO) were purchased from Solarbio (china). Cell Counting Kit-8(CCK-8), 4-t-butyl anisole (BHA), recombinant human insulin, hydrocortisone, l-glutamine, potassium chloride were purchased from USA

Inc (china). Human EGF and FGF-b were purchased from Norrin (China). CCK-8, EdU Apollo 567 and other in vitro kits were purchased from Riobio, China. Rapamycin (rap), XAV-939, 3-MA are available from MedChemexpress (China). Antibodies were purchased from Proteintech (China)

1.2 isolation, culture and characterization of hUC-MSCs

Methods for isolating, identifying and culturing hUC-MSCs are described in the materials and methods section of the cited reference [1 ]. Passage 3-5 cells were used for subsequent experiments.

1.3 cell processing and grouping

The hUC-MSCs were treated with Rap, XA V-939 and 3-MA, respectively, and divided into different groups. Con group (hUC-MSCs untreated group), 1 nRap treatment group (hUC-MSCs received 1nM Rap treatment), 10nM Rap treatment group (hUC-MSCs received 10nM Rap treatment), 20nM Rap treatment group (hUC-MSCs received 20nM Rap treatment), 30nM Rap treatment group (hUC-MSCs received 30nM Rap treatment), Rap + XAV-939 group (10nM Rap and 10mM XAV-939 concurrent hUC-MSCs 48h), Rap +3-MA group (10nM Rap and 5mM 3-MA concurrent hUC-Cs 48 h).

1.4 CCK-8 experiment

Rap was dissolved in DMSO and diluted to appropriate concentration. Cell viability was determined using the CCK-8 kit according to the instructions. Fourth generation hUC-MSCs (P4) were plated in 96-well plates (0.2-0.8 ten thousand cells/well) and treated with different concentrations of Rap (0,1,10,20 and 30nM) for 24, 48, 72 hours, after which cell viability was determined by adding 10. mu.L DMEM/F12 containing 10. mu.L CCK-8 solution as described (methods see "materials and methods" section of citation [2 ]).

1.5 EdU staining

EdU staining detects the proliferation rate of hUC-MSCs. The hUC-MSCs were plated in 48-well plates (0.4-1 ten thousand cells/well) and treated with different concentrations of Rap (0,1,10,20 and 30nM) for 72 hours followed by incubation with 50. mu.M EdU solution for 12 hours. Then, after the incubation is finished, the EdU working solution is discarded, the PBS is used for washing, 4% paraformaldehyde is added for fixing for 30min, the fixing solution is discarded, the PBS is used for washing for 3 times, 5min is carried out each time, 50 mu L of glycine solution with the concentration of 2mg/mL is added into each hole, and the incubation is carried out for 5min at room temperature, so that the residual fixing solution is neutralized. Subsequently, 100. mu.L of 0.5% Triton X-100 prepared with PBS was added to each well after PBS washing, and after 20min incubation at room temperature, 100. mu.L of Apollo staining solution was added to each well and incubated for 30min at room temperature in the dark. Next, the staining solution was removed, washed three times with PBS, DAPI was added for counterstaining, and after 10min DAPI was removed and PBS was added for photography under a fluorescence microscope. Finally, photographs were taken with a fluorescence microscope (olympus, japan), and the number of red (EdU-labeled) and blue (DAPI-labeled) cells was recorded and counted, and graphed.

1.6 Transwell experiment

Cell migration was examined using a Transwell (methods see the materials and methods section of cited reference [3 ]) experiment. Briefly, (0,1,10,20nM) Rap-treated cells were harvested for 72h and tested for migration of hUC-MSCs after 24h incubation in the upper chamber at a density of 2-5 ten thousand cells/well.

1.7 Annexin V/PI staining

After incubating hUC-MSCs (5-18 ten thousand cells) with Rap (0,1,10,20nM) for 72h, the cells were digested with EDTA-free trypsin, 5-8 ten thousand resuspended cells were collected and analyzed for apoptosis using Annexin V/PI apoptosis detection kit (BD Biosciences, USA) (methods see "materials and methods" section of citation [4 ]).

1.8 SA-. beta. -gal staining

hUC-MSCs (5-18 ten thousand cells/well) were incubated in 6-well plates for 24 hours. After treating the cells with Rap (0,1,10,20nM) for 72 hours, the senescence of hUC-MSCs was examined using the SA-. beta. -gal kit (methods see "materials and methods" section of citation [2 ]).

1.9 neural differentiation of hUC-MSCs

Reference is made to the "materials and methods" section of the cited reference [5] for neural differentiation of hUC-MSCs. hUC-MSCs (1-6 ten thousand cells/well) were cultured in 24-well plates for 24 hours until the cells were fully adherent, and after 3 days of Rap (0,1,10,20nM) treatment, hUC-MSCs were pretreated with DMEM/LG medium containing 20% FBS and 10ng/ml bFGF for 24 hours. Subsequently, the cells were replaced with neural induction medium (DMEM/LG medium containing 2% DMSO, 100. mu. MBHA,25nM KCl, 10. mu.M Forskolin, 0.866. mu.M insulin, and 1. mu.M hydrocortisone) for 24 h. Subsequently, differentiation was further maintained for 3 to 4 days with a neural differentiation induction medium (DMEM/F12 medium containing 10ng/mL EGF,10ng/mL bFGF, 10% FBS,1 XB 27,1 XN 2).

1.10 immunofluorescence

The hUC-MSCs (1-6 ten thousand cells/well) were plated on 24-well plates at the P4 generation, and were subjected to neural induction after 3 days of treatment with Rap (0,1,10,20nM), and then to immunofluorescence staining to examine neural differentiation (see "materials and methods" section of citation [2 ]). After washing, fixation, blocking of the cells, incubation with antibodies specific for NeuN (1:200), TuBB3(1:200), NSE (1:200) respectively overnight at 4 ℃ followed by FITC goat anti-rabbit IgG (1:250) and Cy3 goat anti-mouse IgG (1:250) respectively. After that, counterstaining was performed with DAPI (Solarbio, beijing, china). The labeled cells were photographed using an inverted fluorescence microscope (DMi8, come, germany).

1.11 real-time quantitative PCR

hUC-MSCs (5-18 ten thousand cells/well) were cultured in 6-well plates for 24 hours. After treating the cells with Rap (0,1,10,20nM) for 72 hours, total RNA of each group of cells was analyzed by TRIzol reagent extraction. Real-time quantitative PCR was used to detect Ngn1, Ngn2, Mash1,. beta. -IIItubulin and MAP-2mRNA expression (methods see the "materials and methods" section of citation [2 ]).

1.12 Western blotting

hUC-MSCs (5-18 ten thousand cells/well) were cultured in 6-well plates for 24 hours. After treating the cells with Rap (0,1,10,20nM) for 72 hours, proteins were extracted from each group of cells. The total protein load of 25-60 μ g was taken from each group and subjected to SDS-PAGE separation, transferred to PVDF membrane, and then treated with specific primary antibody: wnt3a (1:2000), GSK3 beta (1:3000), beta-catenin (1:2000), P16(1:2000), PCNA (1:2000), Sirt1(1:2000), LC3(1:500), P62(1:500) and beta-actin (1:2000) were incubated overnight. Subsequently, the membrane was incubated with a secondary antibody (1:3000) for 2h, and the protein expression level was calculated using ImageJ software (NIH, Bethesda, Md., USA).

1.13 statistical analysis

Each experiment was repeated 3 times. Data are expressed as mean ± standard deviation and evaluated using Graphpad Prism software. Differences between groups were assessed using one-way analysis of variance (ANOVA) and LSD-t test. P <0.05 is statistically significant for the differences.

2. Results of the experiment

2.1 Rap dose-and time-dependent on the proliferation and migration of hUC-MSCs

The effect of Rap on the proliferation and migration of hUC-MSCs was examined by CCK8, EdU and Transwell experiments, respectively. As can be seen from fig. 1,10nM Rap significantly improved the viability of the hUC-MSCs, increased the percentage of EdU-positive (proliferating) cells, and promoted migration of the hUC-MSCs compared to the CON group (fig. 1, P < 0.05). The results suggest that the activity, proliferation and migration of cells can be improved after the Rap with the concentration of 1-10nM is used for treating the hUC-MSCs. While 20nM Rap significantly inhibited cell viability, EdU positive cells and migration of the hUC-MSCs (figure 1, P < 0.05). Thus, our results indicate that Rap promotes cell proliferation and migration of the hUC-MSCs in a dose and time dependent manner.

In FIG. 1, (A) the cell viability mediated by Rap was analyzed by the CCK-8 method. (B) The different groups had a representative 9 EdU-positive staining pattern. (C) Percent EdU positive cells. (D) The migration of hUC-MSCs was detected by the Transwell method. (E) Each group of migrated cells. Scale bar 200 μm. Data are expressed as mean ± standard deviation. P <0.05 compared to CON group.

2.2 Rap inhibits senescence and apoptosis of hUC-MSCs in a dose-dependent manner

Referring to section A, B in FIG. 2, Rap significantly inhibited senescence of hUC-MSCs, SA- β -Gal positive cell rates were 11.21. + -. 1.0%, 8.63. + -. 2.33%, 12.11. + -. 1.52% in the 1,10 and 20nM Rap-treated groups, respectively, compared to 15.51. + -. 1.03% in the CON group (P < 0.05). western blotting examined the expression of P16, SIRT1, and PCNA. The results show that 1,10 and 20nM Rap decreased the expression of P16, while increasing the expression of SIRT1 and PCNA (FIG. 2, parts C and D, P < 0.05). The P16 gene plays an important role as a cyclin kinase inhibitor gene in the genetic control program of cellular senescence. The P16 gene is over-expressed in senescent cells, and the inhibition of the expression of the P16 gene can not only slow down the senescence speed of cells and prolong the life of the cells, but also slow down the shortening speed of the telomere length of the cells. The SIRT1 gene is an inhibitor in the process of apoptosis, and the expression of the SIRT1 gene under stress conditions can reduce the apoptosis and the senescence of cells and increase the self-repair and survival rate of the cells. PCNA is a gene of proliferating cell nuclear antigen, which participates in the production and repair of cells and can delay the aging rate of cells. Therefore, as shown in the attached figure 2 of the specification, the expression of the P16 gene in the hUC-MSCs can be inhibited through the treatment of the Rap with the concentration of 1-20nM, and the expression of SIRT1 and PCNA is promoted, so that the technical effects of inhibiting the cell senescence of the hUC-MSCs and prolonging the life of the hUC-MSCs are achieved.

Annexin V/PI staining showed that 1nM and 10nM Rap inhibited hUC-MSCs apoptosis, and 20nM Rap promoted hUC-MSCs apoptosis (FIG. 2, part E, P < 0.05). Thus, these results indicate that Rap inhibits senescence and apoptosis in a concentration-dependent manner.

2.3 Rap promotes neural differentiation of hUC-MSCs

To determine whether Rap can induce neuc-MSCs neural differentiation, TuBB3 (early neuronal marker), NSE (neuronal marker) and NeuN (mature neuronal marker) were tested separately. As shown in fig. 3, parts a and B, 1,10 and 20nM Rap significantly increased the expression of TuBB3, NSE and NeuN, and was dose-dependent (P < 0.05). MAP-2 is a microtubule-associated protein that is found primarily in the soma, dendrites, and dendrite spines of neurons in normal brain tissue. Ngn1, Ngn2, and Mash1 are all members of the pre-neuronal basic helix-loop-helix (bHLH) family of transcription factors. High expression of Ngn1 in neuronal progenitor cells and immature neurons plays an important role in neurogenesis. Ngn2 and Mash1 are activated bHLH genes necessary for determining neuronal fate, and increased expression thereof can promote maturation of neural differentiation. qRT-PCR detects neuron-specific gene expression. The results show that Rap decreases expression of Ngn1, while expression of Ngn2, Mash1, TuBB3 and MAP-2 tended to increase in dose (part C of fig. 3, P < 0.05). These data demonstrate that 1,10 and 20nM Rap significantly promoted neural differentiation of the hUC-MSCs.

FIG. 3 Effect of Rap on neural differentiation of hUC-MSCs. (A) Representative immunofluorescent staining for TuBB3, NeuN, NSE in each group. (B) TuBB3+, NeuN +, NSE + cell ratio. (C) The expression of the mRNAs of the groups of Map2, Ngn1, Ngn2, Mash1 and TuBB3 is detected by qRT-PCR. Scale bar 100 μm. Data are expressed as mean ± standard deviation. P <0.05 compared to CON group.

2.4 Rap promoting Wnt/beta-catenin signal channel in hUC-MSCs

1. After 10nM Rap and 20nM Rap are acted for 72h, Western blot detects the expression of Wnt3a, GSK3 beta and beta-catenin. Rap significantly promoted the expression of Wnt3a and beta-catenin in hUC-MSCs, while inhibiting the expression of GSK-3 beta (FIG. 4, P < 0.05). The activated Wnt/beta-catenin signal pathway can regulate the neural differentiation of human umbilical cord mesenchymal stem cells and promote the proliferation and development of nerve cells, so that the Wnt/beta-catenin signal pathway can be obviously activated after 1,10 and 20nM Rap is adopted to act on the human umbilical cord mesenchymal stem cells as shown in figure 4.

FIG. 4 Rap promotes Wnt/beta-catenin signaling pathway in hUC-MSCs. (A) Representative image of Western blotting. (B) And (3) analyzing a Western blotting gray value. Data are expressed as mean ± standard deviation. P <0.05 compared to CON group.

2.5 XAV-939 Reversal of Rap-induced Wnt/beta-catenin Signal activation

To determine whether Rap promotes the proliferation, migration and neural differentiation of hUC-MSCs by activating Wnt/β -catenin signaling, we treated cells with the Wnt/β -catenin pathway inhibitor XA V-939. As shown, 10mM XA V-939 reversed rap-induced activation of Wnt3a and β -catenin and inhibition of GSK3 β by Western blotting assay (FIG. 5, P < 0.05).

FIG. 5 XAV-939 reverses Rap-induced Wnt/β -catenin signaling activation. (A) Immunoblotting and (B) densitometric analysis of Wnt3a, GSK3 β and β -catenin. Data are expressed as mean ± standard deviation. P <0.05 compared to CON group. # P <0.05 compared to Rap group.

2.6 inhibition of the Wnt/beta-catenin signaling pathway attenuates the effect of Rap on hUC-MSCs.

As shown in fig. 6, XAV-939 significantly inhibited Rap-induced proliferation (part a, B of fig. 6), migration (part C, D of fig. 6) and neural differentiation (part E, F of fig. 6) (P < 0.05). XAV-939 reversed the inhibitory effect of Rap on the senescence and apoptosis of hic-MSCs (section G, H, I of fig. 6, P < 0.05).

FIG. 6 XAV-939 attenuates the effect of Rap on hUC-MSCs. (A) EdU staining and (B) quantification of EdU + cells. (C) Transwell assay and (D) quantitative analysis of migrating cells. (E) Immunofluorescence profile of different groups of TuBB3+ cells. (F) TuBB3+ cell ratio. (G) Each group had a representative SA-. beta. -gal staining pattern. (H) Percentage of SA-. beta. -gal positive cells. (I) And detecting the apoptosis condition by using a flow cytometer.

2.7 Rap activation of autophagy in hUC-MSCs

As shown in FIG. 7,1, 10 and 20nM Rap significantly increased the levels of Beclin1 and LC3-II/LC3-I in hUC-MSCs, while decreasing the expression of P62 (FIG. 7, P < 0.05).

FIG. 7Rap activates autophagy of hUC-MSCs. (A) Representative immunoblots and (B) grey value analysis of P62, Beclin1 and LC 3. Data are expressed as mean ± standard deviation. P <0.05 compared to CON group.

2.83-MA Regulation of the Rap on hUC-MSCs

To elucidate the role of autophagy in this process, we added 3-MA to the Rap group. As shown in fig. 8, 3-MA significantly reversed Rap-mediated EdU +, cell migration and neural differentiation, and promoted cellular senescence and apoptosis of Rap-treated hic-MSCs (fig. 8, P < 0.05).

FIG. 83-MA reverses the regulatory effect of Rap on hUC-MSCs (A) EdU staining and (B) quantification of EdU + cells. (C) Transwell assay and (D) quantitative analysis of migrating cells. (E) Different groups of TuBB3+ cell immunofluorescence profiles. (F) TuBB3+ cell ratio. (G) Each group had a representative SA-. beta. -gal staining pattern. (H) Percentage of SA-. beta. -gal positive cells. (I) And detecting the apoptosis condition by using a flow cytometer. Scale bar 200 μm. Data are expressed as mean ± standard deviation. P <0.05 compared to CON group. # P <0.05 compared to Rap group.

Stem cells can be classified into totipotent stem cells, pluripotent stem cells and unipotent stem cells according to their differentiation potential. Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that are present in bone marrow, umbilical cord, placenta, fat, lung, liver, and skin. The stem cells can be differentiated into nerve cells at the damaged part, replace damaged cells, secrete neurotrophic factors, regulate immune response and promote nerve regeneration of Traumatic Brain Injury (TBI) and Alzheimer Disease (AD). However, low migration and neural differentiation abilities are major obstacles to stem cell therapy, and the function of MSCs transplantation in vivo is severely affected. Therefore, how to improve the proliferation and neural differentiation efficiency of the MSCs is a key factor of applying the MSCs to regenerative medicine, and has important significance.

Autophagy plays an important role in cell proliferation, migration, differentiation and apoptosis. Studies have shown that autophagy is involved in a variety of cell differentiation, including monocytes, satellite cells and stem cells, which may provide a new strategy for promoting neural differentiation of regenerative medicine stem cells. Rap is a potent autophagy activator that inhibits the mTOR signaling pathway. Research shows that Rap and ascorbic acid can effectively promote differentiation of embryonic stem cells to cardiac muscle cells. Different stem cell types, passages and different microenvironments may all influence the induction effect of Rap.

The invention discovers that the medium-low dose Rap (1,10nM) can improve the vitality, proliferation and migration of the hUC-MSCs and simultaneously inhibit the senescence and apoptosis of the hUC-MSCs. The 20nM Rap can inhibit the proliferation and migration of hUC-MSCs and induce apoptosis, suggesting that Rap is concentration and time dependent on the self-renewal of hUC-MSCs. Interestingly, 20nM Rap also inhibited senescence of hUC-MSCs, probably because Rap could decoy cells and prevent replicative senescence.

In addition, recent studies demonstrated that rap-induced autophagy promotes differentiation of bone marrow mesenchymal stem cells into neuron-like cells by modulating the Notch1 signaling pathway. And detecting the relationship between the Rap-induced autophagy and neural differentiation in the hUC-MSCs by adopting immunofluorescence and qRT-PCR. The invention finds that the Rap-induced autophagy can enhance the expression of neuron-specific markers, such as TuBB3, NSE and NeuN. In addition, the bHLH transcription factors Ngn1, Ngn2, Mash1 and the like are key proteins for regulating the fate of neural stem cells and are involved in neural differentiation and development of the stem cells. Mash1 probably determines the function of nerve survival and participates in the transition of the neural stem cells to mature differentiated cells. Ngn is a transcriptional agonist of Neuron D. Ngn1, Ngn2 and Mash1 play important roles in determining the nervous system lineage of the central and peripheral nervous systems. In this study, the present inventors found that Rap decreased Ngn1, while Ngn2 and Mash1 increased. Thus, Rap-induced autophagy can promote neural differentiation of the hUC-MSCs. And the autophagy inhibitor 3-MA can obviously inhibit Rap-induced cell proliferation and neural differentiation by reducing EdU +, cell migration and neural differentiation in Rap-treated hUC-MSCs, and promote cell aging and apoptosis.

Wnts is a glycoprotein family, at least comprising three different signal pathways, namely a canonical Wnt/beta-catenin signal pathway, a non-canonical Wnt-Frizzled signal pathway, and two intracellular signal cascade pathways consisting of a Wnt/Ca 2+ pathway and a Wnt/PCP pathway. The Wnt/β -catenin pathway plays a crucial role in the development of stem cells, such as survival, differentiation and apoptosis. In the invention, 1,10 and 20nM Rap is found to significantly activate the Wnt/beta-catenin pathway, the expression of Wnt3a and beta-catenin is high, and the expression of GSK3 beta is reduced, which can be reversed by XAV-939. Further studies have shown that XAV-939 can attenuate Rap-induced proliferation, migration and neural differentiation of hUC-MSCs. XAV-939 can reverse the inhibition effect of Rap on hUC-MSCs senescence and apoptosis.

Rap can promote the proliferation, migration and neural differentiation of hUC-MSCs in a dose-dependent manner, and inhibit senescence and apoptosis by activating the Wnt/beta-catenin pathway and autophagy. These results may provide new options for improving the fate of the hUC-MSCs and the application of stem cell therapy in acute and chronic neurodegenerative diseases (such as brain injury, cerebral ischemia, epilepsy, Parkinson's disease, Alzheimer's disease, etc.).

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

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Claims (7)

1. a method for improving cell viability and/or cell proliferation rate and/or cell migration of human umbilical cord mesenchymal stem cells, which is characterized in that the human umbilical cord mesenchymal stem cells are treated by rapamycin.

2. The method of claim 1, wherein the method for improving the viability of the human umbilical cord mesenchymal stem cell is: human umbilical cord mesenchymal stem cells at a density of 0.2-0.8 ten thousand cells/well are treated with rapamycin at a concentration of 1-10 nM.

3. The method of claim 1, wherein the method for increasing the proliferation rate of human umbilical cord mesenchymal stem cells is: human umbilical cord mesenchymal stem cells at a density of 0.4 to 1 ten thousand cells per well are treated with rapamycin at a concentration of 1 to 10 nM.

4. The method of claim 1, wherein the method for increasing migration of human umbilical cord mesenchymal stem cells is: human umbilical cord mesenchymal stem cells at a density of 2-5 ten thousand cells per well were treated with rapamycin at a concentration of 1-10 nM.

5. Use of rapamycin for increasing the viability, proliferation and migration of human umbilical cord mesenchymal stem cells, wherein the method according to any of claims 1 to 4 is used for increasing the cell viability and/or cell proliferation rate and/or cell migration of human umbilical cord mesenchymal stem cells.

6. A neuron-like cell obtained by culturing a human umbilical cord mesenchymal stem cell treated by the method according to any one of claims 1 to 4.

7. Use of the neuron-like cell of claim 6 in the preparation of a medicament for treating a neurodegenerative disease.

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