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

A method for improving the viability, proliferation and migration of human umbilical cord mesenchymal stem cells and its application

technical field

The application of the present invention belongs to the technical field of stem cell processing, and relates to a method for improving the vitality, proliferation and migration of human umbilical cord mesenchymal stem cells and its application, in particular to a method for applying rapamycin in the field of mesenchymal stem cells and the application thereof. It is suitable for increasing the vitality, proliferation and migration of human umbilical cord mesenchymal stem cells by using rapamycin.

Background technique

Mesenchymal stem cells (MSCs) have high self-renewal ability and multiple differentiation ability, and have broad prospects in the treatment of neurodegenerative diseases such as Alzheimer's disease. However, MSCs cultured in vitro may exhibit replicative senescence, which is mainly manifested by low cell viability, reduced migration and differentiation ability, and directly affects the effect of cell transplantation. There are many methods to induce the differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) into neuron-like cells in vitro. However, these differentiation protocols are less efficient. Therefore, improving the cell viability and neural differentiation rate of MSCs is the key to improve the effect of stem cell transplantation.

Autophagy is an intracellular catabolic mechanism negatively regulated by mTOR signaling, and plays an important role in maintaining stem cell homeostasis and fate through various mechanisms such as self-renewal and differentiation. Rapamycin (Rap) is an autophagy activator that also inhibits the mTOR signaling pathway. Studies have shown that Rap can effectively regulate the cardiac differentiation of mouse embryonic stem cells, the chondrogenic differentiation, adipose differentiation and liver differentiation of hepatic progenitor cells. Furthermore, autophagy was activated during neuronal differentiation of MSCs. Previous studies have shown that autophagy promotes the differentiation of bone marrow mesenchymal stem cells into neurons by regulating the Notch1 signaling pathway. However, the role of Rap-induced autophagy in the proliferation and neural differentiation of hUC-MSCs and its mechanism remain unclear.

The Wnt/β-catenin signaling pathway plays an important role in regulating stem cell fate, such as self-renewal and differentiation. Normally, cytoplasmic β-catenin binds to APC, Axin, GSK-3β, and CKIε, resulting in the degradation of β-catenin. Once the Wnt/β-catenin signaling pathway is activated, cytoplasmic β-catenin transfers to the nucleus, interacts with the LEF1/TCF family, induces the expression of downstream target genes CyclinD1 and c-Myc, and regulates cell proliferation and differentiation. Studies have shown that autophagy can promote cardiac or liver differentiation by regulating the Wnt/β-catenin signaling pathway. However, whether Rap can promote the neural differentiation of hUC-MSCs through Wnt/β-catenin signaling remains to be further investigated.

In the present invention, the effects of Rap on the proliferation and neural differentiation of hUC-MSCs were studied, and the roles of Wnt/β-catenin signal transduction and autophagy in Rap-induced neural differentiation were further explored. We found that Rap promoted the proliferation, migration and neural differentiation of hUC-MSCs in a dose-dependent manner by inhibiting the Wnt/β-catenin signaling pathway and activating autophagy, while inhibiting senescence and apoptosis.

SUMMARY OF THE INVENTION

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

The method for improving the cell viability of human umbilical cord mesenchymal stem cells in the present invention is as follows: using 1-10 nM concentration of rapamycin for human umbilical cord mesenchymal stem cells per density of 0.2-0.8 million cells/well.

The method for increasing the proliferation rate of human umbilical cord mesenchymal stem cells in the present invention is as follows: rapamycin at a concentration of 1-10 nM is used for human umbilical cord mesenchymal stem cells at a density of 0.4-10,000 cells/well.

The method for improving the migration of human umbilical cord mesenchymal stem cells in the present invention is as follows: the human umbilical cord mesenchymal stem cells with a density of 20,000 to 50,000 cells/well are treated with rapamycin at a concentration of 1-10 nM.

The invention also discloses an application for improving the viability, proliferation and migration of human umbilical cord mesenchymal stem cells, specifically using rapamycin to improve the cell viability and/or cell proliferation rate and/or cell migration of human umbilical cord mesenchymal stem cells Applications.

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

The invention further discloses the preparation of a neuron-like cell and its use in treating neurodegenerative diseases.

Compared with the prior art, the present invention uses rapamycin (Rap) to treat human umbilical cord mesenchymal stem cells, which can respectively increase the viability, proliferation and migration of hUC-MSCs, inhibit the senescence and apoptosis of hUC-MSCs, and activate the hUC-MSCs. Autophagy of hUC-MSCs and induction of neuron-like cell differentiation. Further, in the present invention, in a dose-dependent manner, different doses of rapamycin (Rap) can be used to control hUC-MSCs to different degrees, activate corresponding signaling pathways, and increase or inhibit the expression of different genes, thereby achieving The fate of hUC-MSCs needs to be improved.

Description of drawings

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

Figure 2 shows the regulatory effect of Rap on the senescence and apoptosis of hUC-MSCs.

Figure 3 shows the effect of Rap on neural differentiation of hUC-MSCs.

Figure 4 shows that Rap promotes the Wnt/β-catenin signaling pathway in hUC-MSCs.

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

Figure 6 shows that XAV-939 attenuates the effect of Rap on hUC-MSCs.

Figure 7 shows that Rap activates autophagy in hUC-MSCs.

Figure 8 shows that 3-MA reverses the regulatory effect of Rap on hUC-MSCs.

Detailed ways

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

Example

1. Experimental materials and methods.

1.1 Materials

Inc.(中国)。人EGF和FGF-b购自诺富蛋白(中国)。CCK-8、EdUApollo 567等体外试剂盒购于中国Riobio公司。Rapamycin(Rap)、XAV-939、3-MA购自MedChemExpress(中国)。抗体购自Proteintech(中国)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). CellCounting Kit-8 (CCK-8), 4-tert-butylanisole (BHA), recombinant human insulin, hydrocortisone, l-glutamine, potassium chloride were purchased from the United States

Inc. (China). Human EGF and FGF-b were purchased from Novoprotein (China). In vitro kits such as CCK-8 and EdUApollo 567 were purchased from Riobio, China. Rapamycin (Rap), XAV-939, 3-MA were purchased from MedChemExpress (China). Antibodies were purchased from Proteintech (China)

1.2 Isolation, culture and identification of hUC-MSCs

For the isolation, identification and culture of hUC-MSCs, please refer to the "Materials and Methods" section of citation [1]. Cells at passages 3-5 were used for subsequent experiments.

1.3 Cell Handling 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), 1nMRap 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 simultaneously treated hUC-MSCs for 48h), Rap+3-MA group (10nM Rap and 5mM 3 -MA simultaneously treated hUC-MSCs for 48h).

1.4 CCK-8 experiment

Rap was dissolved in DMSO and diluted to the appropriate concentration. Cell viability was determined using the CCK-8 kit according to the instructions. The fourth generation hUC-MSCs (P4) were plated in 96-well plates (0.2-0.8 million cells/well) and treated with different concentrations of Rap (0, 1, 10, 20 and 30 nM) for 24, 48, and 72 hours. Then, 10 μL of DMEM/F12 containing 10 μL of CCK-8 solution was added to measure cell viability according to the manufacturer's instructions (for methods, refer to the "Materials and Methods" section of citation [2]).

1.5 EdU staining

EdU staining was used to detect the proliferation rate of hUC-MSCs. hUC-MSCs were plated in 48-well plates (0.4-10,000 cells/well) and treated with different concentrations of Rap (0, 1, 10, 20 and 30 nM) for 72 hours, and then incubated with 50 μM EdU solution for 12 hours. Then, after the incubation, discard the EdU working solution, wash with PBS, add 4% paraformaldehyde for fixation for 30 min, discard the fixative, wash 3 times with PBS, 5 min each time, and add 50 μL of 2 mg/mL glycine solution to each well Incubate at room temperature for 5 min to neutralize residual fixative. Next, after washing with PBS, 100 μL of 0.5% Triton X-100 prepared with PBS was added to each well, and after incubation at room temperature for 20 minutes, 100 μL of Apollo staining solution was added to each well and incubated at room temperature for 30 minutes in the dark. Next, remove the staining solution, wash with PBS three times, add DAPI for counterstaining, remove DAPI after 10 min, add PBS, and take pictures under a fluorescence microscope. Finally, photographs were taken with a fluorescence microscope (Olympus, Japan), and the numbers of cells in red (EdU labeling) and blue (DAPI labeling) were recorded and counted, and plotted on Graphpad.

1.6 Transwell experiment

Cell migration was assayed using Transwell (methods refer to the "Materials and Methods" section of citation [3]). Briefly, cells treated with Rap (0, 1, 10, 20 nM) for 72 h were collected and cultured in the upper chamber at a density of 20,000 to 50,000 cells/well for 24 h to detect the migration of hUC-MSCs.

1.7 Annexin V/PI staining

After incubating hUC-MSCs (50,000-180,000 cells) with Rap (0, 1, 10, 20nM) for 72h, the cells were digested with EDTA-free trypsin, 50,000-80,000 resuspended cells were collected, and Annexin V/ Apoptosis was analyzed by PI Apoptosis Detection Kit (BDBiosciences, USA) (for the method, refer to the "Materials and Methods" section of citation [4]).

1.8 SA-β-gal staining

hUC-MSCs (50-180,000 cells/well) were incubated in 6-well plates for 24 hours. After treating cells with Rap (0, 1, 10, 20 nM) for 72 hours, the senescence of hUC-MSCs was detected by SA-β-gal kit (for methods, please refer to the "Materials and Methods" section of citation [2]).

1.9 Neural differentiation of hUC-MSCs

For the neural differentiation of hUC-MSCs, refer to the "Materials and methods" section of citation [5]. hUC-MSCs (10,000-60,000 cells/well) were cultured in a 24-well plate for 24 hours until the cells were completely attached, and after Rap (0, 1, 10, 20 nM) was used to treat the cells for 3 days, hUC-MSCs were treated with 20% FBS. and 10ng/ml bFGF in DMEM/LG medium for 24h. Subsequently, the neural induction medium (DMEM/LG medium containing 2% DMSO, 100 μM BHA, 25 nM KCl, 10 μM Forskolin, 0.866 μM insulin, and 1 μM hydrocortisone) was cultured for 24 h. Subsequently, the differentiation was further maintained with neural differentiation induction medium (DMEM/F12 medium containing 10 ng/mL EGF, 10 ng/mL bFGF, 10% FBS, 1×B27, 1×N2) for 3-4 days.

1.10 Immunofluorescence

P4 generation hUC-MSCs (10,000-60,000 cells/well) were plated on 24-well plates, and cells were treated with Rap (0, 1, 10, 20nM) for 3 days to induce neural induction of hUC-MSCs, and then immunofluorescence staining was used for detection. Neural differentiation (methods refer to the "Materials and methods" section of citation [2]). After rinsing, fixing and blocking, the cells were incubated with specific antibodies against NeuN (1:200), TuBB3 (1:200) and NSE (1:200) at 4°C overnight, and then with FITC goat antibody. Rabbit IgG (1:250) and Cy3 goat anti-mouse IgG (1:250) were incubated. Afterwards, counterstaining was performed with DAPI (Solarbio, Beijing, China). Labeled cells were photographed using an inverted fluorescence microscope (DMi8, Leica, Germany).

1.11 Real-time quantitative PCR

hUC-MSCs (50-180,000 cells/well) were cultured in 6-well plates for 24 hours. After treating cells with Rap (0, 1, 10, 20 nM) for 72 hours, the total RNA of each group of cells was extracted and analyzed by TRIzol reagent. Real-time quantitative PCR was used to detect the mRNA expression of Ngn1, Ngn2, Mash1, β-IIItubulin and MAP-2 (refer to the "Materials and Methods" section of citation [2] for methods).

1.12 Western blotting

hUC-MSCs (50-180,000 cells/well) were cultured in 6-well plates for 24 hours. After treating cells with Rap (0, 1, 10, 20 nM) for 72 hours, the proteins of each group of cells were extracted. 25-60 μg of total protein loading in each group was separated by SDS-PAGE, transferred to PVDF membrane, and then used specific primary antibodies: Wnt3a (1:2000), GSK3β (1:3000), β-catenin (1 :2000), P16 (1:2000), PCNA (1:2000), Sirt1 (1:2000), LC3 (1:500), P62 (1:500) and β-actin (1:2000) were incubated overnight . Subsequently, the membrane was incubated with secondary antibody (1:3000) for 2 h, 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 presented as mean ± standard deviation and evaluated using Graphpad Prism software. Differences between groups were evaluated by one-way analysis of variance (ANOVA) and LSD-t test. P<0.05 means the difference is statistically significant.

2. Experimental results

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

The effects of Rap on the proliferation and migration of hUC-MSCs were detected by CCK8, EdU and Transwell assays, respectively. It can be seen from Figure 1 that compared with the CON group, 1 and 10 nM Rap significantly increased the viability of hUC-MSCs, increased the percentage of EdU-positive (proliferating) cells, and promoted the migration of hUC-MSCs (Figure 1, P<0.05). The results suggest that 1-10 nM concentration of Rap can enhance cell viability, proliferation and migration after treatment of hUC-MSCs. And 20nM Rap significantly inhibited the cell viability, EdU positive cells and migration of hUC-MSCs (Figure 1, P<0.05). Therefore, our results suggest that Rap promotes cell proliferation and migration of hUC-MSCs in a dose- and time-dependent manner.

In Figure 1, (A) Rap-mediated cell viability was analyzed by CCK-8 assay. (B) Representative 9 EdU-positive staining images of different groups. (C) Percentage of EdU positive cells. (D) Transwell assay was used to detect the migration of hUC-MSCs. (E) Migrated cells in each group. Scale bar = 200 μm. Data are presented as mean ± standard deviation. *: Compared with CON group, P<0.05.

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

As shown in Parts A and B in Figure 2, Rap significantly inhibited the senescence of hUC-MSCs, and the SA-β-Gal-positive cell rates in the 1, 10, and 20 nM Rap treatment groups were 11.21±1.0%, 8.63±2.33%, 12.11±10%, respectively. 1.52% compared with 15.51±1.03% in CON group (P<0.05). The expressions of P16, SIRT1 and PCNA were detected by western blotting. The results showed that 1, 10 and 20 nMRap decreased the expression of P16, while increased the expression of SIRT1 and PCNA (Figure 2, Parts C and D, P<0.05). As a cyclin kinase inhibitor gene, p16 gene plays an important role in the genetic control program of cellular senescence. P16 gene is overexpressed in senescent cells, and inhibiting the expression of P16 gene can not only slow down cell senescence and prolong cell life, but also slow down the shortening of cell telomere length. SIRT1 gene is an inhibitory factor in the process of apoptosis. SIRT1 gene expression under stress conditions can reduce apoptosis and senescence, and increase the self-repair and survival rate of cells. PCNA is the gene of proliferating cell nuclear antigen, which is involved in the production and repair of cells and can delay the speed of cell senescence. Therefore, as shown in Figure 2 of the description, Rap treatment at a concentration of 1-20 nM can inhibit the expression of P16 gene in hUC-MSCs, and at the same time increase the expression of SIRT1 and PCNA, thereby inhibiting the senescence of hUC-MSCs and prolonging their life. technical effect.

Annexin V/PI staining showed that 1nM and 10nM Rap inhibited the apoptosis of hUC-MSCs, and 20nM Rap promoted the apoptosis of hUC-MSCs (Part E of Figure 2, P<0.05). Therefore, these results suggest 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 neural differentiation of hUC-MSCs, TuBB3 (early neuronal marker), NSE (neuronal marker) and NeuN (mature neuronal marker) were detected respectively. As shown in Parts A and B of Figure 3, 1, 10 and 20 nMRap significantly increased the expression of TuBB3, NSE and NeuN in a dose-dependent manner (P<0.05). MAP-2 is a microtubule-associated protein, and in normal brain tissue, it mainly exists in the soma, dendrites and dendritic spines of neurons. Ngn1, Ngn2 and Mash1 are all members of the proneural basic helix-loop-helix (bHLH) transcription factor family. Ngn1 is highly expressed in neuronal progenitor cells and immature neurons and plays an important role in neurogenesis. Ngn2 and Mash1 are activating bHLH genes required for neuronal fate determination, and their increased expression promotes the maturation of neural differentiation. Neuron-specific gene expression was detected by qRT-PCR. The results showed that Rap decreased the expression of Ngn1, while the expressions of Ngn2, Mash1, TuBB3 and MAP-2 showed a trend of increasing doses (part C of Figure 3, P<0.05). These data confirmed that 1, 10 and 20 nM Rap significantly promoted the neural differentiation of hUC-MSCs.

Figure 3 The effect of Rap on neural differentiation of hUC-MSCs. (A) Representative immunofluorescence staining of TuBB3, NeuN, and NSE in each group. (B) Proportion of TuBB3+, NeuN+, NSE+ cells. (C) qRT-PCR was used to detect the mRNA expression of Map2, Ngn1, Ngn2, Mash1 and TuBB3 in each group. Scale bar = 100 μm. Data are presented as mean ± standard deviation. *: Compared with CON group, P<0.05.

2.4 Rap promotes Wnt/β-catenin signaling pathway in hUC-MSCs

The expressions of Wnt3a, GSK3β and β-catenin were detected by Western blot after 1, 10, and 20 nM Rap for 72 h. Rap significantly promoted the expression of Wnt3a and β-catenin in hUC-MSCs, while inhibited the expression of GSK-3β (Fig. 4, P<0.05). The activated Wnt/β-catenin signaling pathway can regulate the neural differentiation of human umbilical cord mesenchymal stem cells and promote the proliferation and development of neural cells. As shown in Figure 4, 1, 10, and 20 nM Rap was used to act on the human umbilical cord. Mesenchymal stem cells can significantly activate the Wnt/β-catenin signaling pathway.

Figure 4 Rap promotes Wnt/β-catenin signaling pathway in hUC-MSCs. (A) Representative image of Western blotting. (B) Western blotting grey value analysis. Data are presented as mean ± standard deviation. *: Compared with CON group, P<0.05.

2.5 XAV-939 reverses Rap-induced activation of Wnt/β-catenin signaling

To determine whether Rap promotes the proliferation, migration and neural differentiation of hUC-MSCs by activating Wnt/β-catenin signaling, we treated the cells with the Wnt/β-catenin pathway inhibitor XA V-939. As shown, 10 mM XA V-939 reversed the rap-induced activation of Wnt3a and β-catenin and inhibition of GSK3β by Western blotting (Fig. 5, P<0.05).

Figure 5 XAV-939 reverses Rap-induced activation of Wnt/β-catenin signaling. (A) Western blot and (B) densitometric analysis of Wnt3a, GSK3β and β-catenin. Data are presented as mean ± standard deviation. *: Compared with CON group, P<0.05. #: P<0.05 compared to the Rap group.

2.6 Inhibition of Wnt/β-catenin signaling pathway attenuated the effect of Rap on hUC-MSCs.

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

Figure 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 migrated cells. (E) Immunofluorescence images of TuBB3+ cells in different groups. (F) Proportion of TuBB3+ cells. (G) Representative SA-β-gal staining plots for each group. (H) Percentage of SA-β-gal positive cells. (I) Cell apoptosis was detected by flow cytometry.

2.7 Rap activates autophagy in hUC-MSCs

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

Figure 7 Rap activates autophagy in hUC-MSCs. (A) Representative immunoblots and (B) grey value analysis of P62, Beclin1 and LC3. Data are presented as mean ± standard deviation. *: Compared with CON group, P<0.05.

2.8 3-MA reverses the regulatory effect of Rap on hUC-MSCs

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

Figure 8 3-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 migrated cells. (E) Immunofluorescence images of TuBB3+ cells in different groups. (F) Proportion of TuBB3+ cells. (G) Representative SA-β-gal staining plots for each group. (H) Percentage of SA-β-gal positive cells. (I) Cell apoptosis was detected by flow cytometry. Scale bar = 200 μm. Data are presented as mean ± standard deviation. *: Compared with CON group, P<0.05. #: P<0.05 compared to the Rap group.

Stem cells can be divided 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 exist in bone marrow, umbilical cord, placenta, fat, lung, liver and skin. Stem cells can differentiate into nerve cells at the site of injury, replace damaged cells, secrete neurotrophic factors, regulate immune responses, and promote nerve regeneration in traumatic brain injury (TBI) and Alzheimer's disease (AD). However, low migratory ability and neural differentiation ability are the main obstacles to stem cell therapy, which seriously affects the function of MSCs in vivo transplantation. Therefore, how to improve the proliferation and neural differentiation efficiency of MSCs is a key factor for the application of MSCs in regenerative medicine, which is of great 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 stem cells for regenerative medicine. Rap is a potent autophagy activator that inhibits the mTOR signaling pathway. Studies have shown that Rap combined with ascorbic acid can effectively promote the differentiation of embryonic stem cells into cardiomyocytes. Different stem cell types, passages and different microenvironments may affect the induction of Rap.

The present invention finds that low-dose Rap (1, 10 nM) can improve the viability, proliferation and migration of hUC-MSCs, while inhibiting the senescence and apoptosis of hUC-MSCs. 20nM Rap could 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, 20 nM Rap also inhibited the senescence of hUC-MSCs, which may be because Rap can trick the cells into preventing replicative senescence.

Furthermore, recent studies confirmed that rap-induced autophagy promotes the differentiation of BMSCs into neuron-like cells by regulating the Notch1 signaling pathway. Immunofluorescence and qRT-PCR were used to detect the relationship between Rap-induced autophagy and neural differentiation in hUC-MSCs. The present invention found that Rap-induced autophagy can enhance the expression of neuron-specific markers, such as TuBB3, NSE and NeuN. In addition, bHLH transcription factors Ngn1, Ngn2, Mash1, etc. are key proteins regulating neural stem cell fate and are involved in neural differentiation and stem cell development. Mash1 may determine the function of neural survival and participate in the transition of 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. Therefore, Rap-induced autophagy can promote the neural differentiation of hUC-MSCs. While the autophagy inhibitor 3-MA significantly inhibited Rap-induced cell proliferation and neural differentiation by reducing EdU+, and promoted cell senescence and apoptosis in Rap-treated hUC-MSCs.

Wnts are a family of glycoproteins, including at least three different signaling pathways: the canonical Wnt/β-catenin signaling pathway, the non-canonical Wnt-Frizzled signaling pathway, which consists of the Wnt/Ca 2+ pathway and the Wnt/PCP pathway. composed of intracellular signaling cascades. The Wnt/β-catenin pathway plays crucial roles in the development of stem cells, such as survival, differentiation and apoptosis. In the present invention, it was found that 1, 10 and 20 nM Rap significantly activated the Wnt/β-catenin pathway, with high expression of Wnt3a and β-catenin, and decreased GSK3β expression. This phenomenon can be reversed by XAV-939. Further studies showed that XAV-939 could attenuate Rap-induced proliferation, migration and neural differentiation of hUC-MSCs. XAV-939 reversed the inhibitory effect of Rap on senescence and apoptosis of hUC-MSCs.

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

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Citation:

1 Ma S, Liang S, Jiao H, et al.Human umbilical cord mesenchymal stemcells inhibit C6 glioma growth via secretion of dickkopf-1(DKK1)[J].Mol CellBiochem,2014,385(1-2):277

2 Wang X,Ma S,Meng N,et al.Resveratrol Exerts Dosage-DependentEffects on the Self-Renewal and Neural Differentiation of hUC-MSCs[J].MolCells,2016,39(5):418

3 Zhang T, Wang P, Liu Y, et al.Overexpression of FOXQ1 enhances anti-senescence and migration effects of human umbilical cord mesenchymal stemcells in vitro and in vivo[J].Cell Tissue Res,2018,373(2):379

4 Zhu X, Zhang Y, Li Q, et al.β-Carotene Induces Apoptosis in HumanEsophageal Squamous Cell Carcinoma Cell Lines via the Cav-1/AKT/NF-κB Signaling Pathway[J].J Biochem Mol Toxicol,2016,30( 3):148

5 Karahuseynoglu S, Cinar O, Kilic E, et al. Biology of stem cells inhuman umbilical cord stroma: in situ and in vitro surveys[J]. Stem Cells, 2007, 25(2): 319

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.

Images