Research On The Effect And Mechanism Of Ginsenoside Rg1 On The Proliferation Of Murine Bone Marrow Endothelial Progenitor Cells In Vitro

Objective Establishing the endothelial progenitor cell(EPC)line from the bone marrow of Mus musculus as a cell model;to explore the molecular mechanism of ginsenoside Rg1 on the proliferation of EPCs,RNA-Seq and bioinformatics were used to analyze the differentially expressed genes and their main significance of the mechanism,so as to provide the basis of molecular biology evidence for the treatment of endothelial injury diseases with ginsenoside Rg1,at the same time to search for the key targets to promote the growth of EPCs in vitro,and provides new ideas for the cultivation of sufficient and energetic EPCs.Methods 1.The EPCs of murine bone marrow were separated and purified by the method of whole bone marrow attachment and differential passage culture.The cells of EPCs were identified by morphological observation and antigen expression rate detection.2.In the process of EPCs culture,the stable cell lines were established by natural selection,and then the monoclonal cell lines were screened by dilution culture.The cell line MBMME2 was identified by mor phological observation,growth characteristic observation,cell surface antigen detection,karyotype analysis and in vitro angiogenesis experiment.3.The subculture murine bone marrow EPCs(MBMEPCs)and cell line MBMME2 of mouse bone marrow EPCs were divided into 7 groups,4 ginsenoside Rg1 treatment groups(final concentration 5,10,15,20 μmol/L),general subculture control group,solvent control group(1.25 ‰ ethanol)and positive control group(10-8 μmol/L estrogen),after being cultured for 96 h,the cell proliferation level was detected by MTT method.Subcultured MBMEPCs cells were collected and divided into 7 groups according to the above,and cultured for 3 weeks to observe the colony forming ability.MBMEPCs and MBMME2 cells were divided into 10 μmol/L ginsenoside Rg1 group and solvent control group,and cell cycle was analyzed by flow cytometry after being cultured for 96 h.4.MBMME2 cells were divided into four groups,three ginsenoside Rg1 treated groups(the final concentration was 5 μmol/L,10 μmol/L,20 μmol/L)and solvent control group,96 h later,the total RNA was collected to construct c DNA library,hiseq sequencing,and established transcriptome database.Through differential expression analysis,differential expression genes(DEGs)were screened out,and DEGs were used as the object for gene function annotation,signal pathway enrichment analysis,and protein interaction network analysis.According to the results of RNA-Seq analysis,20 DEGs were selected for RT-q PCR verification.Results 1.The isolated and purified MBMEPCs grew well,and most of the cells were short spindle or oval shape.After subcultured,the cells formed colonies in the early stage,grew up gradually,and then converged.In the center of larger colonies,the cells overlapped,and at the edge of the colonies,the cells were monolayer with typical cobblestone-like mor phology.The positive cells rates of CD90,CD133,CD34,CD29 and CD45 of EPC cells were 97.1%,83.4%,90.8%,23.5% and 8.8% respectively.2.In this study,the established cell line MBMME2 is a continuous cell line with the general biological characteristics of EPCs.Cell adherent growth rate was high.And 5% FBS in the growth medium can maintain the rapid proliferation of cells.The cell proliferation index was 44.8% ~ 47.9%.Flow cytometry showed that the positive cell rates of CD90,CD133,CD34,CD31 and CD45 were 99.7%,36.4%,96.8%,97.5% and 0.1% respectively,which were supported by the results of immunofluorescence staining.Most of the karyotypes are triploid.In vitro angiogenesis experiments showed that the cells could form a network like structure with multiple crosssections.The cell line is easy to propagate,and the cells floating on the large colony and fusion layer can be inoculated and cultured.3.After being cultured for 96 h in the condition of ginsenoside Rg1 concentration of 5-20 μmol/L,the MTT results of the subculture cells and MBMME2 cells showed that the absorbance values of the two cells increased first and then decreased with the increase of ginsenoside Rg1 concentration.The MTT absorption value of 10 μmol/L ginsenoside Rg1 group was the largest,and the MTT absorption value of each group in the range of 5-15 μmol/L was significantly higher than that of the control group.There was no significant difference between the 20 μmol/L concentration group and the control group.The colony forming experiment showed that the EPC colony forming ability of 5-20 μmol/L ginsenoside Rg1 groups were significantly higher than that of the control group;the colony forming rate of 10 μmol/L group was the highest,significantly higher than that of other groups.The proliferation index of 10 μmol/L ginsenoside Rg1 group was significantly higher than that of the solvent control group.4.RNA-Seq data showed that compared with the control group,the three ginsenoside Rg1 treatment groups had many significant differences in gene expression.The analysis of the function enrichment of DEG showed that DEGs were mainly related to cell communication,molecular function regulation,development process,cell membrane receptor,extracellular matrix,ion transport,etc.The main signal pathways involved include cell cycle,PI3 K Akt,Wnt,MAPK,estrogen,cell differentiation,etc.RT-q PCR results showed that Celsr3、Mapk4、Col3a1、Nos3、Mmp9、Cdkl3、Notch1、Grk4、Bnip3、Mapk7、Mapk11、Mapk8、Stat5a、Gper1、Bnip1、Cdk20 genes were significantly up-regulated,P21、Casp12、Casp4、Casp6 were significantly down regulated,which supported the results of RNA-Seq.These genes were closely related to the regulation of cell apoptosis and proliferation.Protein interaction network analysis showed that proteins encoded by differential genes such as Cdc20,CDK4,Cdk6,p21 and Bub1 b were important in some signal transduction networks,which were mainly related to cell cycle,cell aging,apoptosis,cell differentiation and cancer.Conclusion 1.The murine bone marrow endothelial progenitor cell line MBMME2 was successfully established for the first time by the combination of cell clone selection with long-term natural subculture.2.Under the condition of culture in vitro,ginsenoside Rg1 can promote the proliferation of murine bone marrow endothelial progenitor cells and their cell line in a concentration dependent manner;when the concentration is too high,the effect of ginsenoside Rg1 on proliferation is weakened.3.Transcriptome study revealed that the mechanism of ginsenoside Rg1 on the growth of MBMME2 cells in vitro involves up-regulation and down-regulation of many genes,which are mainly involved in JAK-stat,MAPK,Wnt,PI3K-Akt,cell differentiation and estrogen signaling pathways.