Experimental Study on the Promotion of Bone Marrow Mesenchymal Stem Cell Migration by Ruanjian Qingmai Granules
Objective:To investigate the effects of the serum containing Ruanjian Qingmai granules on the proliferation of bone marrow mesenchymal stem cells(BMSCs)and their chemotaxis towards vascular endothelial cells under the condition of glucose-oxygen deprivation,and to explore the possible mechanism of the granules in the treatment of atherosclerosis obliterans(ASO).Methods:Serum pharmacology was used to prepare the serum containing Ruanjian Qingmai granules.BMSCs were isolated and divided into six groups:normal group,model group,blank serum group,and low,medium and high dose groups of Chinese medicine.Cell proliferation toxicity assay(CCK-8)method was used to detect the effects of different concentrations of the serum containing the granules on the proliferation of BMSCs cells cultured under glyco-oxygen deprivation at 6,12,18,and 24 h.Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR)and Western blot were used to detect the effects of different concentrations of the drug-containing serum on the expression of CCND1,MYC,ERK1/2 mRNAs and proteins in BMSCs cultured under the condition of glycogen-oxygen deprivation.Transwell assay was performed to detect the migratory ability of BMSCs.BMSCs and human umbilical vein endothelial cells(HUVECs)were added to mouse serum of the blank serum group and the serum of medium-dose group of traditional Chinese medicine under the condition of glucose-oxygen deprivation,and the cell migration was detected after 24 h of treatment.Real-time PCR method was used to detect the expressions of chemokines PDGFB,CXCL12,CCL21mRNA on the cell surface of HUVECs and chemokine receptors PDGFRB,CXCR4,CCR7mRNA on the cell surface of BMSCs under the same conditions.Results:BMSCs cells were successfully isolated with 95%purity.1)CCK-8 results showed:Compared with the normal group,the cell proliferation ability of BMSCs in the model group increased significantly at 6 and 12 h(P＜0.01);compared with the normal group,the cell proliferation survival rate of BMSCs in the model group decreased evidently at 24 h(P＜0.01);compared with the blank serum group,each dosage group of the granules-containing serum improved the cell proliferation ability of hypoxic-ischemic BMSCs to varying degrees at 24 h(P＜0.01).2)The results of qRT-PCR and Western blot showed that:Compared with the normal group,the relative expressions of CCND1,MYC,ERK1/2 mRNA and protein in cells of the model group were noticeably reduced(P＜0.01);compared with the blank serum group,each dose group of the granules-containing serum could increase the relative expressions of CCND1,MYC,ERK1/2 mRNA and protein(P＜0.01).3)Transwell results displayed:Compared with the control group,the co-addition group could significantly promote the migration of BMSCs cells to HUVECs cells(P＜0.01).4)Real-time PCR results presented:Compared with the blank serum group,the Chinese medicine medium-dose group could elevate the expressions of chemokines PDGFB and CCL21 on the surface of HUVECs(P＜0.01);compared with the blank serum group,the Chinese medicine medium-dose group could raise the expression of chemokine receptors PDGFRB and CXCR4 on the surface of BMSCs(P＜0.01).Conclusion:The serum containing Ruanjian Qingmai granules may promote the proliferation of BMSCs cells through ERK pathway,and at the same time achieve homing by promoting the expression of chemokines in HUVECs and the expression of chemokine receptors in BMSCs cells.
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