Study On The Effect And Mechanism Of Chinese Herbal Medicine Mixture On Anti HCMV Infection Positive Glioma Stem Cells

Objective1.To observe the effect of proliferation and apoptosis of HCMV infected U251 GSCS by this Chinese herbal medicine mixture in vitro.2.To explore the effect of angiogenesis and growth of HCMV infected U251 GSCS by Chinese herbal medicine mixture with ganciclovir on the in vivo.Methods1.According to the anti-tumor properties of Curcuma,rubescens,Hedyotis diffusa,then made anti-glioma formula.Chinese medicine extracts were dissolved in culture medium and preared different concentrations of mixture.2.HCMV strain were multiplied in Human embryonic lung fibroblasts,and titer was determined by Plaque quantification.3.GSCS were isolated from glioma cell line U251 by monoclonal culture,the ratio of CD133+ U251 GSCS was detected by flow cytometry.4.U251 GSCS inoculated with HCMV(MOI=1),experimental groups were added with different concentrations of mixture,control group was added with equal volume of culture medium.After 48 h,use MTT to detect the activity of the cells in each group.5.Use RT-PCR to detect the expressions of HCMV IE-1,tumor suppression gene Bcl-2and oncogene Bax m RNA and use Western blot to detect the expressions of Bcl-2 and Bax proteins in the cells of each group.6.HCMV infected U251 GSCS were transplanted into nude mice brain after 14 days,mice were stochastic divided into control group,mixture group,ganciclovir group and mixture with ganciclovir group.After 14 days,killed mice and the tumor tissues were taken for pathological examination.7.Use immunohistochemical to detect the expression of IE and CD34 proteins in tumors and calculated microvessel density(MVD).The expression of Bcl-2 and Bax m RNA in tumors were detected by RT-PCR.Reslts1.U251 GSCS were suspended in serum-free medium,flow cytometry showed the number of CD133+ GSCS in suspension cells was more than 95%.2.MTT results showed that the mixture could inhibit cell proliferation,and the activity of HCMV infected U251 GSCS decreased according to the increase of concentration and time(P<0.05).3.Soft agar colony formation assay showed that mixture could inhibit the colony formation of HCMV infected U251 GSCS,the effect was more remarkable along with the increase of drug concentration(P<0.01).4.RT-PCR showed HCMV infected U251 GSCs with different concentrations of mixture co-culture 48 h,Bax m RNA expression was up-regulated and IE-1,Bcl-2 m RNA expression was down-regulated in cells(P<0.05).5.Western blot showed HCMV infected U251 GSCS in different concentrations of mixture for 48 h,the expression of Bax protein was up-regulated and Bcl-2 protein expression was down-regulated in each group(P<0.05).6.Chinese herbal medicine mixture and ganciclovir could reduce the angiogenesis of HCMV infected U251 GSCS transplanted tumor(P<0.05).The efficacy of combined drugs in inhibiting angiogenesis of transplanted tumor was better than that in single group,and there was no interaction between the two groups(P>0.05).7.RT-PCR showed that Chinese herbal medicine mixture and ganciclovir can inhibit the expression of Bcl-2 m RNA(P<0.05)and promote the expression of Bax m RNA in transplanted tumor cells(P < 0.05).The relative expression of Bcl-2 and m RNA in the combination group was significantly lower than that in single group(P<0.05).The relative expression of Bax and m RNA in the combination group was significantly higher than that in single group(P<0.05).Conclusions1.This Chinese herbal medicine mixture can inhibit the proliferation and induce apoptosis of HCMV infected U251 GSCS.2.This Chinese herbal medicine mixture can inhibit the growth of HCMV infected U251 GSCS orthotopic transplantation tumor by inhibiting the angiogenesis and inducing the apoptosis of transplanted tumor.