| PART ONEObjective:To observe the effects of crocin on human bladder cancer BIU-87 cell line and to explore the anti-cancer mechanism.Methods: BIU-87 cells of human bladder carcinoma were cultured by cell culture technique.The MTT assay was used to evaluate the inhibitory effect of crocin for the growth of BIU-87 cells.Flow cytometry was used to measure the cell cycle.RT-PCR and Western-blot were used to detect cyclin D1 and P27kip1 expression.Results:The growth of BIU-87 cells was inhibited remarkably in the dosage dependence and time dependence way .Flow cytometry profiles revealed that cells were blocked at G0/G1 phase,the cell proliferation was inhibited obviously.After be treated with 3.2mmol/L crocin,the expression of CyclinD1mRNA was down-regulated strikingly,the expression of P27kip1 mRNA was not with significant difference detected by RT-PCR technique. Cyclin D1 protein expression was down-regulated and P27kip1 protin was increased detected by Western blot.Conclution: Crcin can inhibite the proliferation of BIU-87 cells effactivelly,and block cells at G0/G1 phase .The mechanisms may be changing tumor cell cycles by down-regulating the expression of CyclinD1 gene and protin,up-regulating the expression of P27kip1protin.PART TWOObjective: To investigate the effects of crocin on proliferation regulation after silencing PLCεgene expression by RNAi interference in human bladder cancer cells BIU-87. To explore the treatment on bladder cancer by traditional chinese medicine combined with gene treatment,and provide theoretical foundation.Methods: BIU-87 cells were transfected with plasmid pGenesil-PLCε.The experiment was designed as five groups to compared with each other , they were non-transfected cells as control group, pGenesil-NP negative plasmid transfected group, pGenesil-PLCεplasmid transfected group, crocin group, pGenesil-PLCεplasmid transfected together with crocin group. The influence on proliferation of every group was determined by methyl thiazolyl tetrazolium (MTT).The distribution of cell cycle were analyzed by flow cytometry (FCM).The expression of PLCεmRNA of BIU-87 cells after transfection with the plasmid pGenesil-PLCεwas observed by RT-PCR. RT-PCR and Western-blot were used to detect cyclin D1,CyclinE,P27 and PCNA expression.Results: 78.06% mRMA of PLCεwas reduced clearly after transfected with plasmid pGenesil-PLCεin BIU-87 cells. pGenesil-PLCεplasmid transfected together with crocin group could enhance the inhibitive effect of crocin on proliferation up to 45.30% at 48h, which was strikingly compared with the pGenesil-PLCεplasmid transfected group and the crocin group (P<0.01). Flow cytometry profiles revealed that cells were blocked at G0/G1 phase,the cell proliferation was inhibited obviously. Both RT-PCR and western-blot indicated that the expression of PCNA,CyclinE both in mRNA and protein were markedly decreased in the RNAi together with crocin group compared with the control group (P<0.05),and the decrease was distinguished compared with the pGenesil-PLCεplasmid transfected group and the crocin group; The expression of P27 both in mRNA and protein were markedly raised in the RNAi together with crocin group compared with the control group (P<0.05), and the raise was striking compared with the pGenesil-PLCεplasmid transfected group and the crocin group; The expression of Cyclin D1 in protein was markedly decreased in the RNAi together with crocin group compared with the control group (P<0.05), and the decrease was distinguished compared with the pGenesil-PLCεplasmid transfected group and the crocin group ,while the expression of Cyclin D1 mRNA in the RNAi together with crocin group was of significance compared with the pGenesil-PLCεplasmid transfected group (P<0.05) and without significance compared with the crocin group(P>0.05).Conclution:RNAi PLCεtogether with crocin can enhance the inhibit effect of crocin, and blocked more cells at G0/G1 phase ,Further reduce PCNA,CyclinD1 and CyclinE expression and improve the P27kip expression were the mechanism.
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