榄香烯乳联合更昔洛韦对C6脑肿瘤干细胞侵袭能力及TIMP-1 mRNA表达的影响

Effect of Elemene Emulsion Injection Combined with Ganciclovir on Invasive Ability and TIMP-1 mRNA Expression in C6 Brain Tumor Stem Cells

目的:研究榄香烯乳联合更昔洛韦对C6脑肿瘤干细胞(BTSCs)侵袭能力及组织金属蛋白酶抑制物1(TIMP-1)mRNA表达的影响。方法:从大鼠C6恶性胶质瘤细胞株中分离培养传代3次的细胞,采用免疫细胞化学法检测其中干细胞标志物CD133、Nestin蛋白表达情况。将20只大鼠随机分为空白对照组(生理盐水)、更昔洛韦组(ig,216 mg/kg)、榄香烯乳组(尾iv,36 mg/kg)和联用组(ig更昔洛韦216 mg/kg+尾iv榄香烯乳36 mg/kg),每组5只,每天给药1次,连续给药10 d。于末次给药后2 h采集各组大鼠血液并分离血清,分别与C6 BTSCs在体外培养基中共同培养24 h。采用Boyden侵袭试验检测C6 BTSCs的细胞侵袭能力,实时荧光定量聚合酶链式反应(RT-PCR)法测定C6 BTSCs的TIMP-1 mRNA表达情况。结果:传代3次后的细胞中CD133、Nestin蛋白有明显表达,表明其为BTSCs。与空白对照组比较,更昔洛韦组、榄香烯乳组和联用组C6 BTSCs的细胞侵袭率均明显下降、TIMP-1 mRNA表达均明显增强,差异有统计学意义(P<0.05);与更昔洛韦组和榄香烯乳组比较,联用组C6 BTSCs的细胞侵袭率明显下降、TIMP-1 mRNA表达明显增强,差异有统计学意义(P<0.05)。结论:榄香烯乳联合更昔洛韦可明显抑制C6 BTSCs的细胞侵袭,其作用机制可能与促进TIMP-1的生成有关,且联用效果强于单用。

OBJECTIVE：To study the effect of Elemene emulsion combined with ganciclovir on invasive ability and TIMP-1 mRNA expression in C6 brain tumor stem cells. METHODS：Cells with 3 generations were isolated and cultured from rats＇ C6 malignant glioma cell lines,and immunocytochemistry was adopted to detect the protein expressions of stem cell markers CD133,Nestin. 20 rats were randomly divided into blank control group（normal saline）,ganciclovir group（intragastrically administrated,216 mg/kg）,Elemene emulsion injection group（intravenously injected in tail,36 mg/kg）and combination group（intragastrically administrated 216 mg/kg of ganciclovir ＋ intravenously injected 36 mg/kg of Elemene emulsion injection in tail）,5 in each group,once every day,for 10 d. After 2 h of last administration,the blood of rats in each group was collected,serum was isolated and cocultured 24 h with C6 BTSCs in in vitro culture medium. Boyden invasion test was used to detect the invasive ability of C6 BTSCs,and real-time quantitative polymerase chain reaction（RT-PCR）method was used to determine the TIMP-1 mRNA expression of C6 BTSCs. RESULTS：Cells with 3 generations had obvious protein expressions in CD133 and Nestin,indicating they were BTSCs. Compared with blank control group,the cell invasion rates of C6 BTSCs in ganciclovir group,Elemene emulsion injection group and combination group were obviously decreased,TIMP-1 mRNA expression was obviously enhanced,with statistical significances（P0.05）. Compared with ganciclovir group and Elemene emulsion injection group,the cell invasion rate of C6 BTSCs in combination group was obviously decreased,TIMP-1 mRNA expression was obviously enhanced,with statistical significances（P0.05）. CONCLUSIONS：Elemene emulsion injection combined with ganciclovir can obviously inhibit the cell invasion of C6 BTSCs,the mechanism may be associated with promoting the TIMP-1 generation,and combination use shows better effect than single use.