基于Cx32探讨六味地黄丸增效自杀基因抗肝癌的缝隙连接机制

Mechanism of Liuwei Dihuangwan in Enhancing Hepatocarcinoma Suicide Gene Therapy from Cx32

目的:探讨六味地黄丸对大鼠肝癌细胞株CBRH7919缝隙连接蛋白(connexin,Cx)32及间隙连接细胞间通讯(gap junction intercellular communication,GJIC)的影响,进一步研究其增强自杀基因旁杀伤效应的机制。方法:六味地黄丸(32g·kg·d^-1)与等体积的生理盐水灌胃大鼠,取血制备含药血清及空白血清,采用不同浓度的六味地黄丸含药血清及空白血清分别对CBRH7919细胞进行处理。实验分为4组,即空白组(体积分数为10%空白鼠血清),六味地黄丸含药血清高、中、低浓度组(10%,5%,2.5%含药鼠血清)。应用间接免疫荧光法、实时荧光定量聚合酶链式反应(Real-time PCR)检测肝癌细胞Cx32蛋白及mRNA表达,应用光漂白恢复技术检测各组肝癌CBRH7919细胞GJIC功能。结果:(1)间接免疫荧光检测结果显示,与空白组比较,六味地黄丸含药血清各浓度组对CBRH7919细胞Cx32的表达具有浓度依赖性上调作用(P<0.05,P<0.01),尤其在细胞膜上的表达增多。(2)Real-timePCR结果表明,六味地黄丸各剂量组均可提高Cx32mRNA表达(P<0.05,P<0.01),且具有一定的量效关系;(3)光漂白恢复技术检测结果表明,六味地黄丸各组的平均荧光恢复率高于空白组,并呈现出一定的浓度依赖趋势。结论:六味地黄丸增强自杀基因旁杀伤效应的机制与缝隙连接有关,可能是通过增加CBRH7919细胞Cx32在细胞膜上的定位,提高Cx32mRNA及蛋白水平的表达,从而增强GJIC功能而达到增效作用。

Objective: To investigate the effect of Liuwei Dihuangwan on connexin 32(Cx32) in hepatoma cell line CBRH7919 and its gap junction intercellular communication(GJIC),and furthermore study its mechanism of enhancing the bystander killing effect of suicide gene therapy. Method: Liuwei Dihuangwan(32 g·kg·d-1) and the same volume of normal saline were given to the rats by intragastrical administration. Blood was taken to prepare the medicated serum of Liuwei Dihuangwan and blank control serum,respectively. The hepatoma cell line CBRH7919 were treated by control serum and medicated serum of Liuwei Dihuangwan in different concentrations. There were four groups in experiment: the blank control group(volume fraction of 10%),medicated serum high dose group of Liuwei Dihuangwan(the volume fraction of 10%),medicated serum middle dose group of Liuwei Dihuangwan(the volume fraction of 5%),and medicated serum low dose group of Liuwei Dihuangwan(the volume fraction of 2. 5%). The expression levels of Cx32 protein and mRNA in hepatoma cell line CBRH7919 were detected by indirect immunofluorescence assay(Ⅱ A) and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) assay. The fluorescence redistribution after photobleaching(FRAP) method was used to detect the function of GJIC of hepatoma cell line CBRH7919. Result:(1) The indirect immunofluorescence assay(Ⅱ A) analysis indicated that as compared with the blank control group,the cx32 expression of CBRH7919 cells was up-regulated in a concentration-dependent manner in each dose group of the serum containing Liuwei Dihuangwan(P < 0. 05 in low dosage group;P < 0. 01 in middle dosage and high dosage group),especially on cell membrane.(2) The results of Real-time PCR assay indicated that as compared with the blank control group,the expression of Cx32 mRNA was increased in each dose group(P < 0. 05 in low dose group,P < 0. 01 in middle and high dose group) in a dose-effect relationship.(3) The result of fluorescence redistribution after photobleaching(FRAP) analysis indicated that the average fluorescence recovery rates of drug groups were significantly higher than that in blank control group,and also showed a certain concentration-dependent trend.Conclusion: The mechanism of medicated serum of Liuwei Dihuangwan in enhancing the bystander killing effect of suicide geneis related to gap junction. Liuwei Dihuangwan may enhance the function of GJIC by increasing the localization of cx32 on the cell membrane of CBRH7919 cells and increasing the expression of cx32 mRNA and protein to achieve the synergistic action.