基因治疗联合六味地黄丸对肝癌荷瘤小鼠缝隙连接蛋白表达的影响

目的:探讨六味地黄丸联合基因治疗对小鼠移植性肝癌缝隙连接蛋白(connexin,Cx)表达的影响,初步阐明六味地黄丸对肝癌HSV-tk/GCV自杀基因治疗的增效作用机制是否与缝隙连接相关。方法:昆明种小鼠90只,随机分为模型对照组、六味地黄丸治疗组(ig六味地黄丸10 g·kg-1·d-1)、自杀基因治疗组(ip丙氧鸟苷100 mg·kg-1·d-1)、联合治疗组(ig六味地黄丸10 g·kg-1.d-1+ip丙氧鸟苷100 mg·kg-1.d-1);选用H22与H22/tk细胞株皮下接种建立小鼠肝癌模型,取荷瘤小鼠肿瘤组织进行常规病理制片,采用SABC免疫组化法检测各组肿瘤组织Cx26,Cx32,Cx43蛋白表达。结果:阳性表达定位于细胞浆和细胞膜,Cx43在癌旁组织,特别是有纤维组织浸润部位表达呈强阳性,而在癌结节中肿瘤细胞表达相对较弱;Cx26,Cx32在肿瘤细胞特别是脂肪组织浸润附近的癌组织中呈现强阳性表达。与肿瘤模型组相比,联合治疗组则能增强Cx26,Cx43,Cx32蛋白表达,差异有统计学意义(P<0.05);与单独六味地黄丸和自杀基因治疗组相比,联合治疗组能明显提高Cx32蛋白表达,差异有统计学意义(P<0.05,P<0.01)。结论:六味地黄丸联合自杀基因治疗能增强Cx26,Cx43,Cx32蛋白表达,尤其是促进Cx32的表达,提示六味地黄丸增强自杀基因旁观者效应的机制与缝隙连接相关。

Joint Therapeutic Effect of Hepatocarcinoma Suicide Gene TherapyCombined with Liuwei Dihuangwan on Expression of Connexin in vitro

Objective: To investigate the joint therapeutic effect of Liuwei Dihuangwan (LD) combined with hepatocarcinoma HSV-tk/GCV suicide gene on the exp ression of connexin (Cx),clarity the therapeutic mechanism of LD. Method: Ninety Kunming mice were randomized into 4 groups:model group,LD group (10 g ·kg^-1 ·d^-1), suicide gene therapy group (intraperitoneal injection of guanosine ganciclovior) and the combined group.The transplanted tumor mice model of H22 hepatocarcinoma was established by subcutaneous inoculation of H22 and H22/tk cells.We selected the tumor tissue of hepatocarcinoma tumor-bearing mice for conventional preparation,carried out biopsy of DAB immunohistochemical staining,and used the SABC immunohistochemical assay to detect the expression of Cx26,Cx43,Cx32. Result: The positive expression was found in cytoplasm and cell membrane under the microscope. Cx43 expression appeared in tumor adjacent tissues,especially where infiltrated by fibrous tissue had strongly positive expression,but cancer cells in the tumor nodules had relatively weak expression. We found the expression of Cx26 and Cx32 were in tumor cell,especially near the tumor tissues where infiltrated by adipose tissue showed strong positive expression. The expression of connexin in LDB treatment group and suicide gene therapy combined with LD treatment group was obviously stronger than that in tumor model group. The differences of expression of Cx between suicide gene therapy combined with LDB treatment group and showed statistical significance (P<0.05). Conclusion: Suicide gene therapy combined with LD in treatment group can significantly improve the expression of Cx26,Cx43,Cx32,especially Cx32.It indicates that the enhancing effect of LD on suicide gene bystander is related to gap junction.