Du Biao-yan, Guo Yu-Rong, Yi Hua, Tan Yu-Hui, Wu Ying-ya
Department of Pathology, Guangzhou University of Traditional Chinese Medicine, Guangzhou 510006, China.
Zhongguo Zhong Xi Yi Jie He Za Zhi. 2013 May;33(5):651-8.
To study the gap junction mechanisms for synergistic effects of liuwei di-huang pill (LDP) containing serum on HSV-tk/GCV suicide gene therapy of mouse malignant melanoma B16 cells.
The LDP containing serum (2.5% LDP serum group, 5.0% LDP serum group, and 10.0% LDP serum group) and the control serum group were set up. The effects of LDP on mRNA expressions of Cx26 and Cx43 in mouse malignant melanoma B16 cells were detected by RT-PCR assay. The effects of LDP on protein expressions of Cx26 and Cx43 in B16 cells were detected using Western blot and indirect immunofluorescence assay. The interference efficiencies of Cx26-309, Cx26-337, Cx26-367, Cx26-2098 SiRNA on Cx26 gene in B16 cells were detected using RT-RCR technique. Cx26-2098 SiRNA, due to the optimal interference efficiency, was chosen to interfere Cx26 gene, and the bystander effect of LDP + HSV-tk/GCV was observed. The effects of the gap junctional intercellular communication (GJIC) inhibitor glycyrrhetinic acid on the killing of LDP + HSV-tk/GCV system to B16 cells were detected by MTT assay. In this experiment, the control serum group, 2.5% LDP serum group, 5. 0% LDP serum group, 10.0% LDP serum group, the control serum combined GCV group, 2.5% LDP serum combined GCV group, 5.0% LDP serum combined GCV group, 10.0% LDP serum combined GCV group, 2.5% LDP serum combined GCV + glycyrrhetinic acid group, 5.0% LDP serum combined GCV + glycyrrhetinic acid group, 10.0% LDP serum combined GCV + glycyrrhetinic acid group were set up. The final concentration of GCV was 20 micromol/L. The final concentration of glycyrrhetinic acid was 50 micromol/L.
LDP containing serum could increase the protein and mRNA expressions of Cx43 in B16 cell in a dose-dependent manner. It had bi-directional regulation on the Cx26 protein and mRNA expressions. The low dose LDP had inhibition while high dose LDP could up-regulate its expression. After SiRNA interfered Cx26 gene, there was no obvious change in the bystander effect of LDP combined suicide gene therapy between before and after interference. There was significant reduction in the inhibition rate between before (48.75%, 59.39%, and 69.28%) and after blockage (29.14%, 38.71%, and 58.13%) of glycyrrhetinic acid in the 2. 5%, 5.0%, 10.0% LDP serum combined GCV groups (P <0.01).
The synergistic effects of LDP containing serum on HSV-tk/GCV suicide gene therapy in mouse malignant melanoma B16 cells were correlated with the gap junction mechanisms, which might be achieved through increasing the expressions of Cx26 and Cx43.
研究六味地黄丸含药血清对小鼠恶性黑色素瘤B16细胞单纯疱疹病毒胸苷激酶/丙氧鸟苷（HSV-tk/GCV）自杀基因疗法协同作用的缝隙连接机制。
设立六味地黄丸含药血清组（2.5%含药血清组、5.0%含药血清组、10.0%含药血清组）和对照血清组。采用逆转录聚合酶链反应（RT-PCR）法检测六味地黄丸对小鼠恶性黑色素瘤B16细胞中Cx26和Cx43 mRNA表达的影响。采用蛋白质免疫印迹法和间接免疫荧光法检测六味地黄丸对B16细胞中Cx26和Cx43蛋白表达的影响。采用RT-RCR技术检测Cx26-309、Cx26-337、Cx26-367、Cx26-2098小干扰RNA（SiRNA）对B16细胞中Cx26基因的干扰效率。因Cx26-2098 SiRNA干扰效率最佳，故选用其干扰Cx26基因，并观察六味地黄丸+HSV-tk/GCV的旁观者效应。采用噻唑蓝（MTT）比色法检测缝隙连接细胞间通讯（GJIC）抑制剂甘草次酸对六味地黄丸+HSV-tk/GCV体系杀伤B16细胞的影响。本实验设立对照血清组、2.5%含药血清组、5.0%含药血清组、10.0%含药血清组、对照血清联合丙氧鸟苷组、2.5%含药血清联合丙氧鸟苷组、5.0%含药血清联合丙氧鸟苷组、10.0%含药血清联合丙氧鸟苷组、2.5%含药血清联合丙氧鸟苷+甘草次酸组、5.0%含药血清联合丙氧鸟苷+甘草次酸组、10.0%含药血清联合丙氧鸟苷+甘草次酸组。丙氧鸟苷终浓度为20 μmol/L。甘草次酸终浓度为50 μmol/L。
六味地黄丸含药血清可呈剂量依赖性增加B16细胞中Cx43蛋白和mRNA表达。其对Cx26蛋白和mRNA表达具有双向调节作用。低剂量六味地黄丸具有抑制作用，高剂量六味地黄丸可上调其表达。SiRNA干扰Cx26基因后，六味地黄丸联合自杀基因疗法的旁观者效应在干扰前后无明显变化。2.5%、5.0%、10.0%含药血清联合丙氧鸟苷组中，甘草次酸阻断前抑制率分别为48.75%、59.39%、69.28%，阻断后分别为29.14%、38.71%、58. = 13%，差异有统计学意义（P <0.01）。
六味地黄丸含药血清对小鼠恶性黑色素瘤B16细胞HSV-tk/GCV自杀基因疗法的协同作用与缝隙连接机制相关，可能是通过增加Cx26和Cx43表达实现的。
