六味地黄丸含药血清对自杀基因治疗黑色素瘤增效作用的缝隙连接机制初探

The Preliminary Research on the Gap Junction Mechanisms for Synergistic Effects of Liuwei Dihuang Pill Containing Serum on Suicide Gene Therapy of Melanoma

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DOI：10.7661/CJIM.2013.05.0651

中文关键词: 恶性黑色素瘤基因治疗自杀基因六味地黄丸血清药理学缝隙连接

英文关键词:malignant melanoma gene therapy suicide gene Liuwei Dihuang Pill serum pharmacology gap junction

基金项目:国家自然科学基金资助项目（No. 30572433；No81072906）

作者	单位	E-mail
杜标炎	广州中医药大学基础医学院病理教研室（广州510006）	dubybill@sohu.com
郭玉荣,易华,谭宇蕙

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中文摘要:

目的探讨六味地黄丸含药血清对HSV-tk/GCV自杀基因系统杀伤小鼠黑色素细胞瘤B16细胞增效作用的缝隙连接机制。方法制备六味地黄丸含药血清（分别为2.5%、5.0%、10.0%六味血清）及10.0%对照血清。采用RT-PCR法检测六味地黄丸含药血清对B16细胞株的缝隙链接蛋白（Cx）26和Cx43 mRNA的影响，Western blot法和间接免疫荧光法检测其对B16细胞Cx26和Cx43蛋白表达的影响；RT-PCR检测Cx26－309、Cx26－337、Cx26－367、Cx26－2098 SiRNA对Cx26的干扰效率，选用干扰效率最高的Cx26－2098 SiRNA干扰Cx26基因后观察六味地黄丸联合HSV-tk/GCV的旁观者效应；MTT法检测细胞间缝隙连接通讯功能（gap junctional intercellular communication， GJIC）抑制剂甘草次酸（GA）对六味地黄丸联合tk/GCV系统对B16细胞杀伤作用的影响，实验设为对照血清组，2.5%、5.0%、10.0%六味血清组（简称低、中、高剂量组）及对照血清联合GCV组，低、中、高剂量联合GCV组，低、中、高剂量联合GCV加GA组。GCV终浓度为20 mol/L，GA终浓度为50 mol/L。结果六味地黄丸含药血清能提高B16细胞中Cx43 mRNA及蛋白的表达，且具有量效依赖关系；对Cx26 mRNA与蛋白的表达具有双向调节作用，低剂量时具有抑制作用而高剂量是能上调其表达；SiRNA干扰Cx26基因表达后，六味地黄丸联合自杀基因治疗的旁观者效应与没有干扰前比较无明显的变化；低、中、高剂量联合GCV组在GA阻断前细胞抑制率（48.75%、59.39%、69.28%）与阻断后细胞抑制率（29.14%、38.71%、58.13%）比较，显著降低，差异有统计学意义（P<0.01）。结论六味地黄丸含药血清对HSV-tk/GCV自杀基因系统杀伤恶性黑色素瘤B16细胞的增效作用与缝隙连接机制有关，可能是通过提高Cx26和Cx43等缝隙连接蛋白表达而达到协同增效作用。

英文摘要:

Objective To study the gap junction mechanisms for synergistic effects of Liuwei Dihuang Pill （LDP） containing serum on HSV-tk/GCV suicide gene therapy of mouse malignant melanoma B16 cells. Methods 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 20μmol/L. The final concentration of glycyrrhetinic acid was 50μmol/L. Results 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）. Conclusion 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.

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