六味地黄丸含药血清调控黑色素瘤B16细胞株缝隙连接蛋白表达作.docVIP

六味地黄丸含药血清调控黑色素瘤B16细胞株缝隙连接蛋白表达作 　 作者：杜标炎，张小贺，谭宇蕙，吴映雅，易华，郭玉荣 【摘要】 　　【目的】观察六味地黄丸含药血清调控小鼠黑色素瘤B16细胞株缝隙连接蛋白（Cx）表达的作用。【方法】采用SD大鼠灌胃8d制备六味地黄丸含药血清（药物剂量为32g·kg-1·d-1）和对照血清（灌胃等容积生理盐水）；选择对B16细胞生长和形态影响不大的血清浓度（体积分数为10%）作为大鼠血清的工作浓度；采用Western blot技术和间接免疫荧光法及流式荧光法检测六味地黄丸含药血清对黑色素瘤B16细胞株缝隙连接蛋白（Cx26/30/32）表达的影响。【结果】Western blot检测显示空白组（无鼠血清）3种Cx几乎不表达，而对照血清和六味地黄丸各浓度含药血清均能提高Cx蛋白的表达，但含药血清的作用更为明显且有显著的量效关系；间接免疫荧光法和流式荧光法检测显示类似的结果：对照血清组和中、高剂量含药血清组的Cx蛋白荧光强度均较空白组显著增加（P＜001），但与对照血清组比较，只有高剂量含药血清组荧光强度显著增加（P＜005），低剂量含药血清组Cx蛋白表达反而下降(P＜001)。【结论】六味地黄丸含药血清具有调控小鼠黑色素瘤B16细胞株缝隙连接蛋白表达的作用。 【关键词】 肿瘤/中西医结合疗法；六味地黄丸/药理学；黑色素瘤；基因治疗；肿瘤细胞，培养的 　　Abstract: Objective To investigate the regulatory effect of medicated serum of Liuwei Dihuang Bolus (LDB) on the expression of connexins (Cxs) in mice melanoma B16 cells.MethodsMedicated serum was obtained from SD rats which were treated with LDB at the dose of 32g·kg-1·d-1 and nonmedicated serum was obtained from rats receiving normal saline by gavage for 8 days. The rat serum at the concentration of 10% (V/V) served as the working solution. The expression of connexins (Cx26/30/32) was detected by using western blot method，indirect immunofluorescence assay and fluorescence activated cell sorter (FACS) assay.ResultsThe results of western blot assay showed that Cx26/30/32 was undetectable in the blank group(without use of rat serum)，while the amount of Cx26/30/32 in B16 cells were upregulated by rat control serum and the medicated serum，the upregulation of medicated serum being obvious and in a doseeffect manner.Indirect immunofluorescence assay and FACS assay showed similar results: the fluorescence intensity in the groups treated with rat control serum and medicated serums was higher than that in the blank group(P＜001)；the fluorescence intensity in the group of highdose medicated serum was higher than that in the control serum group(P＜005)，and Cx expression was decreased in the group of lowdose medicated serum (P＜001).Conclusion Medicated serum of LDB exerts regul