摘要
目的探讨薯蓣皂苷(Dioscin,Dio)对人肾癌786-0细胞缝隙连接(Gap junction,GJ)功能的影响及其作用机制。方法 MTT法测Dio对786-0细胞生长的影响,荧光显微镜观察及流式细胞术结合荧光示踪法分析GJ功能变化,RT-PCR和Western blot分析连接蛋白基因表达。结果 0～2.5μmol.L-1 Dio处理786-0细胞48h不影响其存活率;用0、0.1、0.5、1和2μmol.L-1 Dio处理细胞48 h后,荧光显微镜下观察,Dio能明显提高786-0细胞Calcein传递,流式细胞术分析对照组和实验组的绿色荧光细胞(G4)与双阴性受体细胞(G3)比值(G4/G3)分别是0.13±0.01、0.23±0.01、0.30±0.01、0.56±0.02和1.15±0.02,各实验组G4/G3值明显高于对照组(P<0.01);RT-PCR和Western blot分析结果显示,用0、0.1、0.5、1和2μmol.L-1 Dio处理细胞48 h对其Cx43、Cx32和Cx26表达无明显影响。结论体外较低浓度Dio能够有效促进786-0细胞GJ功能,且具有明显的剂量效应关系。但Dio促进GJ机制并不是通过上调Cx43、Cx32和Cx26蛋白表达途径。
Aim To investigate the effects of Dioscin on the GJ function in a human renal carcinoma cell line 786-0.Methods Effects of Dio on the proliferation of 786-0 cells were determined by MTT.The gap junction function was analyzed by Fluorescent tracer with flow cytometry and fluorescence microscopy.The expression of connexins was detected by RT-PCR and Western blot.Results No effect was found on the survival of 786-0 cells treated with 0-2.5 μmol·L-1 Dio after 48 h.786-0 cells treated with 0,0.1,0.5,1 and 2 μmol·L-1 Dio was after 48h,the normalized enhancement of the Calcein spread treated with Dio was observed under a fluorescence microscope.Flow cytometry analysis showed the ratio of Calcein labeled cell group（G4） to double negative group（G3） was 0.13±0.01,0.23±0.01,0.30±0.01,0.56±0.02 and 1.15±0.02 respectively,and the G4/G3 of experimental groups was significantly higher than that of the control（P0.01）.The RT-PCR and Western bolt results showed no distinguishing effect on the expression of Cx43,Cx32 and Cx26 genes within 786-0 cell treated with 0,0.1,0.5,1 and 2 μmol·L-1 Dio after 48 h.Conclusions Low concentration Dio can competently promote the gap junction function of 786-0 cell in a dose-dependent way in vitro,however,its biological mechanism is not targeting the up-regulation of the Cx43,Cx32 and Cx26 expression.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2012年第6期778-782,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81072906
30973811)
广州中医药大学创新基金(No KAB108112K65)
