白花蛇舌草注射液对多发性骨髓瘤细胞株RPMI 8226增殖的影响

Effects of Hedyotis diffusa Willd Injection on the Proliferation of RPMI 8226 Cells

目的探讨白花蛇舌草注射液(HDI)抑制多发性骨髓瘤细胞株RPMI 8226增殖作用及机制。方法采用四甲基偶氮唑(MTT)比色法检测HDI对RPMI 8226抑制作用并筛选研究浓度;通过流式细胞技术使用Annexin V-PI检测细胞凋亡、PI单染检测细胞周期分布、FITC-CD44、PE-CD49d双标检测黏附分子表达;ELISA检测细胞上清IL-6、VEGF含量;RT-PCR检测Bax、Bcl-2、Caspase-3、Survivin、IL-6、VEGF mRNA的表达。结果 HDI可抑制RPMI8226增殖,同时诱导该细胞株早期凋亡,将细胞周期阻滞于G1期,且呈浓度依赖。经0、20、40、60μL/mL HDI作用后RPMI 8226上清VEGF含量降低且呈浓度依赖(P<0.01),IL-6含量增高(P<0.01),细胞表面CD44、CD49d表达水平呈浓度依赖上调。经40μL/mL HDI作用后,该细胞株Survivin mRNA表达显著下调(P<0.01),Bcl-2、IL-6、VEGF mRNA显著上调(P<0.01),Bax、Caspase-3 mRNA则上调不明显(P>0.05)。结论 HDI可抑制RPMI 8226增殖,其机制可能和诱导细胞早期凋亡,细胞周期G1期阻滞,降低VEGF分泌,下调Survivin转录水平等有关。

Objective To explore the inhibition of Hedyotis diffusa Willd Injection （HDI） on the proliferation of RPMI 8226 cells and its mechanisms. Methods The inhibition of HDI on the proliferation of RPMI 8226 cells was detected by MTT and the drug concentrations for further researches were screened out. The apoptosis rate was detected using Annexin V-PI of flow cytometry. The cell cycle distribution was detected by PI. The ex- pressions of adhesion molecule FITC-CD44 and PE-CD49d were detected. The IL-6 and VEGF concentrations of cell supernatants were tested by ELISA. The mRNA expressions of Bax, Bcl-2, Caspase-3, Survivin, IL-6, and VEGF were detected by RT-PCR. Results HDI could inhibit the proliferation of RPMI 8226 cells. Meanwhile, it induced their early apoptosis, arresting them at G1 phase in a concentration-dependent manner. The VEGF concentrations were down-regulated after acted by 0, 20, 40, and 60 uL/mL HDI in a dose-dependent manner （ P 〈 0.01 ）. The IL-6 content increased （P 〈0.01 ）. The expressions of CD44 and CD49d were up-regulated in a concentration-dependent manner. After acted by 40 uL/mL HDI, the Survivin mRNA level was significantly down- regulated （ P 〈0.01 ）, the mRNA levels of Bcl-2, IL-6, and VEGF were significantly up-regulated （ P 〈0. 01 ）, but the up-regulation of Bax and Caspase- 3 mRNA levels were not so obvious （ P 〉 0.05）. Conclusions HDI could inhibit the proliferation of RPMI 8226 cells. Its mechanisms might be correlated with early apoptosis induction, G1 phase arresting, VEGF secretion lowering, and Survivin mRNA transcription level down-regulating.