血瘀证食管鳞癌患者血清对EC9706细胞增殖和细胞周期的影响

Effect of Serum of Blood-stasis Esophageal Squamous Cell Carcinoma Patients on Proliferation and Cell Cycle of EC9706 Cells

【目的】观察血瘀证食管鳞癌患者血清对食管鳞癌EC9706细胞增殖和细胞周期的影响,探讨血瘀证食管鳞癌患者血液微环境对食管癌细胞的作用。【方法】将EC9706细胞以含胎牛血清(FCS)的RPMI-1640培养液置于37℃、体积分数5%饱和湿度的CO2培养箱中孵育24 h,再以不含血清的培养液孵育24 h后,分别加入血瘀证、脾气虚证食管鳞癌患者血清和健康人血清,四甲基偶氮唑盐(MTT)法检测细胞增殖情况,光镜下观察细胞形态变化,流式细胞术观察细胞周期变化。【结果】血瘀证、脾气虚证食管鳞癌患者血清刺激细胞增殖率为50%时的浓度(PI50)分别为71.1μL/m L和118μL/m L;2组人血清刺激细胞增殖的峰值均出现在时间点48 h。光镜观察结果显示,血瘀证组、脾气虚证组血清刺激细胞呈长梭形或多角形改变,血瘀证组细胞数目较脾虚证组增多。细胞周期结果显示,与脾气虚组比较,血瘀证组患者血清刺激的G1增殖期细胞百分率明显减少,S增殖期细胞百分率明显增高(均P<0.05)。【结论】血瘀证食管鳞癌患者血清微环境较脾气虚证食管鳞癌患者更有利于食管癌细胞的增殖,其作用机制可能与调节细胞周期有关。

Objective To investigate the effect of serum of esophageal squamous cell carcinoma （ESCC） patients with blood stasis syndrome （BSS） on proliferation and cycle of EC9706 cells, and to explore the action of blood micro-environment of ESCC patient with BSS on EC9706 cells. Methods Human EC9706 cells were cultured in an incubator with RPMI-1640 medium containing fetal bovine serum （ FBS） , at 37℃ and under 5% saturated humidity for 24 h. After EC9706 cells were starved in serum-free medium for another 24h, the three experimental groups were treated with serum of ESCC patients with BSS, serum of ESCC patients with spleen-qi deficiency syndrome （SQDS）, and serum from healthy volunteers, respectively. Cell proliferation was determined by methyl thiazolyl tetrazolium （ MTT） assay, EC9706 cell morphology was observed under light microscope, and cell cycle was measured by flow cytometer （FCM）. Results The serum concentrations of ESCC patients with BSS and ESCC patients with SQDS for obtaining 50 percent cell proliferation rates （ PI50） were 71.1 μL/mL and 118 μL/mL, respectively. And the proliferation of EC9706 cells in the both groups all arrived to the peak values at culturing hour 48. The light microscopy results showed that the feature of EC9706 cells in both groups presented as spindle-like or polygon-like shape, and cell count in BSS group was larger than SQDS group. FCM assay results for EC9706 cell cycle showed that the percentage of G1-phase EC9706 was decreased and the percentage of S-phase EC9706 was increased in BSS group as compared with those in SQDS group （ P〈0.05）. Conclusion Serum micro -environment in ESCC patients with BSS is more beneficial to EC9706 cells proliferation than ESCC patients with SQDS, and the mechanism may be related to the regulation of cell cycle.