RASSF1A基因对食管癌细胞EC9706生长抑制作用的研究

Suppression of RASSF1A gene on human esophageal carcinoma cells:experiments in vitro and in vivo

目的观察 RASSF1A 基因对食管癌细胞 EC9706生长的抑制。方法将包含RASSF1A 基因的质粒转染 RASSF1A 表达缺失的 EC9706细胞,建立稳定转染细胞系,通过 MTT 法检测细胞活性和增殖能力、流式细胞仪检测其对细胞周期的影响、裸鼠移植瘤实验检测转染细胞的体内成瘤特性。结果稳定表达 RASSF1A 的 EC9706细胞较对照组细胞的 RASSF1A 蛋白表达增加;细胞生长速度减慢(P<0.01);细胞周期中 G_1期比例增加,S 期比例减少(G_1期:RASSF1A 组细胞:68.53%±3.34%;空质粒组:54.25%±4.61%;未转染组:53.41%±5.60%。S 期:RASSF1A 组细胞:(23.19±5.04)%;空质粒组:(31.81±2.05)%;未转染组:32.09%±1.99%)(P<0.01);同时裸鼠致瘤能力也被抑制(P<0.05)。结论 RASSF1A 基因的外源性表达能显著抑制食管癌细胞在体内外的生长,提示该基因在食管癌的发生发展中发挥重要作用。

Objective To investigate the effect of Ras association domain family 1A （RASSF1A） gene, a new tumor suppressor gene （TSG）, on tumorigenesis of human esophageal carcinoma cells. Methods pcDNA3. 1 （ ＋ ）-RASSF1A, a plasmid containing RASSF1A gene, and the blank plasmid pcDNA3.1 （ ＋ ） were transfected into human esophageal carcinoma cells of the line EC9706. The expression of RASSF 1A protein was examined by Western blotting. The changes of cell cycle of stablytransfected cells were determined by flow cytometry （ FCM）, and the cellular proliferation was analyzed by MTT assay. Fifteen nude mice were randomly divided into 3 groups to be inoculated subcutaneously with EC9706 cells transfected with pcDNA3.1 （ ＋ ） -RASSF1 A, EC9706 cells transfected with pcDNA3.1 （ ＋ ）, and untransfected EC9706 cells respectively. Other 5 nude mice were used as controls. Four weeks later, the mice were killed to take out the carcinoma tissues. FCM was used to analyze the cell cycle. Results Western blotting showed that RASSF1A protein was expressed highly in the stably transfected cells. The cell viability and growing speed were decreased obviously in the cells expressing of RASSF1A （both P 〈 0.01 ） ; FCM showed that the proportion of cells at the G1 phase of the EC9706 cells expressing RASSF1A was significantly higher than those in the blank plasmid group and untransfected group （ both P 〈 0.01 ）. The size of the EC9706 cells obtained from the nude mice inoculated with the EC9706 cells transfected with pcDNA3.1 （ ＋ ）-RASSF1A was significantly smaller than those of the pcDNA3. 1 （ ＋ ） group and blank plasmid group （ both P 〈 0.05）. Conclusion Expression of exogenous RASSF1A inhibits the progression of human esophageal carcinoma cells in vitro and in vivo. As a tumor suppressor gene, it plays an important role in origination, progression and metastasis of esophageal carcinoma. [ Key words]