摘要
目的探讨E1 A基因与喉癌Hep-2细胞生长和增殖的关系。方法将Ad-E1 A和对照空载体组Ad-β-gal在2 9 3细胞中扩增后提取、纯化并滴定,将其转染至喉癌Hep-2细胞,转染后将Hep-2细胞分为空白组(PBS组)、对照组(Ad-β-gal)和实验组(Ad-E1A组)。经RT-PCR鉴定,采用胎盘兰染色并计算细胞数及MTT法绘制细胞生长曲线并计算倍增时间;采用流式细胞术计算分析细胞周期。结果RT-PCR结果显示E1 A已整合到Ad-E1 A转染的细胞基因组中并且稳定表达。实验组(E1 A组)细胞生长减慢,倍增时间分别是空白组(PBS组)、对照组(Ad-β-gal组)的1.7 2倍和1.7 0倍。流式细胞术结果显示转染E1 A的细胞出现S期减少和G2/M期被阻滞。结论①E1 A基因可以抑制喉癌Hep-2细胞在体外的生长,延长其倍增时间。②E1 A基因可以改变喉癌Hep-2细胞的细胞周期,使S期细胞减少,G2/M期被阻滞。
Objective To investigate the effect of E 1 A gene on the growth and multiplication of human laryngeal carcinoma cell line （Hep-2 cells ）. Methods The Ad- E 1 A gene and blank vector Ad- β- gal were amplified in Hek293 cells, extracted by repeated freezing （ -80℃ ） and thawing （37 ℃ ） （3 times ） , purified by the method of density gradient of CsCl, and titrated by the plaque assay. The purified product was transfected into Hep-2 cells and then authenticated by the RT-PCR. Hep-2 cells were divided into blank group, control group, and experimental group. Tile in vitro growth rates of Hep-2 cells transfected and not transfected with E1 A were studied by cellular count and MTT, and the cell cycle was analyzed by the flow eytometry. Results ① The results of RT- PCR showed that E 1 A gene was successfully integrated into the genome of Ad- E 1 A- transfeeted Hep-2 cells with stable expression.② The in vitro growth of E 1 A-transfected Hep-2 cells （ experimental group ） was inhibited , and the cell- doubling time increased 1 . 7 2 and 1 . 7 0 times of that in the blank group and control group respectively. ③ Flow cytometry showed reduction of S- phase and blockage of G2 / M phase in the cell cycle of E 1 A-transfected Hep-2 cells. Conclusion E 1 A gene can inhibit the in vitro growth and prolong the doubling time of Hep-2 cells. ② E1A gene can change the cell cycle of Hep-2 cells.
出处
《中国耳鼻咽喉颅底外科杂志》
CAS
2009年第2期103-105,110,共4页
Chinese Journal of Otorhinolaryngology-skull Base Surgery
