摘要
目的研究Fas基因转染对舌鳞癌Tca8113细胞的体外抑制作用,构建Fas基因真核表达质粒pBK-Fas并将其转导入舌鳞癌Tca8113细胞,为舌鳞癌的Fas基因治疗提供实验基础。方法采用RT-PCR反转录成cDNA扩增Fas基因全长,亚克隆到真核表达载体pBK-CMV内,构建出重组真核表达质粒pBK-Fas,酶切鉴定并测序。脂质体法将pBK-Fas转染入Tca8113细胞,RT-PCR检测FasmRNA表达及半定量分析,琼脂糖凝胶电泳检测Fas转染细胞凋亡,流式细胞仪检测Fas蛋白表达。通过细胞生长曲线描记和软琼脂集落形成实验研究Fas基因的体外抑瘤效应。结果PCR扩增后的产物在约1008bp处出现明显的特异性条带,重组质粒pBK-Fas经酶切和测序鉴定,表明均含有大小正确的Fas基因片段。经检测Fas基因转染能提高FasmRNA表达水平、Fas蛋白表达强度,以及细胞凋亡指数。Fas基因转染能提高Fas蛋白的表达强度,由转染前的34.01±4.76上升到转染后的55.72±7.33,差异有统计学意义(P<0.01)。Fas转染细胞株在Fas抗体作用下能够有效启动Fas介导的细胞凋亡途径。MTT法检测细胞增殖抑制率的结果显示PBK-Fas转染组Tca8113细胞的增殖抑制作用最明显,表明Fas基因转染能增加舌鳞癌Tca8113细胞的增殖抑制率,结果与细胞生长曲线测定(计数法)结果具有一致性。软琼脂集落形成实验结果显示,与未转染细胞相比,重组质粒转染的Tca8113细胞群体倍增时间延长,克隆形成能力降低。结论Fas基因片段被成功插入真核表达载体pBK-Fas质粒的多克隆位点。Fas基因转染能有效的抑制体外培养的舌鳞癌Tca8113细胞的生长。
Objective To evaluate the inhibitory effects of Fas gene transduction on squamous cell carcinoma of tongue Tca8113 in vitro. To provide experimental basis for further study on Fas gene transfection on squamous cell carcinoma of tongue Tca8113 cells. Methods Total RNA was isolated from human Lymphoid tissue. Full-length Fas fragment was amplified by reverse transcription-polymerase chain reaction（RT-PCR）. The plasmids were prepared by double digestion;then the DNA fragment was extracted, purified and ligated in the plasmid pBK-CMV to form pBK-Fas. Plasmid including Fas gene was transfected into Tea8113 cells by lipofectamine kit, We to evaluated the inhibitory effects of expression of Fas mRNA in Fas-transfected, Fas protein expression in tumor cells was detected by flow cytometry. We evaluated the inhibitory effects of Fas gene transduction on squamous cell carcinoma of tongue Tca8113 cells by cell growth curve and plating efficiency test. Results Product of RT-PCR showed a clear specific band at 1 008 bp. It showed that there was the correct size of the Fas gene fragments in eukaryotic expression vector pBK-Fas by digestion and PCR. The transfection in Fas gene could increase the inhibitory effects of expression of Fas mRNA in Fas-transfected and the apoptosis in cells. The expression and intensity of Fas protein was increased in no-transfected and Fas-transfected Tea8113 cells from 34. 01 ±4.76 to 55.72 ±7.33（P 〈0.01 ）. Fas-transfeeted ceils could stimulate apoptosis pathway by Fas antibody. Inhibitory effect of Tca8113 cells by MTT showed that the transfection in Fas gene could increase the inhibitory effects of cells growth. Cell growth curve and plating efficiency test revealed that Fas-transfeeted Tca8113 cells had longer population and lower plating efficiency than the control cells. Conclusion DNA （Fas）is successfully constructed and transformed into pBK-Fas, the insgenie expression of Fas gene can effectively inhibit the increase of squamous cell carcinoma of tongue Tca8113 cells.
出处
《安徽医科大学学报》
CAS
北大核心
2009年第4期437-442,共6页
Acta Universitatis Medicinalis Anhui
基金
安徽省教育厅自然科学研究重点项目(编号:KJ2007A026)
关键词
舌肿瘤
癌
鳞状细胞
转染
基因疗法
细胞凋亡Fas基因
真核表达质粒
tongue neoplasms
carcinoma, squamous cell
transduction
gene therapy
apoptosis
fas gene
eukaryotic expression plasmid
