【作者】 李彦；

【导师】 姜政；

【作者基本信息】 重庆医科大学 ， 消化内科， 2008， 硕士

【摘要】 目的:通过构建pBud-EGFP-TRAIL质粒,在体内外观察可溶性人TRAIL蛋白(sTRAIL)对人低分化胃癌细胞株BGC-823的作用。方法:以EFGP为报告基因,sTRAIL为目的基因,构建pBud-EGFP-TRAIL重组质粒,并通过酶切和测序对重组体进行鉴定。经脂质体转染法转染体外培养的胃癌细胞BGC-823,RT-PCR及Western blot检测sTRAIL mRNA及其蛋白的表达,用荧光显微镜观察EGFP的表达,MTT法检测转染后对胃癌细胞的生长抑制率,TUNEL法观察细胞的凋亡情况,流式细胞仪分析转染后胃癌细胞的凋亡率。然后,将重组质粒pBud-EGFP-TRAIL注入胃癌细胞BGC-823裸鼠皮下移植瘤内,通过比较肿瘤体积及H.E染色观察肿瘤组织病理变化,TUNEL染色检测细胞凋亡情况,研究其在体内环境下对肿瘤细胞生长的影响。结果:测序结果证实pBud-EGFP-TRAIL载体构建成功,质粒经脂质体转染后,通过表达sTRAIL蛋白对胃癌细胞发挥作用。MTT法显示转染该质粒的细胞的生长抑制率明显高于对照组,TUNEL法显示细胞核固缩,核染色质聚集或断裂,形成凋亡小体。流式细胞仪结果表明转染该质粒的细胞的的凋亡率明显高于对照组。体内实验表明,注射pBud-EGFP-TRAIL质粒的裸鼠移植瘤体积明显小于对照组,肿瘤组织切片H.E染色可见核大量破碎、溶解,肿瘤细胞大量死亡,并出现大量纤维化结构包裹肿瘤组织,TUNEL染色显示pBud-EGFP-TRAIL组有较多细胞凋亡,而对照组无此明显变化。结论:在体内外初步证实sTRAIL的重组真核表达质粒pBud-EGFP-TRAIL能诱导胃癌细胞BGC-823凋亡,为进一步开展胃癌的基因治疗奠定了基础。更多还原

【Abstract】 Objective: Constructing a plasmid of pBud-EGFP-TRAIL to study the effect of soluble TRAIL protein (sTRAIL)on the gastric cancer cell line of BGC-823 in vitro and vivo.Methods: EGFP was as the report gene while sTRAIL as the target gene, to form a recombinated plasmid of pBud-EGFP-TRAIL,which was then identified by the techonology of restriction digest and sequencing. After it had been transfected into gastric cancer cells of BGC-823 by LipofectamineTM2000, the expression of EGFP was observed by fluorescent microscope , RT-PCR and Western Blot were used to detected the expression of sTRAIL. The inhibition of cell proliferation was examined by MTT. The method of TUNEL and flow cytometry were selected to detect apoptosis. Cells of BGC-823 were injected into nude mice hypodermically to form a transplantation tumor, and the plasmid of pBud-EGFP-TRAIL was then injected into the tumors. The changes of sTRAIL were detected by RT-PCR and Western blot. The size of tumors were compared between the experimental group and the control ones. H.E dyeing and TUNEL were used to detect the pathological change and apoptosis in the tumors. Results: Sequencing result showed that the recombinant plasmid of pBud-EGFP-TRAIL had been constructed successfully. After the transfection of it into the BGC-823 cells, sTRAIL protein was expressed and cells were induced to apoptosis. The inhibition rate was higher than that of the control ones though MTT, and the apoptosis rate was detected higher through the flow cytometry. Karyopycnosis, nuclear chromosomal condensation and segmentation were observed by TUNEL. In vivo, the results showed that the volume of tumors in the pBud-EGFP-TRAIL group is much smaller than that of the control ones, and more cell death and apoptosis were detected through H.E dyeing and TUNEL.Conclusion: It has been initially proved that the recombinated eukaryotic expressing plasmid of pBud-EGFP-TRAIL can induce apoptosis of gastric cancer cell of BGC-823 both in vitro and vivo ,which is a basic foundation for the genetic therapy of gastric cancer in future.更多还原