摘要
目的构建携带人可溶性肿瘤坏死因子相关凋亡诱导配体(sTRAIL)基因的自身失活型慢病毒载体,观察sTRAIL对人胃癌SGC-7901细胞的诱导凋亡作用,探讨其基因治疗功效。方法构建含人sTRAIL基因及内部核糖体进入位点序列(IRES)和绿色荧光蛋白(GFP)基因的双顺反子慢病毒载体。采用脂质体转染法将慢病毒三质粒包装系统包装出的携带人sTRAIL的重组慢病毒,在体外感染人胃癌SGC-7901细胞、正常人肝细胞HL-7702、人胚肺成纤维细胞HLF-1。应用流式细胞术(FCM)检测细胞凋亡率,酶联免疫吸附实验(ELISA)评价sTRAIL蛋白在胃癌细胞中的表达。结果成功构建了慢病毒表达质粒pXZ208-sTRAIL,包装的重组慢病毒滴度可达106 IU/mL,可有效感染体外培养的细胞;含sTRAIL的重组慢病毒感染SGC-7901细胞,细胞凋亡率随病毒感染复数(MOI)的增加而升高,呈剂量依赖性;在MOI为6.0、作用24h时,细胞凋亡率最高,可达(29.12±2.87)%,细胞上清中sTRAIL表达量为(34.08±3.43)ng/mL;而在HL-7702、HLF-1等人正常细胞未见明显细胞凋亡。结论含有sTRAIL基因的重组慢病毒载体在体外能够有效地感染胃癌SGC-7901细胞并使其分泌有生物活性的sTRAIL蛋白,可诱导SGC-7901细胞的凋亡,而对正常细胞无显著影响。
Objecitve To construct self-inactivating （SIN） lentiviral vector carrying human soluble tumor necrosis factor-related apoptosis-inducing ligand （sTRAIL） gene and to observe the effects of sTRAIL gene on apoptosis in SGC-7901 cells, so as to assess the value of sTRAIL in gene therapy for gastric cancer. Methods The bicistronic SIN lentiviral transfer plasmid containing sTRAIL gene and internal ribosomal entry site-green fluorescent protein gene （IRES-GFP） was constructed. Recombinant lentivirus containing sTRAIL were packaged using liposome by lentiviral packing system. Several cell lines including HL-7702, HLF-1 and SGC-7901 cells were infected with the viral supernatant. Flow cytometry was used to measure apoptosis of cells and recombinant sTRAIL protein was assayed by EIASA at 24 h after infection. Results The lentiviral transfer plasmid pXZ208-sTRAIL was constructed, and the virus titres were above 106 IU/mL in the supernatant. SGC-7901 cells were efficiently infected by recombinant virus. The apoptosis rate of SGC-7901 cells was increased with the virus multiplicity of infection （MOI） increasing. When the MOI was ≥0.5, a dose-dependent apoptosis rate was observed. At MOI of 6.0, the highest apoptosis rate （29.12±2.87）M was observed, while the expression of sTRAIL in the supernatant was only （34.08±3. 43） ng/mL. However, no significant apoptosis was observed in HL-7702 cells and HLF-1 cells. Conclusion Recombinant lentivirus carrying human sTRAIL gene can efficiently infected SGC-7901 cells, which induced apoptosis in SGC-7901 cells in vitro by secreting bioactive sTRAIL protein. However, this effect is not seen in normal cells.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2013年第3期348-351,392,共5页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(No.81100349)
江苏省卫生厅青年科研课题(No.H201063)资助
