CAJ | 학술논문

目的探讨转染肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因的骨髓间充质干细胞(BMSC)基因表达情况及其功能.方法实验分三组,即转染TRAIL基因组、转染空载体组及空白对照组.用脂质体法将TRAIL转入绿色荧光蛋白(GFP)-BMSC中,反转录PCR法检测BMSC的TRAILmRNA水平,Western blot、免疫荧光法检测TRAIL蛋白的表达；将携带有TRAIL的GFP-BMSC同大鼠C6胶质瘤细胞共培养,通过四甲基偶氮唑蓝比色法检测其对肿瘤细胞的旁观者效应,Hochest-PI双染色法观察TRAIL转染的BMSC对C6细胞凋亡的影响.结果免疫荧光检测显示,转染TRAIL 24、48 h的GFP-BMSC细胞质和细胞膜有TRAIL蛋白的表达,24h比48 h荧光强,空白对照组及空载体组细胞未见表达.反转录PCR、Western blot显示转染TRAIL基因组细胞TRAIL mRNA及蛋白高表达,空白对照组及空载体组未见表达.转染TRAIL的GFP-BMSC明显抑制C6细胞存活,抑制率为(62.7±0.1)％,高于空载体组的(16.7±0.1)％(P＜0.05),同时转染TRAIL基因的BMSC可促进C6细胞的凋亡.结论转染TRAIL的BMSC能够稳定表达目的基因,且能促进大鼠C6胶质瘤细胞凋亡,具有明显的旁观者效应.
목적 탐토전염종류배사인자상관조망유도배체(TRAIL)기인적골수간충질간세포(BMSC)기인표체정황급기공능.방법 실험분삼조,즉전염TRAIL기인조、전염공재체조급공백대조조.용지질체법장TRAIL전입록색형광단백(GFP)-BMSC중,반전록PCR법검측BMSC적TRAILmRNA수평,Western blot、면역형광법검측TRAIL단백적표체；장휴대유TRAIL적GFP-BMSC동대서C6효질류세포공배양,통과사갑기우담서람비색법검측기대종류세포적방관자효응,Hochest-PI쌍염색법관찰TRAIL전염적BMSC대C6세포조망적영향.결과 면역형광검측현시,전염TRAIL 24、48 h적GFP-BMSC세포질화세포막유TRAIL단백적표체,24h비48 h형광강,공백대조조급공재체조세포미견표체.반전록PCR、Western blot현시전염TRAIL기인조세포TRAIL mRNA급단백고표체,공백대조조급공재체조미견표체.전염TRAIL적GFP-BMSC명현억제C6세포존활,억제솔위(62.7±0.1)％,고우공재체조적(16.7±0.1)％(P＜0.05),동시전염TRAIL기인적BMSC가촉진C6세포적조망.결론 전염TRAIL적BMSC능구은정표체목적기인,차능촉진대서C6효질류세포조망,구유명현적방관자효응.
Objective To investigate the gene expression in bone mesenchymal stem cells transferred by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its effect on C6 glioma cells in vitro.Methods The experiment was divided into three groups,the test group transfected with TRAIL gene to BMSC,control group of BMSC transfected with empty liposomal vector,and blank control of BMSC alone.After transfering the TRAIL into GFP-BMSC with Liposomes,the expression of TRAIL was detected by RTPCR,Western blot,and immunofluorescence.After co-culturing C6 glioma cells with GFP-BMSC-TRAIL,the bystander effect of TRAIL was detected by MTT assay,and C6 cells apoptosis was detected by immunohistochemical method.Results GFP-BMSC-TRAIL vector was successfully constructed,with stable expression of TRAIL.The immunofluorescence showed the increased expression of TRAIL in GFP-BMSC cells at 48 h than 24 h.Western blot and PCR further revealed TRAIL expression in transfected cells,but no expression was found in control cells.In addition,compared with the blank control group and control group,GFP-BMSC-TRAIL significantly inhibited the C6 cell proliferation,with a lower survival rate (62.7 ± 0.1) ％ and higher apoptosis rate.Conclusion BMSC transfected with TRAIL gene can inhibit the proliferation of C6 glioma cell,suggesting BMSC may be an effective vector for gene therapy.