CAJ | 학술논문

目的通过体内外研究人端粒酶逆转录酶单位(hTERT)启动子和存活因子启动子在实现肿瘤基因治疗靶向性中的作用并探讨其应用前景.方法 PCR方法扩增hTERT启动子片段和存活因子启动子片段,并构建包含重组可溶性肿瘤坏死因子相关细胞凋亡诱导配体(TRAIL)的重组腺相关病毒载体.体外转染四株肝细胞癌细胞株及人原代正常肝细胞(PHH),通过EGFP表达及MTT检测启动子活性.体内建立SMMC-7721皮下移植瘤模型,观察瘤内注射rAAV病毒对肿瘤生长的影响.结果两启动子驱动的外源基因表达具有肿瘤细胞特异性,且它们驱动的TRAIL表达能降低HCC的存活率,而不能降低PHH存活率.以rAAV为载体,hTERT启动子驱动的TRAIL能显著抑制皮下肿瘤生长.结论肿瘤特异启动子是实现以rAAV为载体的肿瘤靶向基因治疗的潜在手段.
목적 통과체내외연구인단립매역전록매단위(hTERT)계동자화존활인자계동자재실현종류기인치료파향성중적작용병탐토기응용전경.방법 PCR방법확증hTERT계동자편단화존활인자계동자편단,병구건포함중조가용성종류배사인자상관세포조망유도배체(TRAIL)적중조선상관병독재체.체외전염사주간세포암세포주급인원대정상간세포(PHH),통과EGFP표체급MTT검측계동자활성.체내건립SMMC-7721피하이식류모형,관찰류내주사rAAV병독대종류생장적영향.결과 량계동자구동적외원기인표체구유종류세포특이성,차타문구동적TRAIL표체능강저HCC적존활솔,이불능강저PHH존활솔.이rAAV위재체,hTERT계동자구동적TRAIL능현저억제피하종류생장.결론 종류특이계동자시실현이rAAV위재체적종류파향기인치료적잠재수단.
Objective To investigate the effects of human telomerase reverse transcriptase(hTERT)promoter and survivin promoter in tumor-specific gene therapy.Methods hTERT promoter and survivin promoter were obtained by PCR using Jurkat genomic DNA.Recombinant adeno-associated virus(AAV)vectors containing exogenous TRAIL gene and hTERT promoter or survivin promoter were constructed and designated as rAAV-hTERT-TRAIL(h/TRAIL)or rAAV-survivin-TRAIL(s/TRAIL).rAAV particles were obtained after packing and purification and the virus titer was calculated by real-time PCR.Human hepatocellular carcinoma(HCC)cells of the lines SMMC-7721,BEL-7402,HepG2,and Hep3B.and primary human hepatocytes(PHHs)were transfected with h/TRAIL or s/TRAIL.Flow cytometry was used to detect the expression of the reporter gene enhanced green fluorescent protein(EGFP),so as to examine the activitV of the two promoters.MTT method was used to detect the activity of the cells.Fifteen BalB/C mice underwent subcutaneous injection of SMMC-7721 cells so as to establish tumor models and then were randomly divided into 3 groups to undergo intra-tumor injection of h/TRAIL,s/TRAIL,or PBS.The growth of tumor was observed for 5 weeks,and then peripheral blood samples were collected to examine the serum AST and ALT levels.TUNEL was used to detect the apoptosis if the tumor cells.Results All the SMMC-7721,BEL-7402,HepG2,and Hep3B cells driven by both h/TRAIL and s/TRAIL showed EGFP expression,however,no fluorescence could be seen in the PHHs transfected with h/TRAIL and s/TRAIL.MTT method showed that 72 hours after the transfection of h/TRAIL and s/TRAIL the survival rates of the SMMC-7721,BEL-7402,and HepG2 cells all decreased,however,the survival rate of the Hep3B cells and PHHs did not changed significantly.The size of the subcutaneous tumor of the mice of the h/TRAIL group was 625 mm3,significantly smaller than that of the PBS group(1500 mm3,P＜0.05),however.the tumor size of the s/TRAIL group was 1117 mm3,not significantly different from that of the PBS group(P＞0.05).The AST and ALT levels of all mice did not change significantly 5 weeks after the intratumor injection.Conclusion Tumor-specific promoters are promising candidates in targeted tumor gene therapy.