泸州医学院学报
瀘州醫學院學報
로주의학원학보
JOURNAL OF LUZHOU MEDICAL COLLEGE
2013年
4期
350-355
,共6页
曾永秋%曹洋%余红%赵矫%税青林
曾永鞦%曹洋%餘紅%趙矯%稅青林
증영추%조양%여홍%조교%세청림
Survivin启动子%hTERT基因%RNA干扰%肿瘤靶向性
Survivin啟動子%hTERT基因%RNA榦擾%腫瘤靶嚮性
Survivin계동자%hTERT기인%RNA간우%종류파향성
Survivin promoter%hTERT gene%RNAi%Tumor specific
目的：构建带有肿瘤特异性Survivin启动子、hTERT基因靶向的shRNA表达载体pYr-Survivinp-miR30-hTERT，探讨该载体的肿瘤靶向性及其RNAi作用效率，为RNAi技术在肿瘤基因治疗中的进一步优化进行有益的探索。方法：化学合成hTERT基因shRNA寡核苷酸序列，克隆至质粒pYr-CMV-Mir30-shRNA，构建重组质粒pYr-CMV-Mir30-hTERT；克隆Survivin启动子序列，取代该质粒原有的CMV启动子，构建重组质粒pYr-Survivinp-miR30-hTERT，同时构建阴性对照质粒pYr-CMV-Mir30-NC，以上质粒均进行酶切及测序鉴定。将以上载体分别转染人乳腺癌MCF-7细胞、人宫颈癌Hela细胞及293T细胞，荧光显微镜下观察细胞转染情况，并采用半定量RT-PCR、western blot 技术检测MCF-7细胞和Hela 细胞中hTERT mRNA和蛋白的表达。结果：重组质粒经酶切及测序鉴定证实均符合设计要求，构建成功；荧光检测结果表明，CMV组在三种细胞中均有荧光蛋白的表达，而Survivin组质粒仅在MCF-7细胞和Hela细胞中表达绿色荧光，转染效率均能达到70%以上，而在293T细胞中则未见荧光；RT-PCR和Western blot的结果均显示MCF-7细胞和Hela细胞的Survivin组和CMV组中hTERT mRNA和蛋白的表达均受到明显抑制（P<0.05），两组之间无明显差异（P>0.05）。结论：Survivin启动子调控的hTERT基因RNAi载体能有效抑制Survivin阳性肿瘤细胞中hTERT的表达，而对正常细胞不产生影响。
目的：構建帶有腫瘤特異性Survivin啟動子、hTERT基因靶嚮的shRNA錶達載體pYr-Survivinp-miR30-hTERT，探討該載體的腫瘤靶嚮性及其RNAi作用效率，為RNAi技術在腫瘤基因治療中的進一步優化進行有益的探索。方法：化學閤成hTERT基因shRNA寡覈苷痠序列，剋隆至質粒pYr-CMV-Mir30-shRNA，構建重組質粒pYr-CMV-Mir30-hTERT；剋隆Survivin啟動子序列，取代該質粒原有的CMV啟動子，構建重組質粒pYr-Survivinp-miR30-hTERT，同時構建陰性對照質粒pYr-CMV-Mir30-NC，以上質粒均進行酶切及測序鑒定。將以上載體分彆轉染人乳腺癌MCF-7細胞、人宮頸癌Hela細胞及293T細胞，熒光顯微鏡下觀察細胞轉染情況，併採用半定量RT-PCR、western blot 技術檢測MCF-7細胞和Hela 細胞中hTERT mRNA和蛋白的錶達。結果：重組質粒經酶切及測序鑒定證實均符閤設計要求，構建成功；熒光檢測結果錶明，CMV組在三種細胞中均有熒光蛋白的錶達，而Survivin組質粒僅在MCF-7細胞和Hela細胞中錶達綠色熒光，轉染效率均能達到70%以上，而在293T細胞中則未見熒光；RT-PCR和Western blot的結果均顯示MCF-7細胞和Hela細胞的Survivin組和CMV組中hTERT mRNA和蛋白的錶達均受到明顯抑製（P<0.05），兩組之間無明顯差異（P>0.05）。結論：Survivin啟動子調控的hTERT基因RNAi載體能有效抑製Survivin暘性腫瘤細胞中hTERT的錶達，而對正常細胞不產生影響。
목적：구건대유종류특이성Survivin계동자、hTERT기인파향적shRNA표체재체pYr-Survivinp-miR30-hTERT，탐토해재체적종류파향성급기RNAi작용효솔，위RNAi기술재종류기인치료중적진일보우화진행유익적탐색。방법：화학합성hTERT기인shRNA과핵감산서렬，극륭지질립pYr-CMV-Mir30-shRNA，구건중조질립pYr-CMV-Mir30-hTERT；극륭Survivin계동자서렬，취대해질립원유적CMV계동자，구건중조질립pYr-Survivinp-miR30-hTERT，동시구건음성대조질립pYr-CMV-Mir30-NC，이상질립균진행매절급측서감정。장이상재체분별전염인유선암MCF-7세포、인궁경암Hela세포급293T세포，형광현미경하관찰세포전염정황，병채용반정량RT-PCR、western blot 기술검측MCF-7세포화Hela 세포중hTERT mRNA화단백적표체。결과：중조질립경매절급측서감정증실균부합설계요구，구건성공；형광검측결과표명，CMV조재삼충세포중균유형광단백적표체，이Survivin조질립부재MCF-7세포화Hela세포중표체록색형광，전염효솔균능체도70%이상，이재293T세포중칙미견형광；RT-PCR화Western blot적결과균현시MCF-7세포화Hela세포적Survivin조화CMV조중hTERT mRNA화단백적표체균수도명현억제（P<0.05），량조지간무명현차이（P>0.05）。결론：Survivin계동자조공적hTERT기인RNAi재체능유효억제Survivin양성종류세포중hTERT적표체，이대정상세포불산생영향。
Objectives: To construct tumor specific Survivin promoter regulated hTERT gene-targeted RNAi expression vector (pYr-Survivinp-miR30-hTERT) and investigate its tumor-targeting gene suppression effects and efficiency for the improvement of RNAi technology. Methods: The hTERT gene-targeted RNAi oligonucleotides sequence was synthesized and cloned to the plasmid pYr-CMV-Mir30-shRNA to construct the recombinant plasmid pYr-CMV-Mir30-hTERT; the CMV promoter in the plasmid was then replaced by the Survivin promoter, to construct the plasmid pYr-Survivinp-miR30-hTERT. The negative control plasmid pYr-CMV-Mir30-NC was constructed by the same method and these new recombinant vectors were confirmed by enzyme digestion and sequencing. Next,they were transfected into three kinds of cells (human breast cancer MCF-7 cells, human cervical carcinoma Hela cells,and human embryo renal cell-293T cells) and the fluorescent microscope was used to observe the cell transfection situation. Finally, semi-quantitative RT-PCR and western blot were performed to evaluate the expression of hTERT gene in MCF-7 and Hela cells. Results: Enzyme digestion and sequencing confirm that the recombinant plasmid was constructed successfully; the results of fluorescence microscopy showed that in CMV group all three kinds of cells had fluorescent protein expression, while in Survivin group green fluorescent only in MCF-7 and Hela cells , the transfection efficiency were both more than 70%;in 293T cells were not seen fluorescent cells. The results of RT-PCR and western blot showed that the expression hTERT mRNA and protein were significantly inhibited in MCF-7 and Hela cells of both Survivin and CMV groups of (P<0.05), while there was no significant difference in these two groups (P>0.05). Conclusions:The hTERT gene-targeted RNAi expression vector mediated by Survivin promoter can inhibit expression of hTERT gene in Survivin positive tumor cells effectively , but did not affect the normal cells.
