【作者】 李朝阳；

【导师】 郭德银；

【作者基本信息】 武汉大学 ， 微生物学， 2005， 博士

【摘要】 RNA干扰(RNAi)是一种由小干扰RNA(siRNA)介导的靶序列特异性RNA降解机制,现在它不仅被当作一种研究工具,还可以用作一种抑制病毒感染的基因治疗策略。在哺乳动物细胞里,基于小发夹RNA(shRNA)的RNA干扰技术可以成功下调细胞或者病毒基因的表达。 我们在HeLa,293T以及U373-MAGI-CCR5e细胞中表达靶向细胞内cyclin T1蛋白的shRNA表达质粒后,发现表达质粒CT-4可以特异的下调细胞内cyclin T1蛋白的表达。同时,我们的结果显示cyclin T1蛋白的下调水平跟转染的shRNA表达质粒的剂量以及转染的时间都有一定的正相关性。 CDK9/cyclin T1这一复合物在细胞内的活性不依赖于细胞周期,其作用是参与转录的延伸。这表明作为这个复合物的一个亚单位的cyclin T1对细胞的存活是至关重要的。但是,我们的实验表明在培养的细胞系中,下调cyclin T1蛋白的表达既没有诱导宿主细胞产生凋亡,甚至没有影响细胞周期的变化。我们的结果与刚刚报道的下调P-TEFb的表达没有引起细胞死亡相一致。在细胞水平,cyclin T1将会是一个很好的抑制HIV-1复制的细胞内靶位点。更多还原

【Abstract】 RNA interference (RNAi), a sequence-specific RNA degradation mechanism mediated by small interfering RNA (siRNA), can be used not only as a research tool but also as a therapeutic strategy for viral infection. RNAi with short hairpin RNA (shRNA) has been successfully utilized in a number of recent studies in cultured mammalian cells to reduce the expression of specific cellular and viral genes.Here, intracellularly expressed shRNA based on plasmid vector were utilized in 293T, HeLa and U373-MAGI-CCR5e cells. Our results indicated that intracellularly generated shRNAs could specifically down-regulate cyclin T1 expression. Similar phenomena were demonstrated in 293T and U373-MAGI-CCR5e cells. Additionally, our results indicated that the inhibition of cyclin Tl expression by shRNAs was both dose- and time-dependent.CDK9/cyclin T directs its activity in a cell cycle independent manner and was involved in transcription during the elongation steps. This means that cyclin Tl is important for cell survival. Surprisingly, knockdown of cyclin Tl protein neither influenced the cell cycle nor induced apoptosis in cultured cell lines, and this was consistent with a previous report that knockdown of P-TEFb led to inhibition of HIV-1 replication but not resulted in cell death. Our data indicated that down regulation ofcyclin Tl did not induce significant apoptosis of transfected cells and cyclin Tl could be used as a potential target for inhibiting HIV-1 replication.We have also shown that knockdown of cyclin Tl expression by RNA interference inhibited HIV-1 replication specifically and effectively. Furthermore, we observed the correlation between the levels of down-regulation of cyclin Tl expression and the inhibition of HIV-1 replication, indicating that the replication level of HIV-1 may be dependent on the amount of cyclin Tl in cultured cells.In previous reports about RNAi with HIV-1 replication and infection, chemically synthesized siRNAs were most commonly used, however, synthetic siRNAs do not persist for long period in cells and are not feasible to be delivered into the vast number of target cells like T lymphocytes and macrophages. Our result renders a possibility to deliver the anti-HIV shRNA-encoding genes by viral vectors into target cells and stably express the shRNAs.Taken together, this study demonstrated the cyclin Tl could be a promising cellular target for suppression of HIV-1 replication by DNA-based shRNAs. Now, We are focusing on the delivery of shRNA-encoding genes into primary cells, such as T lymphocytes, monocytes and macrophages by lentiviral vectors and examine the feasibility to use cyclin Tl as target in genetic therapy of AIDS.更多还原