瞭解細胞核運送的分子機制以及細胞如何藉由細胞核質間運送的方式調控蛋白質的活性，將能提供我們對於一些疾病有更進一步的認知。細胞中大分子物質的進出細胞核通常被其本身所帶有的標的訊號所控制，且此標的訊號受一群專一的入核與出核的運送因子所辨識。幾個不同種類的細胞核定位訊號，包刮來自於SV40 large T-antigen，nucleoplasmin，hnRNP A1以及p53，幫助許多蛋白質運送入細胞核中。核糖核酸干擾術具有極大的潛力做為一個實驗上的工具，提供生物性的發現、分析與治療。在本研究中，我們即試圖利用RNA干擾術這個技術來探討細胞核定位訊號與細胞核運送因子間的關係。目前，我們已針對SV40 large T-antigen，nucleoplasmin，hnRNP A1以及p53四個蛋白質，建構其細胞核定位訊號與單一的綠色螢光蛋白，兩個綠色螢光蛋白，和兩個綠色螢光蛋白以及glutathione S-transferase(GST)的融合表現載體。同時，我們也針對幾個主要的細胞核運送因子，包刮六個人類的importin alpha (1，3，4，5，6，7)，三個importin beta (1，2，3)以及Ran，篩選了有效的siRNA和shRNA。藉由共同轉染細胞核定位訊號的表現載體與針對細胞核運送因子的shRNA，我們發現針對importin beta和Ran的shRNA相較於importin alpha，對於細胞核定位訊號的入核效力具有較大的影響。特別的是，我們很驚訝地發現針對importin beta 1和Ran的shRNA對於p53的細胞核定位訊號的入核效力具有非常顯著的影響。重要的是，這個發現不只可進一步地藉由共同轉染p53基因的表現載體與這些shRNA觀察到，也可利用免疫螢光染色法於內生性的p53上得到一致性的結果。在另一個研究中，我們將幾個針對細胞生存基因的shRNA進行組合，包刮Bcl-2，survivin，Akt1，Erk2，cyclinE，NFκB，發現即使組合至六個shRNA之多，也不會影響組合中的各個單一的shRNA對其標的基因的抑制能力。甚至，當我們進一步地將這些shRNA轉染入HeLa細胞中，我們有趣地觀察到他們能誘發不同程度的細胞計畫性死亡。由於一種細胞核定位訊號通常藉由不只一種細胞核運送因子的幫助進入細胞核中，所以我們試圖利用此套shRNA的組合表現載體將針對細胞核運送因子的shRNA進行組合，使我們能利用各種針對細胞核運送因子的組合shRNA進一步更深入地探討細胞核運送的分子機制。目前，我們已針對三個importin beta (1，2，3)以及Ran成功地建構了各種可能的shRNA組合表現載體，提供我們在未來能進一步地探討細胞核運送的分子機制。

Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity. Transport of macromolecules into and out of the nucleus is generally controlled by targeting signals that are recognized by specific members of the importin/exportin transport receptor family. Several distinct types of nuclear localization signals (NLSs), from SV40 large T-antigen, nucleoplasmin, hnRNP A1, and p53, mediate passage of cargo proteins into the nucleus. RNA interference (RNAi) has shown great potential for use as a tool for biological discovery, analysis and therapeutics. In this study, we try to dissect the relationship between NLSs and nuclear transport receptors by using RNAi-based techniques. Until now, we have established the expression vectors of NLSs, including SV40 large T-antigen, nucleoplasmin, hnRNP A1, and p53, fused with one or two copies of enhanced green fluorescent protein (EGFP; EGFP2), or two copies EGFP plus glutathione S-transferase (EGFP2-GST). Simultaneously, we have screened the effective siRNAs/shRNAs against nuclear transport receptors, including human importin alpha (1,3,4,5,6,7), beta (1,2,3) family and Ran. By
co-transfection of those NLS-expression vectors and shRNAs against nuclear transport receptors, we found that the shRNAs against importin beta family and Ran contrast to alpha have larger effect on the efficacy of NLS-mediated nuclear import. In particular, we were surprised to find that shRNAs against importin beta 1 and Ran individually have most dramatic effect on the efficacy of p53-NLS1-mediated nuclear import. Importantly, this result could be further observed not only by co-transfection of p53 gene-expression vector and these shRNAs but also on the endogenous level by using immunofluorescence staining. In another study, we used several shRNAs against cell survival genes, including Bcl-2, survivin, Akt1, Erk2, cyclinE, NFκB for combination and found that the combination of at most six shRNAs would not affect their silencing efficiency. Furthermore, when these shRNA-expression vectors were transfected into HeLa cell line, we interestingly observed that these shRNAs would induce different level of apoptosis. Because each type of NLS was recognized more than one type of nuclear transport receptors, this combination RNAi expression system was used to further analyze the nuclear localization mechanism by combining those shRNAs against nuclear transport receptors. Until now, we have established all possible types of combination expression vectors from those shRNAs against
importin beta family and Ran, and these shRNA combination expression vectors can provide further analysis for nuclear transport mechanism in the future.

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