本文選題：Livin　切入點(diǎn)：膀胱癌　出處：《中南大學(xué)》2014年博士論文

【摘要】：凋亡抑制蛋白(Inhibitor of Apoptosis Protein, IAPs)是生物體內(nèi)一類包含高度保守的BIR結(jié)構(gòu)域的內(nèi)源性抗細(xì)胞凋亡因子。Livin是近年來IAPs家族最新發(fā)現(xiàn)的成員,其分子結(jié)構(gòu)包含1個BIR結(jié)構(gòu)域和1個RING指結(jié)構(gòu)域。Livin在正常成人組織中基本不表達(dá),在許多腫瘤中高表達(dá),被認(rèn)為是腫瘤預(yù)后不良及藥物抵抗的重要影響因素。研究表明Livin不但具有抗細(xì)胞凋亡作用,其分子結(jié)構(gòu)包含的RING指結(jié)構(gòu)域還賦予其泛素結(jié)合酶樣活性,在細(xì)胞周期的調(diào)節(jié)上也有重要作用。國內(nèi)關(guān)于Livin在膀胱癌中的表達(dá)情況,對其預(yù)后和化療藥物抗性的研究目前仍然較少。Livin的表達(dá)對于膀胱癌細(xì)胞凋亡及細(xì)胞周期的影響有待進(jìn)一步研究。 目的： (1)設(shè)計(jì)針對Livin的高效特異性siRNA并進(jìn)行篩選,作為進(jìn)一步實(shí)驗(yàn)研究的工具。(2)通過對人膀胱癌T24細(xì)胞系中Livin基因表達(dá)的RNA干擾,抑制Livin基因的表達(dá),觀察Livin基因表達(dá)下降對于膀胱癌細(xì)胞凋亡及細(xì)胞周期的影響。 方法： (1)通過siRNA設(shè)計(jì)軟件及siRNA數(shù)據(jù)庫等工具設(shè)計(jì)出Livin基因特異性的siRNA。通過RT-PCR和Western blot等方法觀察其抑制Livin基因在人膀胱癌T24細(xì)胞系中表達(dá)的效率,篩選出具有高效性特異性的siRNA。(2)質(zhì)粒轉(zhuǎn)染高特異性siRNA干擾Livin基因的表達(dá),通過雙變量流式細(xì)胞儀計(jì)數(shù),檢測Livin基因表達(dá)下降對人膀胱癌T24細(xì)胞凋亡的影響。(3)質(zhì)粒轉(zhuǎn)染高特異性siRNA干擾Livin基因的表達(dá),用過流式細(xì)胞儀計(jì)數(shù),檢測抑制Livin基因表達(dá)下降對人膀胱癌T24細(xì)胞的細(xì)胞周期的影響。 結(jié)果： 1. Livin特異性siRNA的篩選 使用siRNA設(shè)計(jì)軟件和siRNA數(shù)據(jù)庫獲得3種siRNA,通過RT-PCR和Western blot對每種siRNA在T24細(xì)胞中干擾效率進(jìn)行檢驗(yàn),結(jié)果表明3種siRNA均能較顯著的抑制Livin基因在T24細(xì)胞中的表達(dá),其中1種能夠顯著的降低Livin基因的表達(dá)。 2. Livin基因表達(dá)下調(diào)對人膀胱癌T24細(xì)胞凋亡的影響 雙變量流式細(xì)胞計(jì)數(shù)顯示,Livin基因表達(dá)下降,導(dǎo)致T24細(xì)胞自發(fā)性凋亡出現(xiàn)明顯增高。 3. Livin基因表達(dá)下調(diào)對人膀胱癌T24細(xì)胞周期的影響 流式細(xì)胞計(jì)數(shù)顯示Livin基因表達(dá)下降,導(dǎo)致T24細(xì)胞周期明顯停滯于G2/M期。 結(jié)論： 1. Livin基因在T24細(xì)胞中的表達(dá)抑制,可以提高T24細(xì)胞自發(fā)性凋亡的發(fā)生率。 2. Livin基因在T24細(xì)胞中的表達(dá)抑制,可以有效的抑制腫瘤細(xì)胞的增殖,減緩腫瘤的發(fā)展。
[Abstract]:Inhibitor of apoptosis protein (Inhibitor of Apoptosis Protein, IAPs) is an endogenous organism contains a highly conserved BIR domain anti apoptotic factor.Livin is a member of the IAPs family in recent years, the newly discovered, its molecular structure contains 1 BIR domains and 1 RING domains of.Livin in normal adult tissue no expression, is highly expressed in many tumors, were considered to be important factors affecting prognosis and adverse drug resistance. The results show that Livin not only has anti apoptosis effect and its molecular structure contains the RING finger domain also gives the ubiquitin conjugating enzyme like activity, also has an important role in the regulation of cell cycle. The expression of Livin in bladder cancer, study on the prognosis and chemotherapy drug resistance is still less influence on the expression of.Livin in cell cycle and apoptosis of bladder cancer cells have to be Further study.
Objective:
(1) design for Livin, and specific siRNA were selected as further research tools. (2) by RNA interference on the expression of Livin in human bladder cancer cell line T24 gene, inhibit the expression of Livin gene, Livin gene expression decreased for observation and cell cycle of bladder cancer cell apoptosis.
Method錛，

本文編號：1681063