【摘要】：Survivin 是1997 年Ambrosini 等應(yīng)用EPR-1 基因cDNA 作為探針去篩選人基因組文庫而得到的,是IAP 家族的一個成員,以其獨特的分子結(jié)構(gòu)以及表達特點,在其被發(fā)現(xiàn)后的幾年中得到廣泛的研究與關(guān)注。 本論文中,我們用蛋白合成抑制劑放線菌酮CHX處理宮頸癌細(xì)胞HeLaS3,并用Western-blot 檢測,發(fā)現(xiàn)60min 后Survivin 蛋白的表達量明顯降低。而用PKA 的抑制劑H89 預(yù)先處理細(xì)胞兩個小時后,再用CHX 處理細(xì)胞,則Survivin蛋白量變化不明顯。我們推斷,Survivin 的穩(wěn)定性可能與其潛在的PKA 磷酸化位點的磷酸化狀態(tài)有關(guān)。 進而通過序列比對,我們選擇出三條針對Survivin 基因的siRNA 片段,并構(gòu)建了相應(yīng)的RNAi 載體,轉(zhuǎn)染入HeLaS3 細(xì)胞后,瞬時轉(zhuǎn)染的結(jié)果表明,Survivin 表達量在RNA 和蛋白水平上都得以下調(diào),尤以S3 片段效果最為明顯,并且用流式細(xì)胞術(shù)檢測干涉載體轉(zhuǎn)染對細(xì)胞產(chǎn)生的生物學(xué)效應(yīng),發(fā)現(xiàn)轉(zhuǎn)染干涉載體pTet-U6-S3 后,細(xì)胞凋亡率得到很大的提高。 另外,我們構(gòu)建了Survivin 上潛在的pKA 磷酸化位點Ser81 的突變體S81D、S81A,用來檢測該位點磷酸化不同狀態(tài)對Survivin 穩(wěn)定性的影響。 但是把干涉載體轉(zhuǎn)染至細(xì)胞,并把其培養(yǎng)成穩(wěn)定株后,HeLaS3 細(xì)胞內(nèi)源Survivin 量并沒有被有效地抑制,這是很出乎預(yù)料的。
[Abstract]:Survivin was obtained from 1997 when Ambrosini used EPR-1 gene cDNA as a probe to screen human genome library. It is a member of IAP family with its unique molecular structure and expression characteristics. In the years since its discovery, it has received extensive research and attention. In this paper, we treated cervical cancer cell HeLaS3, with actinomycin CHX, a protein synthesis inhibitor, and detected by Western-blot. It was found that the expression of Survivin protein decreased significantly after 60min. However, when the cells were pretreated with H89, a PKA inhibitor, and then treated with CHX for two hours, the Survivin protein content did not change significantly. We infer that the stability of Survivin may be related to the phosphorylation state of its potential PKA phosphorylation site. Then through sequence alignment, we selected three siRNA fragments targeting Survivin gene and constructed corresponding RNAi vector. After transfection into HeLaS3 cells, the transient transfection results showed that the expression of Survivin was down-regulated at both RNA and protein levels. The effect of S3 fragment was the most obvious, and the biological effect of interference vector transfection on cells was detected by flow cytometry. It was found that after transfection of interference vector pTet-U6-S3, cell apoptosis rate was greatly increased. In addition, we constructed the mutant S81D- S81A of Ser81, a potential pKA phosphorylation site on Survivin, to detect the effect of different phosphorylation states on the stability of Survivin. However, after the interference vector was transfected into the cells and cultured into a stable strain, the amount of endogenous Survivin in HeLaS3 cells was not effectively inhibited, which was unexpected.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：Q78

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