本文選題：重組單純皰疹病毒 + 溶瘤機(jī)制��； 參考：《武漢大學(xué)》2006年博士論文

【摘要】： 我們曾經(jīng)報道構(gòu)建了一株大腸桿菌β-半乳糖苷酶(lacZ)基因插入凋亡抑制基因(神經(jīng)毒基因)icp34.5位點使之失活的選擇性復(fù)制的重組單純皰疹病毒mtHSV,它能選擇性地殺死腫瘤細(xì)胞,而不殺死正常細(xì)胞。mtHSV的這種選擇性溶瘤效果,在多種腫瘤細(xì)胞系和瘤內(nèi)注射的荷Hep3B的裸鼠模型上得到了證實,然而,它的溶瘤機(jī)制尚未確定。 Farassati F.等研究證明,HSV-1易感染轉(zhuǎn)染了癌基因v-erbB、激活了sos或者ras基因的NIH3T3細(xì)胞。這意味著HSV-1通過Ras信號途徑進(jìn)入細(xì)胞。我們以前的研究表明RTN家族中的一個重要成員RTN3(RTN3即HAP,ASYIP, ASY的同源相互作用蛋白)是內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)的靶基因,過表達(dá)的RTN3能導(dǎo)致內(nèi)質(zhì)網(wǎng)超載反應(yīng)。我們以前的研究也顯示RTN3的一個重要特征是它定位在內(nèi)質(zhì)網(wǎng)上,但是關(guān)于RTN3與Ras的關(guān)系以及與mtHSV感染Hela細(xì)胞的關(guān)系尚未見報道。 在本研究中,我們著眼于研究mtHSV選擇性溶瘤的分子機(jī)制,即mtHSV侵染Hela細(xì)胞與Ras和RTN3的關(guān)系。研究表明,HeLa細(xì)胞能被mtHSV有效殺死。流式細(xì)胞術(shù)和Western blot實驗表明,mtHSV感染HeLa細(xì)胞48小時后,隨著內(nèi)質(zhì)網(wǎng)中RTN3蛋白上調(diào),細(xì)胞質(zhì)膜上的Ras蛋白明顯下調(diào),而細(xì)胞中的Ras蛋白總量沒有變化。用人法尼基轉(zhuǎn)移酶的α亞基的siRNA抑制劑siFTa和RTN3的siRNA抑制劑siRTN3分別轉(zhuǎn)染HeLa細(xì)胞48小時后,細(xì)胞質(zhì)膜上的Ras蛋白和細(xì)胞中的Ras蛋白總量分別被下調(diào),siFTa和siRTN3能有效地殺死HeLa細(xì)胞,有效地抑制mtHSV感染HeLa細(xì)胞。我們進(jìn)一步用激光共聚焦和免疫共沉淀實驗研究了Ras和RTN3的關(guān)系。研究結(jié)果表明:Ras和RTN3能在內(nèi)質(zhì)網(wǎng)上相互作用,Ras/RTN3是mtHSV侵染HeLa細(xì)胞的一個重要因素。Ras和RTN3分子間的相互作用進(jìn)一步提升了我們對Ras和RTN3在mtHSV感染HeLa細(xì)胞過程中功能的理解。 為了進(jìn)一步研究mtHSV侵染Hela細(xì)胞與P53以及細(xì)胞凋亡的關(guān)系,分別用流式細(xì)胞術(shù)和Western blot方法檢測了P53蛋白的表達(dá)。結(jié)果表明:mtHSV感染Hela細(xì)胞后24h,P53蛋白的表達(dá)無明顯變化。DNA ladder分析、Hoechst 33258染色觀察、Annexin V/PI雙染法檢測表明:mtHSV不誘導(dǎo)Hela細(xì)胞早期凋亡。在Hela細(xì)胞中,p53基因序列正常,HPV-16被整合到Hela細(xì)胞中,P53蛋白功能失常,因此mtHSV不誘導(dǎo)Hela細(xì)胞早期凋亡。上述結(jié)果表明:mtHSV引起Hela細(xì)胞死亡與P53無關(guān),mtHSV通過裂解細(xì)胞導(dǎo)致Hela細(xì)胞死亡。 為了進(jìn)一發(fā)現(xiàn)mtHSV進(jìn)入細(xì)胞的入口,以便透徹理解mtHSV侵染細(xì)胞的分子機(jī)制。用mtHSV去篩選噬菌體十五肽文庫,經(jīng)過四輪淘洗,篩選到一個特異性很強(qiáng)的十五肽片段。經(jīng)推斷出的氨基酸序列APFSRLLFPDFRSFV與RasGRP2具有84%的同源性。構(gòu)建了重組質(zhì)粒AcBacmid-RasGRP2和重組AcNPV vAc-RasGRP2,在昆蟲Sf9細(xì)胞中表達(dá)了RasGRP2蛋白,表達(dá)的蛋白分子量約為68KD。生物軟件"DAS"  Transmembrane Prediction server對RasGRP2蛋白的跨膜區(qū)預(yù)測結(jié)果表明:RasGRP2蛋白是跨膜蛋白,RasGRP2蛋白297-314處連續(xù)有18個疏水氨基酸(LGVHLKDLVALQLALPDW),為RasGRP2蛋白的跨膜區(qū)。 為了確定mtHSV的相互作用蛋白RasGRP2在mtHSV和HSV-1感染細(xì)胞過程中的作用,構(gòu)建了能表達(dá)RasGRP2蛋白的真核表達(dá)載體pAdtrackCMV RasGRP2 ,以gfp作(綠色熒光蛋白)作為報道基因,以確定RasGRP2蛋白在細(xì)胞中的表達(dá)。用pAdtrackCMV RasGRP2轉(zhuǎn)染mtHSV和HSV-1的非允許細(xì)胞系NIH3T3 48小時后,分別用mtHSV和HSV-1感染NIH3T3細(xì)胞。病毒吸附感染實驗結(jié)果表明:表達(dá)RasGRP2蛋白的NIH3T3細(xì)胞可以被mtHSV和HSV-1感染。Stone JC等報道:RasGRP2位于Ras信號通路上且位于Ras的上游,我們關(guān)于mtHSV的相互作用蛋白RasGRP2介導(dǎo)mtHSV和HSV-1對非允許細(xì)胞NIH3T3感染的研究結(jié)果同時證明了,RasGRP2是mtHSV和HSV-1進(jìn)入細(xì)胞的新的入口,Ras信號途徑是mtHSV進(jìn)入細(xì)胞的重要門戶。上述研究結(jié)果將為mtHSV用于癌癥治療提供理論依據(jù)。
[Abstract]:We have reported that a recombinant herpes simplex virus mtHSV , which has been constructed , inserted into an apoptotic suppressor gene ( neurotoxic gene ) icp34 . 5 , has been constructed to selectively kill tumor cells without killing normal cells . This selective dissolving effect of mtHSV is confirmed on the nude mouse model of Hep3B injected in a variety of tumor cell lines and tumors , however , its tumor - dissolving mechanism has not yet been determined .






Farassati F . et al . have shown that HSV - 1 is susceptible to infection with the oncogene v - erbB , activating the cells of the cell line of sos or ras . This means that HSV - 1 is an important member of the RTN family that is the target gene of the endoplasmic reticulum stress response . Previous studies have shown that one of the important features of the RTN family is that it is located on the endoplasmic reticulum , but the relationship between RTN3 and Ras and the relationship with mtHSV - infected Hela cells has not yet been reported .






In this study , we focus on studying the molecular mechanism of mtHSV selective dissolving , that is , the relationship between Ras and RTN3 in HeLa cells infected by mtHSV . The expression of Ras protein and Ras protein in the cytoplasm of HeLa cells were downregulated by flow cytometry and Western blot . The results showed that Ras and RTN3 could effectively kill HeLa cells . The interaction between Ras / RTN3 and Ras / RTN3 was an important factor in the invasion of HeLa cells .






The expression of P53 protein was detected by flow cytometry and Western blot . The results showed that mtHSV did not induce the early apoptosis of Hela cells .






涓轟簡榪涗竴鍙戠幇mtHSV榪涘叆緇嗚優(yōu)鐨勫叆鍙，

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