本文選題：卡波濟(jì)肉瘤相關(guān)皰疹病毒(KSHV) + 腫瘤轉(zhuǎn)化基因K12��； 參考：《南京醫(yī)科大學(xué)》2005年碩士論文

【摘要】：本研究旨在探索胞嘧啶脫氨酶(cytosine deaminase,CD)自殺基因聯(lián)合5-氟胞嘧啶(5-fluorocytosine,5-FC)(cD/5-FC系統(tǒng))對(duì)卡波濟(jì)肉瘤相關(guān)皰疹病毒(Kaposi's sarcoma-associated herpesvirus,KsHV)腫瘤轉(zhuǎn)化基因K12誘導(dǎo)NIH3T3轉(zhuǎn)化細(xì)胞的殺傷作用。首先分別構(gòu)建含KSHV K12基因和CD自殺基因Fcy∶∶Fur的重組逆轉(zhuǎn)錄病毒表達(dá)質(zhì)粒LZRS-K12和pLXSN-FF,并將重組質(zhì)粒LZRS-K12單獨(dú)或與pLXSN-FF一起穩(wěn)定轉(zhuǎn)染MH3T3細(xì)胞,獲得穩(wěn)定表達(dá)卡波濟(jì)蛋白(kaposin)的N/K12和N/K12/FF細(xì)胞。然后通過(guò)普通倒置顯微鏡、透射電子顯微鏡觀察和軟瓊脂集落形成實(shí)驗(yàn)以及裸鼠成瘤實(shí)驗(yàn)驗(yàn)證K12基因能夠體外誘導(dǎo)細(xì)胞轉(zhuǎn)化、體內(nèi)誘導(dǎo)腫瘤的形成。結(jié)果顯示,K12基因轉(zhuǎn)染后的NIH3T3細(xì)胞與對(duì)照細(xì)胞相比其生長(zhǎng)特性和形態(tài)結(jié)構(gòu)均發(fā)生明顯改變,表現(xiàn)為失去生長(zhǎng)接觸抑制而呈多層生長(zhǎng)、出現(xiàn)異型核及生長(zhǎng)依賴(lài)性降低而能夠在軟瓊脂培養(yǎng)基上形成細(xì)胞集落。動(dòng)物實(shí)驗(yàn)結(jié)果表明,K12轉(zhuǎn)化的NIH3T3細(xì)胞接種至裸鼠頸背部皮下2～3w后均可在接種部位形成腫瘤。瘤組織經(jīng)石蠟切片后HE染色,鏡下可見(jiàn)血管豐富,瘤細(xì)胞呈梭形,排列紊亂,核分裂旺盛,伴有異型核和病理性核分裂。5-FC使用后,采用MTT法、流式細(xì)胞儀和免疫組化(IHC)染色等方法分別檢測(cè)細(xì)胞存活率、凋亡率及凋亡相關(guān)蛋白的表達(dá)。結(jié)果顯示,隨著5-FC作用濃度的提高,N/K12/FF細(xì)胞存活率明顯下降。流式細(xì)胞儀
[Abstract]:The aim of this study was to investigate the killing effect of cytosine deaminase (CDCD) suicide gene combined with 5-fluorocytosine 5-fluorocytosine (5-fluorocytosine 5-FC system) on K12 induced NIH3T3 transformation cells induced by Kaposiella sarcoma-associated herpesvirusKsHV. Firstly, the recombinant retroviral expression plasmids LZRS-K12 and pLXSN-FFF containing KSHV K12 gene and CD suicide gene Fcy::Fur were constructed, and the recombinant LZRS-K12 was stably transfected into MH3T3 cells either alone or together with pLXSN-FF to obtain N/K12 and N/K12/FF cells stably expressing kaposin. Then the normal inverted microscope, transmission electron microscope, soft Agar colony formation test and nude mice tumorigenesis experiment proved that K12 gene can induce cell transformation in vitro and tumor formation in vivo. The results showed that the growth characteristics and morphological structure of the NIH3T3 cells transfected with K12 gene were obviously changed compared with the control cells. The abnormal nuclei and growth dependence were decreased, and cell colonies could be formed on soft Agar medium. The results of animal experiments showed that the transformed NIH3T3 cells could form tumor at the inoculation site after inoculation to the subcutaneous of the neck and back of nude mice for 3 weeks. The tumor tissue was stained with HE after paraffin section. The tumor cells were fusiform, disordered, mitotic and accompanied with abnormal nucleus and pathological mitosis. MTT method was used. The survival rate, apoptosis rate and the expression of apoptosis-related protein were detected by flow cytometry and immunohistochemical staining. The results showed that the survival rate of N- / K12 / FF cells decreased with the increase of 5-FC concentration. Flow cytometry
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 朱建中,盧春;人皰疹病毒8型K12基因在大腸桿菌中的克隆和表達(dá)[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2002年04期

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本文編號(hào)：1942042