發(fā)布時間：2018-03-19 21:00

  本文選題：ING4　切入點：OSM　出處：《蚌埠醫(yī)學院》2013年碩士論文　論文類型：學位論文

【摘要】：目的：研究腺病毒載體介導的雙基因共表達（Ad-ING4-OSM）對鼻咽癌CNE-2細胞體外抑制作用及機制。 方法：將基因重組腺病毒載體反復感染HEK293t細胞，經(jīng)反復擴增取病毒上清冷凍備用；利用不同劑量攜帶有綠色熒光蛋白(GFP)的空腺病毒載體（Ad-GFP）感染鼻咽癌CNE-2細胞確定最佳感染復數(shù)(MOI)；RT-PCR檢測外源性ING4和OSM基因的轉(zhuǎn)錄；PI單染流式細胞儀（FCM）檢測各實驗組鼻咽癌CNE-2細胞凋亡效應；MTT檢測各實驗組鼻咽癌CNE-2細胞生長抑制效應；Hoechst33285核染色觀察各實驗組鼻咽癌CNE-2細胞凋亡核形態(tài)學改變；RT-PCR檢測分析鼻咽癌CNE-2細胞的細胞周期和凋亡相關(guān)基因P53、P27、P21、Survivin的表達。 結(jié)果：經(jīng)反復感染HEK293t細胞后，獲得病毒滴度約為1.3~2.7×109pfu/ml；腺病毒載體（Ad-GFP）感染鼻咽癌CNE-2細胞最佳感染復數(shù)為100MOI；RT-PCR檢測確定腺病毒介導的ING4和OSM基因可在鼻咽癌CNE-2細胞中轉(zhuǎn)錄，且在對照組和空白組未檢測出內(nèi)源性ING4和OSM基因轉(zhuǎn)錄；PI單染流式細胞儀（FCM）檢測鼻咽癌CNE-2細胞凋亡效應，雙基因共表達（Ad-ING4-OSM）組明顯明顯高于單基因Ad-ING4組和Ad-OSM組（P＜0.05），單基因組之間無明顯差異，但優(yōu)于空白組和空病毒組(P＜0.05)，空白組和空病毒組間無統(tǒng)計學意義(P0.05)；MTT檢測發(fā)現(xiàn)雙基因共表達（Ad-ING4-OSM）組鼻咽癌CNE-2細胞生長抑制效應明顯優(yōu)于單基因ING4組和OSM組（P＜0.05），單基因組之間無明顯差異(p0.05)，但優(yōu)于空白組和空病毒組(P＜0.05)；Hoechst33285核染色觀察到雙基因共表達（Ad-ING4-OSM）組鼻咽癌CNE-2細胞中細胞核和細胞質(zhì)內(nèi)可見濃染致密的顆粒塊狀藍色熒光及明顯核形態(tài)變化；RT-PCR檢測分析顯示Ad-ING4-OSM雙基因共表達可明顯上調(diào)P53、P27、P21和下調(diào)Survivin等細胞周期和凋亡相關(guān)基因表達。 結(jié)論：1、與單基因相比AD-ING4-OSM雙基因共表達可在體外顯著抑制鼻咽癌CNE-2細胞生長，促進凋亡，具有抑瘤增效作用，，其作用機制可能是上調(diào)了P53、P27、P21和下調(diào)Survivin等細胞周期和凋亡相關(guān)基因的表達。 2、腺病毒可作為鼻咽癌基因治療的安全有效的載體。
[Abstract]:Aim: to study the inhibitory effect of adenovirus vector mediated double gene coexpression of Ad-ING4-OSMon nasopharyngeal carcinoma (NPC) CNE-2 cells in vitro and its mechanism. Methods: the recombinant adenovirus vector was repeatedly infected with HEK293t cells. CNE-2 cells infected with nasopharyngeal carcinoma (NPC) infected by empty adenovirus vector Ad-GFP with different doses of green fluorescent protein (GFP) were used to determine the best infection number of CNE-2 cells. RT-PCR was used to detect the transcription of exogenous ING4 and OSM gene by flow cytometry (FCM) to detect the nasal nose of each experimental group. Apoptosis effect of CNE-2 cells in pharyngeal carcinomas the growth inhibition effect of CNE-2 cells in each experimental group was observed by Hoechst33285 nuclear staining the morphological changes of apoptotic nuclei of nasopharyngeal carcinoma CNE-2 cells in each experimental group were observed and the cell cycle and regulation of CNE-2 cells in nasopharyngeal carcinoma were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of survivin gene P53, P27, P 27 and P 21 1. Results: after repeated infection of HEK293t cells, the virus titer was about 1.32.7 脳 109pfur / ml, and the best number of CNE-2 cells infected by adenovirus vector was 100MOI RT-PCR. The results showed that the ING4 and OSM genes mediated by adenovirus could be transcripted in CNE-2 cells of nasopharyngeal carcinoma (NPC). The apoptotic effect of nasopharyngeal carcinoma (NPC) CNE-2 cells was detected by flow cytometry (FCM), and no endogenous ING4 and OSM gene transcription Pi single staining flow cytometry were detected in the control group and blank group. The co-expression of two genes in Ad-ING4-OSM group was significantly higher than that in single gene Ad-ING4 group and Ad-OSM group (P < 0.05). But it was better than that of blank group and empty virus group (P < 0.05). There was no significant difference between blank group and empty virus group (P < 0.05). It was found that the growth inhibition effect of CNE-2 cells in the group of double gene co-expression of Ad-ING4-OSMM was significantly better than that in the group of single gene ING4 and OSM (P < 0.05), and the single genome was significantly better than that in the group of single gene ING4 and group OSM (P < 0.05). There was no significant difference between the two groups, but it was better than that of blank group and empty virus group (P < 0.05) Hoechst33285 nuclear staining showed that dense and dense granular blue fluorescence and obvious nuclear morphosis could be observed in the nucleus and cytoplasm of nasopharyngeal carcinoma (NPC) CNE-2 cells with double gene coexpression (Ad-ING4-OSM). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the co-expression of Ad-ING4-OSM double genes could significantly up-regulate the expression of cell cycle and apoptosis-related genes such as P53, P27, P21, and down-regulate the expression of Survivin and other apoptosis-related genes. Conclusion compared with single gene, AD-ING4-OSM double gene coexpression can significantly inhibit the growth of nasopharyngeal carcinoma (NPC) CNE-2 cells in vitro, promote apoptosis, and have a synergistic effect on tumor growth. The mechanism may be to up-regulate the expression of cell cycle and apoptosis-related genes such as P53, P27, P21 and Survivin. 2, adenovirus can be used as a safe and effective vector for gene therapy of nasopharyngeal carcinoma.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R739.63

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