AD-ING4-OSM对鼻咽癌CNE-2细胞体外抑制作用实验研究

发布时间：2018-03-19 21:00

本文选题：ING4　切入点：OSM　出处：《蚌埠医学院》2013年硕士论文　论文类型：学位论文

【摘要】：目的：研究腺病毒载体介导的双基因共表达（Ad-ING4-OSM）对鼻咽癌CNE-2细胞体外抑制作用及机制。方法：将基因重组腺病毒载体反复感染HEK293t细胞，经反复扩增取病毒上清冷冻备用；利用不同剂量携带有绿色荧光蛋白(GFP)的空腺病毒载体（Ad-GFP）感染鼻咽癌CNE-2细胞确定最佳感染复数(MOI)；RT-PCR检测外源性ING4和OSM基因的转录；PI单染流式细胞仪（FCM）检测各实验组鼻咽癌CNE-2细胞凋亡效应；MTT检测各实验组鼻咽癌CNE-2细胞生长抑制效应；Hoechst33285核染色观察各实验组鼻咽癌CNE-2细胞凋亡核形态学改变；RT-PCR检测分析鼻咽癌CNE-2细胞的细胞周期和凋亡相关基因P53、P27、P21、Survivin的表达。结果：经反复感染HEK293t细胞后，获得病毒滴度约为1.3~2.7×109pfu/ml；腺病毒载体（Ad-GFP）感染鼻咽癌CNE-2细胞最佳感染复数为100MOI；RT-PCR检测确定腺病毒介导的ING4和OSM基因可在鼻咽癌CNE-2细胞中转录，且在对照组和空白组未检测出内源性ING4和OSM基因转录；PI单染流式细胞仪（FCM）检测鼻咽癌CNE-2细胞凋亡效应，双基因共表达（Ad-ING4-OSM）组明显明显高于单基因Ad-ING4组和Ad-OSM组（P＜0.05），单基因组之间无明显差异，但优于空白组和空病毒组(P＜0.05)，空白组和空病毒组间无统计学意义(P0.05)；MTT检测发现双基因共表达（Ad-ING4-OSM）组鼻咽癌CNE-2细胞生长抑制效应明显优于单基因ING4组和OSM组（P＜0.05），单基因组之间无明显差异(p0.05)，但优于空白组和空病毒组(P＜0.05)；Hoechst33285核染色观察到双基因共表达（Ad-ING4-OSM）组鼻咽癌CNE-2细胞中细胞核和细胞质内可见浓染致密的颗粒块状蓝色荧光及明显核形态变化；RT-PCR检测分析显示Ad-ING4-OSM双基因共表达可明显上调P53、P27、P21和下调Survivin等细胞周期和凋亡相关基因表达。结论：1、与单基因相比AD-ING4-OSM双基因共表达可在体外显著抑制鼻咽癌CNE-2细胞生长，促进凋亡，具有抑瘤增效作用，，其作用机制可能是上调了P53、P27、P21和下调Survivin等细胞周期和凋亡相关基因的表达。 2、腺病毒可作为鼻咽癌基因治疗的安全有效的载体。
[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.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.63

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