本文選題：斜紋夜蛾細(xì)胞系　切入點(diǎn)：細(xì)胞凋亡　出處：《華中師范大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

【摘要】：由于近年來大氣中臭氧層的破壞,輻射到地面的UV增多,故UV對人類的健康威脅越來越大,過量的紫外線照射以成為危害人類健康的重要環(huán)境因素之一。研究發(fā)現(xiàn)UV不僅能引發(fā)日光性皮炎、皮膚老化、免疫抑制、白內(nèi)障和皮膚癌等疾病,還能誘導(dǎo)細(xì)胞發(fā)生凋亡,從而消除受損的細(xì)胞避免癌癥的發(fā)生。UV誘導(dǎo)凋亡的機(jī)制具有細(xì)胞特異性,對于其誘導(dǎo)細(xì)胞凋亡機(jī)制的研究成為研究的重點(diǎn)。但在昆蟲細(xì)胞中的研究比較少。本研究以斜紋夜蛾細(xì)胞SL-1細(xì)胞為實(shí)驗(yàn)材料,對紫外線誘導(dǎo)SL-1細(xì)胞的凋亡進(jìn)行了初步的研究。完成的工作包括以下幾個方面: (1)UVC誘導(dǎo)斜紋夜蛾SL-1細(xì)胞凋亡 UVC處理斜紋夜蛾SL-1細(xì)胞后,可誘導(dǎo)SL-1細(xì)胞產(chǎn)生典型細(xì)胞凋亡。光鏡下可見細(xì)胞膜表面突出或形成小泡,細(xì)胞破裂成凋亡小體。UVC處理24h后,細(xì)胞幾乎全部破裂成凋亡小體。DAPI熒光染色顯示感染細(xì)胞核逐漸形變,直至破裂成小塊而被凋亡小體包裹。SL-1細(xì)胞處理后DNA瓊脂糖凝膠電泳成典型凋亡細(xì)胞DNA梯形電泳譜帶。PI染色法檢測其凋亡率,處理后8hr的凋亡率為47.08%。 (2)細(xì)胞內(nèi)線粒體的變化 流式細(xì)胞儀對線粒體膜電位進(jìn)行檢測,結(jié)果表明,細(xì)胞經(jīng)羅丹明(Rho-123)染色后在流式細(xì)胞儀上檢測,處理1小時后即出現(xiàn)熒光強(qiáng)度下降,說明此時膜電位開始下降,并且細(xì)胞膜電位逐漸下降且呈時間依賴性。Western-blotting檢測調(diào)控蛋白Cyto C在細(xì)胞質(zhì)和線粒體中的含量,結(jié)果顯示SL-1細(xì)胞在處理后4小時線粒體中的細(xì)胞色素C被釋放到了細(xì)胞質(zhì)中,這與很多在哺乳動物細(xì)胞中所做的結(jié)果相同。 (3)Caspase 3活性的檢測 對caspase-3的活性檢測,首先用caspase-3特異性序列人工合成熒光底物AC-DEVD-AFC與所提取的細(xì)胞裂解液成份反應(yīng),置熒光分光光度計上檢測,結(jié)果顯示SfaMNPV感染4小時后即檢測到caspase-3的活性,且其活性呈時間依賴性增強(qiáng)。這提示了cytc在UVC誘導(dǎo)昆蟲細(xì)胞凋亡中可能起重要作用,通過cytc的釋放激活下游的效應(yīng)酶。
[Abstract]:Due to the destruction of the ozone layer in the atmosphere in recent years and the increase of UV radiation to the ground, UV poses an increasing threat to human health. Excessive ultraviolet radiation has become one of the important environmental factors harmful to human health. Studies have found that UV can not only cause sunburn, skin aging, immunosuppression, cataract and skin cancer, but also induce apoptosis of cells. In order to eliminate the damage of cells to avoid the occurrence of cancer. UV induced apoptosis mechanism has cell specificity, The research on the mechanism of apoptosis induced by Spodoptera litura has become the focus of research, but there are few studies in insect cells. In this study, SL-1 cells of Spodoptera litura were used as experimental materials. The apoptosis of SL-1 cells induced by ultraviolet radiation was studied in this paper. The work accomplished includes the following aspects:. Apoptosis of Spodoptera litura SL-1 cells induced by UVC. After treated with UVC on SL-1 cells of Spodoptera litura, typical apoptosis of SL-1 cells was induced. Under light microscope, the surface of cell membrane protruded or vesicles were formed, and the cells ruptured into apoptotic bodies for 24 hours. Almost all the cells ruptured into apoptotic bodies. DAPI fluorescence staining showed that the infected nuclei gradually deformed. The apoptotic rate of typical apoptotic cells was detected by DNA agarose gel electrophoresis (DNA agarose gel electrophoresis) with DNA ladder electrophoresis band. Pi staining method was used to detect the apoptotic rate. The apoptotic rate was 47.08 after 8hr treatment. Changes of mitochondria in cells. The mitochondrial membrane potential was detected by flow cytometry. The results showed that the fluorescence intensity decreased after one hour treatment, which indicated that the membrane potential began to decrease after the cells were stained with Rho-123). Furthermore, the cell membrane potential decreased gradually and was time-dependent. Western-blotting was used to detect the content of regulatory protein Cyto C in cytoplasm and mitochondria. The results showed that cytochrome C in mitochondria of SL-1 cells was released into the cytoplasm 4 hours after treatment. This is the same as many results in mammalian cells. Detection of Caspase 3 activity in Caspase-3. The activity of caspase-3 was detected by caspase-3 specific sequence synthetic fluorescent substrate AC-DEVD-AFC and the extracted cell lysate component. The results showed that the activity of caspase-3 was detected 4 hours after SfaMNPV infection. This suggests that cytc may play an important role in UVC induced insect cell apoptosis and activate downstream effector enzymes through the release of cytc.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R363;Q967

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 廖紹裕;徐漢虹;李文歐;;UVA對斜紋夜蛾SL細(xì)胞損傷研究[J];激光生物學(xué)報;2008年06期

相關(guān)博士學(xué)位論文 前1條

1 鄭桂玲;鱗翅目昆蟲胚胎細(xì)胞系的建立和高蛋白表達(dá)細(xì)胞克隆株的研究[D];山東農(nóng)業(yè)大學(xué);2010年

相關(guān)碩士學(xué)位論文 前2條

1 陳默;紫外線誘導(dǎo)家蠶卵巢細(xì)胞（BmN-SWU1）凋亡的研究[D];西南大學(xué);2008年

2 趙丹;滅多威誘導(dǎo)MDEC-07114細(xì)胞凋亡的實(shí)驗(yàn)研究[D];遵義醫(yī)學(xué)院;2012年

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本文編號：1620560