紫外线诱导斜纹夜蛾细胞SL-1凋亡的研究

发布时间：2018-03-16 15:39

本文选题：斜纹夜蛾细胞系　切入点：细胞凋亡　出处：《华中师范大学》2006年硕士论文　论文类型：学位论文

【摘要】：由于近年来大气中臭氧层的破坏,辐射到地面的UV增多,故UV对人类的健康威胁越来越大,过量的紫外线照射以成为危害人类健康的重要环境因素之一。研究发现UV不仅能引发日光性皮炎、皮肤老化、免疫抑制、白内障和皮肤癌等疾病,还能诱导细胞发生凋亡,从而消除受损的细胞避免癌症的发生。UV诱导凋亡的机制具有细胞特异性,对于其诱导细胞凋亡机制的研究成为研究的重点。但在昆虫细胞中的研究比较少。本研究以斜纹夜蛾细胞SL-1细胞为实验材料,对紫外线诱导SL-1细胞的凋亡进行了初步的研究。完成的工作包括以下几个方面: (1)UVC诱导斜纹夜蛾SL-1细胞凋亡 UVC处理斜纹夜蛾SL-1细胞后,可诱导SL-1细胞产生典型细胞凋亡。光镜下可见细胞膜表面突出或形成小泡,细胞破裂成凋亡小体。UVC处理24h后,细胞几乎全部破裂成凋亡小体。DAPI荧光染色显示感染细胞核逐渐形变,直至破裂成小块而被凋亡小体包裹。SL-1细胞处理后DNA琼脂糖凝胶电泳成典型凋亡细胞DNA梯形电泳谱带。PI染色法检测其凋亡率,处理后8hr的凋亡率为47.08%。 (2)细胞内线粒体的变化流式细胞仪对线粒体膜电位进行检测,结果表明,细胞经罗丹明(Rho-123)染色后在流式细胞仪上检测,处理1小时后即出现荧光强度下降,说明此时膜电位开始下降,并且细胞膜电位逐渐下降且呈时间依赖性。Western-blotting检测调控蛋白Cyto C在细胞质和线粒体中的含量,结果显示SL-1细胞在处理后4小时线粒体中的细胞色素C被释放到了细胞质中,这与很多在哺乳动物细胞中所做的结果相同。 (3)Caspase 3活性的检测对caspase-3的活性检测,首先用caspase-3特异性序列人工合成荧光底物AC-DEVD-AFC与所提取的细胞裂解液成份反应,置荧光分光光度计上检测,结果显示SfaMNPV感染4小时后即检测到caspase-3的活性,且其活性呈时间依赖性增强。这提示了cytc在UVC诱导昆虫细胞凋亡中可能起重要作用,通过cytc的释放激活下游的效应酶。
[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.
【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363;Q967

【引证文献】