The effect of cytokines on programd cell death (apoptosis) in ovary.

细胞凋亡是细胞死亡的一种生理模式，在组织的组织和更替或卵泡闭锁等过程中观察到。细胞凋亡的一个基本生化机制是染色质结构的改变，通常与DNA由于内源性内切酶的激活而断裂成约n×185个碱基对的寡核小体片段有关。经凝胶电泳法和溴化乙锭染色后可见明显的DNA梯形条带。但溴化乙锭染色法需要大量的DNA。因此，另一位工作人员报道了放射自显影的方法，利用末端脱氧转移酶(TDT)在DNA的3‘端标记32P-ddATP。但减少放射性同位素方法的使用提供了安全和环境效益，简化了法规遵从性，并降低了存储和处置的成本。我们尝试建立微量非放射性方法来定性和定量分析…更多的凋亡性DNA片段化。该程序利用TDT将地高辛配基(Dig)-ddUTP均匀地添加到DNA片段的3‘端。凝胶电泳后，根据Southern blotting将标记的DNA转移到尼龙膜上。然后将膜与抗地高辛碱性磷酸酶偶联物孵育。清洗后，用CDP-Star(化学发光底物)孵育5min，密封在显影文件夹中。孵育3或5小时后，将X光片放在显影文件夹上，曝光30秒或2分钟。这种方法只需要200 ng的细胞DNA，并且比广泛使用的溴化乙锭染色方法提高了至少20倍的灵敏度。利用图像分析软件对X线片进行分析，可以定量地确定低分子DNA组分中DIG标记的总量，并用来判断DNA片段的凋亡程度。该方法对于分析微量组织和培养细胞中的细胞凋亡应该是有价值的。较少

Apoptosis is a physiological mode of cell death, observed during procceses such as tissue organization and turnover, or follicle atresia. A fundamental biochemical mechanism of apoptosis is a change in chromatin structure, often associated with fragmentation of DNA into oligonucleosomal fragment of about n x 185 base pairs due to the activation of an endogenous endonuclease. These apoptotic DNA fragments can be visualized as a distinct ladder of DNA band following gel electrophoresis and ethidium bromide staining. But large amount of DNA required for ethidium bromide staining method. So another workers reported autoradiographic method, the DNA is radiolabeled with 32P-ddATP on 3'-ends using terminal deoxy transferase enzyme (TdT). But reduced radioisotope method use offers safety and environmental benefits, simplifies regulatory compliance, and reduce costs for storage and disposal. We tried to establish microscale non-radioactive method for the qualitative and quantitative analysis of … More apoptotic DNA fragmentation. This procedure utilized TdT to uniformly add digoxigenin (DIG) -ddUTP to the 3'-end of DNA fragments. Following gel electrophoresis, labeled DNA was transferred to nylon membrane according to the southern blotting. The membrane was then incubated with the anti-DIG alkaline phosphatase conjugate. After washing the membrane was incubate with CDP-Star (chemiluminescence substrate) for 5 min, sealed in development folder. After 3 or 5 hours incubation, the X ray film was placed on the development folder and exposed 30 sec or 2 min. This method required as little as 200ng of cellular DNA and increase sensitivity of apoptotic DNA detection by at least 20 fold over the widely used ethidium bromide staining method. The total amount of DIG label incorporated into low molecular weight DNA fraction can be quantified and used to estimate the degree of apoptotic DNA fragment by analyzing the X ray film with image analysis software. The procedure should prove valuable for the analysis of apoptosis in minute quantities of tissues and cultured cells. Less