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

细胞因子对卵巢程序性细胞死亡（细胞凋亡）的影响。

• 批准号: 07671761
• 负责人: FURUTA Itsuko
• 金额: $ 1.34万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07671761/
• 关键词: APOTOSIS; CYTOKINE; OVARY

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

细胞凋亡是细胞死亡的一种生理模式，发生于组织的形成、更新或卵泡闭锁等过程中。细胞凋亡的基本生物化学机制是染色质结构的变化，通常与由于内源性内切核酸酶的活化而将DNA片段化成约n × 185个碱基对的寡核小体片段有关。这些凋亡的DNA片段在凝胶电泳和溴化乙锭染色后可以被观察为明显的DNA条带梯状带。但溴化乙锭染色法需要大量的DNA。因此，另一位工作者报道了放射自显影法，用末端脱氧转移酶（TdT）在DNA的3 ′-末端标记32 P-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，并降低了储存和处置成本。本文尝试建立微量非放射性分析方法， ...更多信息 凋亡DNA片段化。该方法利用TdT将地高辛（DIG）-ddUTP均匀地添加到DNA片段的3 '末端。凝胶电泳后，根据Southern印迹法将标记的DNA转移到尼龙膜上。然后将膜与抗DIG碱性磷酸酶缀合物一起孵育。洗涤后，将膜与CDP-Star（化学发光底物）孵育5分钟，密封在显影夹中。孵育3或5小时后，将X射线胶片放在显影夹上，曝光30秒或2分钟。这种方法所需的细胞DNA少至200 ng，并增加了凋亡DNA检测的灵敏度至少20倍，比广泛使用的溴化乙锭染色法。掺入低分子量DNA部分的DIG标记的总量可以通过用图像分析软件分析X射线照片来定量并用于估计凋亡DNA片段的程度。该程序应被证明是有价值的细胞凋亡的分析在微量的组织和培养细胞。少

项目成果

桜木範明: "Turner症候群と卵胞閉鎖の機序" 臨床婦人科産科. 49(11). 1504-1508 (1995)

Noriaki Sakuragi：“特纳综合征和卵泡闭锁的机制”《临床妇产科》49（11）1504-1508（1995）。

N.Sakuragi., I.Furuta., S.Fujimoto.: "The mechanism of follicular atresia and Turner syndrome." Rinsho Fujinka Sanka. 49 (11). 1504-1508 (1995)

N.Sakuragi.、I.Furuta.、S.Fujimoto.：“卵泡闭锁和特纳综合征的机制。”

桜木範明: "Turner症候群と卵胞閉鎖の機序" 臨床婦人科産科. 48(11). 1504-1508 (1995)

Noriaki Sakuragi：“特纳综合征和卵泡闭锁的机制”《临床妇产科》48（11）1504-1508（1995）。