过表达Daxx对Chol：MβCD诱导巨噬细胞凋亡的影响
发布时间：2018-12-09 17:50
【摘要】：目的：观察Daxx对Chol:MβCD诱导RAW264.7细胞荷脂与凋亡的影响，为延缓动脉粥样硬化的病理改变提供理论依据。 方法：人源性Daxx基因重组质粒稳定转染RAW264.7细胞，应用RT-PCR和Western blot检测稳定转染细胞内Daxx的表达。然后用Chol:MβCD与RAW264.7细胞共同孵育48h，建立RAW264.7细胞荷脂模型，油红O染色和酶荧光法检测细胞内荷脂蓄积情况。MTT检测细胞活性，流式细胞术观察细胞凋亡。Westernblot测定ASK1，JNK，Caspase3的表达情况。 结果：转染细胞经G418筛选14天后，RT-PCR和Western blot检测转染细胞内Daxx明显高表达，表明稳定转染成功。Chol:MβCD与RAW264.7细胞共同孵育48h后，结果发现Daxx高表达组细胞胆固醇蓄积明显，并且细胞活性下调，凋亡细胞数目增加；同时Daxx转染细胞组ASK1，JNK，Caspase3蛋白表达明显增高。 结论：Daxx可介导Chol:MβCD诱导的巨噬细胞凋亡，其凋亡通路可能与Daxx上调ASK1表达，继之增加下游JNK活性，，进一步激活Caspase3有关。
[Abstract]:Aim: to observe the effects of Daxx on lipid loading and apoptosis of RAW264.7 cells induced by Chol:M 尾 CD, and to provide a theoretical basis for delaying the pathological changes of atherosclerosis. Methods: the recombinant plasmid of human Daxx gene was transfected into RAW264.7 cells stably. RT-PCR and Western blot were used to detect the expression of Daxx in the transfected cells. Then Chol:M 尾 CD was incubated with RAW264.7 cells for 48h to establish the model of RAW264.7 cell lipids. Oil red O staining and enzyme fluorescence assay were used to detect the accumulation of lipid in the cells. MTT was used to detect cell activity, flow cytometry was used to observe apoptosis. Westernblot was used to detect ASK1,. Expression of JNK,Caspase3. Results: after being screened by G418 for 14 days, the expression of Daxx in transfected cells was detected by RT-PCR and Western blot, indicating that the transfected cells were stably transfected successfully. Chol:M 尾 CD was incubated with RAW264.7 cells for 48 h. The results showed that the accumulation of cholesterol was obvious, the cell activity was down-regulated and the number of apoptotic cells was increased in the high expression group of Daxx. At the same time, the expression of ASK1,JNK,Caspase3 protein was significantly increased in Daxx transfected cells. Conclusion: Daxx can mediate the apoptosis of macrophages induced by Chol:M 尾 CD, and its apoptotic pathway may be related to the up-regulation of ASK1 expression by Daxx, the increase of downstream JNK activity and the further activation of Caspase3.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2369789
[Abstract]:Aim: to observe the effects of Daxx on lipid loading and apoptosis of RAW264.7 cells induced by Chol:M 尾 CD, and to provide a theoretical basis for delaying the pathological changes of atherosclerosis. Methods: the recombinant plasmid of human Daxx gene was transfected into RAW264.7 cells stably. RT-PCR and Western blot were used to detect the expression of Daxx in the transfected cells. Then Chol:M 尾 CD was incubated with RAW264.7 cells for 48h to establish the model of RAW264.7 cell lipids. Oil red O staining and enzyme fluorescence assay were used to detect the accumulation of lipid in the cells. MTT was used to detect cell activity, flow cytometry was used to observe apoptosis. Westernblot was used to detect ASK1,. Expression of JNK,Caspase3. Results: after being screened by G418 for 14 days, the expression of Daxx in transfected cells was detected by RT-PCR and Western blot, indicating that the transfected cells were stably transfected successfully. Chol:M 尾 CD was incubated with RAW264.7 cells for 48 h. The results showed that the accumulation of cholesterol was obvious, the cell activity was down-regulated and the number of apoptotic cells was increased in the high expression group of Daxx. At the same time, the expression of ASK1,JNK,Caspase3 protein was significantly increased in Daxx transfected cells. Conclusion: Daxx can mediate the apoptosis of macrophages induced by Chol:M 尾 CD, and its apoptotic pathway may be related to the up-regulation of ASK1 expression by Daxx, the increase of downstream JNK activity and the further activation of Caspase3.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 李明;丁健;缪泽鸿;;未折叠蛋白反应的信号转导[J];生命科学;2008年02期
本文编号：2369789
本文链接：https://www.wllwen.com/xiyixuelunwen/2369789.html
最近更新
教材专著
