Kazrin参与凋亡并与ARC和BAX相互作用

发布时间：2018-06-05 20:30

本文选题：Kazrin + ARC　；参考：《天津医科大学》2009年博士论文

【摘要】：目的：新近发现的Kazrin蛋白是在进化中非常保守的蛋白,能与桥粒、ARC等蛋白相互作用。我们发现了Kazrin的一种新的异构体,在人的脑胶质瘤细胞(U373MG细胞)株中表达,但它的确切的功能还不清楚,本研究拟运用RNAi技术,探讨Kazrin蛋白的功能,以及发挥这种功能的机制。方法：根据本实验室前期工作中鉴定的新的Kazrin的序列设计Kazrin siRNA,将Kazrin siRNA的cDNA插入载体pSilencer2.1,构建pSilencer/Kazrin-siRNA质粒,将不同剂量的pSilencer/Kazrin-siRNA转染U373MGMG细胞,24h后MTT法检测细胞活性,TUNEL法检测细胞凋亡,比色法检测caspase-3活性的变化。构建Kazrin siRNA作用位点突变但氨基酸序列不变的pcDN A3/Flag-Kazrin-m,转染U373MG细胞,筛选表达Kazrin-m的单克隆细胞株,再用pSilencer/Kazrin-siRNA质粒转染Kazrin-m表达阳性的U373MG细胞,24h后MTT法检测细胞活性。构建cDNA3/Flag-ARC、pcDNA3/Flag-Bax质粒,用免疫共沉淀和免疫荧光共定位的方法,证实Kazrin与ARC、Bax相互作用。结果： 1、pSilencer/Kazrin-siRNA能有效地抑制U373MG细胞中内源性Kazrin的表达,但不能抑制Kazrin siRNA作用位点突变但氨基酸序列不变的pcDNA3/Flag-Kazrin-m的表达。 2、RNAi技术静默Kazrin蛋白的表达后,U373MG细胞的细胞活性和细胞的凋亡与Kazrin siRNA的表达水平有关,caspase-3的活性增强。 3、pcDNA3/Flag-Kazrin-m能拯救pSilencer/Kazrin-siRNA引起的U373MGMG细胞的凋亡。 4、免疫共沉淀和免疫荧光共定位证实Kazrin与ARC、Bax互作用,Kazrin可能通过这两种蛋白而参与凋亡。结论： 1、在细胞凋亡的研究领域,我们用RNAi技术和MTT、TUNEL检测方法,首次证实了一种新的蛋白——Kazrin蛋白与细胞凋亡有关,是一种新的抗凋亡蛋白。 2、通过免疫共沉淀和免疫荧光共定位证实Kazrin与凋亡途径中两种重要的蛋白ARC、BAX相互作用,提示Kazrin可能通过这两种蛋白参与细胞凋亡。
[Abstract]:Aim: the recently discovered Kazrin protein is a very conserved protein in evolution and can interact with desmosome ARC and other proteins. We found a new isoform of Kazrin, which was expressed in human glioma cell line U373MG, but its exact function is not clear. In this study, we intend to use RNAi technique to explore the function of Kazrin protein. Methods: Kazrin siRNAs were designed according to the sequence of Kazrin which was identified in previous work in our laboratory. The cDNA of Kazrin siRNA was inserted into the vector pSilencer2.1 to construct pSilencerr Kazrin-siRNA plasmid. After different doses of pSilencer / Kazrin-siRNA were transfected into U373MGMG cells for 24 hours, the apoptosis of U373MGMG cells was detected by MTT assay and the change of caspase-3 activity by colorimetric assay. Kazrin siRNA interaction site mutation but amino acid sequence invariant pcDN A 3 / Flag-Kazrin-m. transfected U373MG cell line, screened the Kazrin-m expression monoclonal cell line, then transfected Kazrin-siRNA plasmid into Kazrin-m positive U373MG cell line for 24 hours. The construction of pcDNA3 / Flag-Bax plasmid of cDNA3 / Flag-ARCX was used to confirm the interaction between Kazrin and ARCN Bax by immunoprecipitation and immunofluorescence co-localization. Results: 1pSilencer / Kazrin-siRNA could effectively inhibit the expression of endogenous Kazrin in U373MG cells. But it could not inhibit the expression of pcDNA3 / Flag-Kazrin-m with mutation of Kazrin siRNA action site but invariant amino acid sequence. 2The cell activity and apoptosis of U373MG cells were silent after Kazrin protein expression. 3pcDNA3 / Flag-Kazrin-m could increase the activity of Kazrin siRNA. 3 pcDNA3 / Flag-Kazrin-m could not inhibit the expression of Kazrin siRNA. 3pcDNA3 / Flag-Kazrin-m could increase the activity of ppcDNA3 / Flag-Kazrin-m. Rescue pSilencerr / Kazrin-siRNA induced apoptosis in U373MGMG cells. 4. Co-immunoprecipitation and immunofluorescence co-localization confirmed that Kazrin may be involved in apoptosis through these two proteins. Conclusion: 1, in the field of apoptosis, Kazrin may be involved in apoptosis. We demonstrated for the first time that a new protein, Kazrin protein, was associated with apoptosis by RNAi and MTTT-Tunel assay. Kazrin is a new antiapoptotic protein. It is proved that Kazrin interacts with two important proteins in apoptosis pathway by immunoprecipitation and immunofluorescence co-localization, suggesting that Kazrin may participate in apoptosis through these two proteins.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

【共引文献】