TRAF1引起线粒体聚集的抗凋亡生物学效应及其机制研究

发布时间：2018-06-14 12:30

本文选题：TRAF1 + Mfn1　；参考：《华中科技大学》2009年硕士论文

【摘要】：TRAF1属于TNF受体相关因子(TNFR-associated factor TRAF)家族。TRAF家族是一类胞内衔接蛋白,能介导TNF受体和IL-1受体超家族的信号转导引起NF-?B和JNK等的活化,从而对细胞的生存与死亡产生影响。TRAF1不具有TRAF家族其他成员所共有的环指和与之相邻的锌指结构,其可募集一些TNF受体超家族成员,包括TNFR2,CD30,CD27,TRANCE-R等。据报道,TRAF1可抵抗/抑制细胞凋亡:在转基因动物中过表达TRAF1,可抑制抗原诱导的CD8+细胞的凋亡;TRAF1可通过募集cIAP以执行其抗凋亡作用。但也有不同研究结果表明TRAF1可促进细胞凋亡。本室前期工作首次证实高表达TRAF1能与线粒体共定位并引起线粒体聚集,这一现象具有普遍性。而且转染TRAF1的HepG2细胞,其NF-κB活性增强,能抵抗TM-TNF-α的杀伤。转染TRAF1能上调线粒体融合蛋白Mfn2的mRNA水平,提示TRAF1引起的线粒体聚集可能与线粒体融合分子相关。本课题选取Hela细胞作为研究对象,借助Realtime-PCR观察高表达TRAF1线粒体融合蛋白Mfn1转录水平的变化;以化疗药物阿霉素诱导Hela细胞凋亡为模型,观察高表达TRAF1对于阿霉素杀伤作用的影响,并通过检测线粒体膜电位的变化、caspase-9的活化以及I?B的降解,进一步深入探讨TRAF1导致线粒体聚集的生物学效应及其分子机制。一.转染TRAF1使线粒体融合蛋白Mfn1的mRNA水平增高以Realtime-PCR分别观察Hela细胞各实验组:未转染组、转染空载体pDsRed组和转染pDsRed-TRAF1重组质粒组中线粒体融合蛋白Mfn1的mRNA水平的变化。转染TRAF1后,Mfn1分子的mRNA水平明显增高,为未转染组的2.18倍(P0.01),为转染空载体组的2.48倍(P0.01)。提示TRAF1可能通过上调Mfn1的表达而导致线粒体聚集。二.转染TRAF1可抵抗化疗药物阿霉素的杀伤效应以Hela细胞为靶细胞,设未转染组、转染空载体组和转染TRAF1组,以1μM阿霉素分别刺激各组细胞48小时,观察TRAF1对阿霉素杀伤Hela细胞的影响。MTT结果显示:转染TRAF1组表现为对阿霉素杀伤作用的抵抗,其杀伤率与未转染组和转染空载组相比,分别下降了18%和17.21% (P0.05)。提示转染TRAF1可抵抗阿霉素对Hela细胞的杀伤效应。三.转染TRAF1可使线粒体发生聚集以阿霉素未刺激/刺激的Hela细胞为靶细胞,分别设未转染组,转染空载组和转染TRAF1组,1μM阿霉素刺激各组细胞24小时,在荧光显微镜油镜下观察阿霉素刺激前后TRAF1转染所导致的线粒体形态变化。阿霉素未刺激时,3组细胞线粒体结构均呈紧密的管网状结构细胞,而TRAF1转染组与未转染组和转染空载组相比,表现为线粒体聚集。阿霉素刺激后,未转染组与转染空载组线粒体结构松散紊乱,并有片段化的线粒体出现,而转染TRAF1组仍能保持聚集状态。四.转染TRAF1可增强线粒体膜电位稳定性以阿霉素未刺激/刺激的Hela细胞为靶细胞,分别设未转染组,转染空载组和转染TRAF1组,1μM阿霉素刺激各组细胞24小时,观察阿霉素刺激前后各组细胞线粒体膜电位的稳定性。结果显示:转染TRAF1组在阿霉素刺激前后,膜电位的稳定性均比未转染组和转染空载组增强。阿霉素刺激之前,TRAF1组凋亡细胞与对照组和空载组相比,分别下降了19.8%和21.8%;刺激后,TRAF1组凋亡细胞与对照组和空载组相比,分别下降了16.8%和14%。提示TRAF1能维持线粒体膜电位的稳定性。五.转染TRAF1可减少caspase-9的活化以阿霉素未刺激/刺激的Hela细胞为靶细胞,分别设未转染组,转染空载组和转染TRAF1组,1μM阿霉素刺激各组细胞24小时,通过Western blot观察阿霉素刺激前后各组细胞胞浆蛋白中caspase-9的活化。无论阿霉素刺激前后,转染TRAF1均可使caspase-9的活化明显减少。结合上述膜电位的结果,提示TRAF1可能通过抑制线粒体凋亡途径,提高线粒体膜电位的稳定性,减少caspase-9的活化而发挥其抵抗杀伤的作用。六.转染TRAF1可增加IκB的降解以阿霉素未刺激/刺激的Hela细胞为靶细胞,分别设未转染组,转染空载组和转染TRAF1组,1μM阿霉素刺激各组细胞24小时,观察阿霉素刺激前后各组细胞胞浆蛋白中IκB的降解。转染TRAF1后,IκB的降解增加,提示TRAF1可活化NF-κB,从而抵抗凋亡。结论:TRAF1可能通过增加线粒体融合蛋白Mfn1的表达而引起线粒体聚集,此种聚集所导致的生物学效应为抵抗细胞凋亡,表现为转染TRAF1可抵抗化疗药物阿霉素对Hela细胞的杀伤。其抵抗杀伤(凋亡)的机制之一可能是通过抑制凋亡线粒体途径:提高线粒体膜电位的稳定性、减少caspase-9的活化。另一方面,也可能通过NF-κB的活化而促进细胞生存。因此,TRAF1通过影响细胞的生存和死亡两方面的共同作用,最终发挥抵抗细胞凋亡的效应。
[Abstract]:TRAF1 belongs to the TNF receptor related factor (TNFR-associated factor TRAF) family.TRAF family, a class of intracellular cohesive proteins, which mediate the activation of NF-, B and JNK, which mediate the signal transduction of the TNF receptor and IL-1 receptor superfamily, and thus influence the survival and death of the cells. The adjacent zinc finger structure, which can raise a number of TNF receptor superfamily members, including TNFR2, CD30, CD27, TRANCE-R and so on. It is reported that TRAF1 can resist / inhibit apoptosis: the expression of TRAF1 in transgenic animals can inhibit the apoptosis of antigen induced CD8+ cells; TRAF1 can pass through cIAP to perform its anti apoptosis effect. But there are different studies. The results showed that TRAF1 could promote cell apoptosis.
It was first confirmed in the previous work that the high expression of TRAF1 can co localize with mitochondria and cause mitochondrial aggregation. This phenomenon is universal. Moreover, the NF- kappa B activity of HepG2 cells transfected with TRAF1 can resist the killing of TM-TNF- alpha. The transfection of TRAF1 can increase the mRNA level of mitochondrial fusion protein Mfn2, suggesting the mitochondrial aggregation caused by TRAF1. It may be associated with mitochondrial fusion molecules.
In this study, Hela cells were selected as the research object and Realtime-PCR was used to observe the changes in the transcriptional level of the mitochondrial fusion protein Mfn1 with high expression of TRAF1. The effect of high expression of TRAF1 on the killing effect of adriamycin was observed with adriamycin induced apoptosis of Hela cells, and the changes of mitochondrial membrane potential and caspase-9 were detected. Activation and degradation of I? B further explored the biological effects and molecular mechanisms of TRAF1 induced mitochondrial aggregation.
1. Transfection of TRAF1 increased the level of mRNA of mitochondrial fusion protein Mfn1.
The changes in the mRNA level of the mitochondrial fusion protein Mfn1 in the empty carrier pDsRed group and the transfected pDsRed-TRAF1 recombinant plasmid group were observed by Realtime-PCR, respectively. The mRNA level of Mfn1 molecules increased significantly after transfection of TRAF1, which was 2.18 times (P0.01) of the untransfected group and 2.48 times (P0.01) in the transfected space carrier group. It is suggested that TRAF1 may lead to mitochondrial aggregation through up regulation of Mfn1 expression.
Two. Transfection of TRAF1 can resist the killing effect of doxorubicin for chemotherapeutic drugs.
Hela cells were used as target cells, untransfected groups were set, transfected to empty carrier group and transfected TRAF1 group. The effects of TRAF1 on adriamycin killing Hela cells respectively for 48 hours were observed, and the effect of.MTT on adriamycin killing Hela cells showed that the transfection of TRAF1 group was against the killing effect of adriamycin, and its killing rate was compared with that of untransfected group and transfected empty group. It decreased by 18% and 17.21% (P0.05) respectively, suggesting that TRAF1 transfection can resist the killing effect of adriamycin on Hela cells.
Three. Transfection of TRAF1 can induce aggregation of mitochondria
The unstimulated / unstimulated Hela cells of adriamycin were used as target cells, and the untransfected group was set respectively. The cells were transfected to the unloaded group and transfected to the TRAF1 group. The cells of 1 mu M adriamycin stimulated each group for 24 hours. The morphologic changes of mitochondria caused by TRAF1 transfection before and after the stimulation of doxorubicin were observed under the fluorescence microscope oil microscope. The structure of the mitochondria of the 3 groups was all in the absence of adriamycin. Compared with the untransfected group and the unloaded group, the TRAF1 transfected group showed mitochondrial aggregation. After adriamycin stimulation, the mitochondria structure of the untransfected group and the transfected empty group was loosely disturbed, and the fragment of mitochondria appeared, while the transfected TRAF1 group still kept the aggregation state.
Four. Transfection of TRAF1 enhances mitochondrial membrane potential stability.
The untransfected Hela cells were set with adriamycin as the target cells, and the untransfected group was set respectively. The cells were transfected to the empty group and transfected to the TRAF1 group. The cell mitochondrial membrane potential stability of each group was observed before and after the stimulation of adriamycin. The results showed that the stability of the membrane potential in the transfected TRAF1 group before and after adriamycin stimulation was all compared with that of the Hela cells. Before the stimulation of adriamycin, the apoptotic cells in the TRAF1 group decreased by 19.8% and 21.8%, respectively, before the stimulation of adriamycin. After stimulation, the apoptotic cells in the TRAF1 group decreased by 16.8% and 14%., respectively, indicating the stability of the mitochondrial membrane potential of TRAF1.
Five. Transfection of TRAF1 can reduce the activation of caspase-9
With adriamycin unstimulated / stimulated Hela cells as target cells, untransfected groups were set respectively, transfected to unloaded group and transfected TRAF1 group. The cells of 1 mu M adriamycin stimulated each cell for 24 hours. The activation of caspase-9 in the cytoplasm protein of each cell before and after adriamycin stimulation was observed by Western blot. TRAF1 could be used to make caspase-9 before and after adriamycin stimulation. In combination with the results of the above membrane potential, it is suggested that TRAF1 may enhance the stability of mitochondrial membrane potential and reduce the activation of caspase-9 by inhibiting the mitochondrial apoptosis pathway and exerting its anti killing effect.
Six. Transfection of TRAF1 can increase the degradation of I kappa B
Hela cells with adriamycin unstimulated / stimulated as target cells were set up as untransfected cells, transfected to unloaded group and transfected TRAF1 group. The cells of 1 mu M adriamycin stimulated each cell for 24 hours, and the degradation of I kappa B in the cytoplasm proteins of each group before and after adriamycin stimulation. After transfection of TRAF1, the degradation of I kappa B increased, suggesting that TRAF1 can activate NF- kappa B, thus resistance to apoptosis.
Conclusion: TRAF1 may cause mitochondrial aggregation by increasing the expression of mitochondrial fusion protein Mfn1. The biological effect caused by this aggregation is to resist apoptosis, which shows that transfection of TRAF1 can resist the killing of adriamycin against Hela cells. One of the mechanisms of resistance to kill (apoptosis) may be by inhibiting apoptosis mitochondria. Ways: to improve the stability of mitochondrial membrane potential and to reduce the activation of caspase-9. On the other hand, it may also promote cell survival through the activation of NF- kappa B. Therefore, TRAF1 can play an effective role in resistance to cell apoptosis by affecting the two aspects of cell survival and death.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

【共引文献】