發(fā)布時(shí)間：2018-06-14 12:30

  本文選題：TRAF1 + Mfn1��； 參考：《華中科技大學(xué)》2009年碩士論文

【摘要】：TRAF1屬于TNF受體相關(guān)因子(TNFR-associated factor TRAF)家族。TRAF家族是一類胞內(nèi)銜接蛋白,能介導(dǎo)TNF受體和IL-1受體超家族的信號(hào)轉(zhuǎn)導(dǎo)引起NF-?B和JNK等的活化,從而對(duì)細(xì)胞的生存與死亡產(chǎn)生影響。TRAF1不具有TRAF家族其他成員所共有的環(huán)指和與之相鄰的鋅指結(jié)構(gòu),其可募集一些TNF受體超家族成員,包括TNFR2,CD30,CD27,TRANCE-R等。據(jù)報(bào)道,TRAF1可抵抗/抑制細(xì)胞凋亡:在轉(zhuǎn)基因動(dòng)物中過(guò)表達(dá)TRAF1,可抑制抗原誘導(dǎo)的CD8+細(xì)胞的凋亡;TRAF1可通過(guò)募集cIAP以執(zhí)行其抗凋亡作用。但也有不同研究結(jié)果表明TRAF1可促進(jìn)細(xì)胞凋亡。 本室前期工作首次證實(shí)高表達(dá)TRAF1能與線粒體共定位并引起線粒體聚集,這一現(xiàn)象具有普遍性。而且轉(zhuǎn)染TRAF1的HepG2細(xì)胞,其NF-κB活性增強(qiáng),能抵抗TM-TNF-α的殺傷。轉(zhuǎn)染TRAF1能上調(diào)線粒體融合蛋白Mfn2的mRNA水平,提示TRAF1引起的線粒體聚集可能與線粒體融合分子相關(guān)。 本課題選取Hela細(xì)胞作為研究對(duì)象,借助Realtime-PCR觀察高表達(dá)TRAF1線粒體融合蛋白Mfn1轉(zhuǎn)錄水平的變化;以化療藥物阿霉素誘導(dǎo)Hela細(xì)胞凋亡為模型,觀察高表達(dá)TRAF1對(duì)于阿霉素殺傷作用的影響,并通過(guò)檢測(cè)線粒體膜電位的變化、caspase-9的活化以及I?B的降解,進(jìn)一步深入探討TRAF1導(dǎo)致線粒體聚集的生物學(xué)效應(yīng)及其分子機(jī)制。 一.轉(zhuǎn)染TRAF1使線粒體融合蛋白Mfn1的mRNA水平增高 以Realtime-PCR分別觀察Hela細(xì)胞各實(shí)驗(yàn)組:未轉(zhuǎn)染組、轉(zhuǎn)染空載體pDsRed組和轉(zhuǎn)染pDsRed-TRAF1重組質(zhì)粒組中線粒體融合蛋白Mfn1的mRNA水平的變化。轉(zhuǎn)染TRAF1后,Mfn1分子的mRNA水平明顯增高,為未轉(zhuǎn)染組的2.18倍(P0.01),為轉(zhuǎn)染空載體組的2.48倍(P0.01)。提示TRAF1可能通過(guò)上調(diào)Mfn1的表達(dá)而導(dǎo)致線粒體聚集。 二.轉(zhuǎn)染TRAF1可抵抗化療藥物阿霉素的殺傷效應(yīng) 以Hela細(xì)胞為靶細(xì)胞,設(shè)未轉(zhuǎn)染組、轉(zhuǎn)染空載體組和轉(zhuǎn)染TRAF1組,以1μM阿霉素分別刺激各組細(xì)胞48小時(shí),觀察TRAF1對(duì)阿霉素殺傷Hela細(xì)胞的影響。MTT結(jié)果顯示:轉(zhuǎn)染TRAF1組表現(xiàn)為對(duì)阿霉素殺傷作用的抵抗,其殺傷率與未轉(zhuǎn)染組和轉(zhuǎn)染空載組相比,分別下降了18%和17.21% (P0.05)。提示轉(zhuǎn)染TRAF1可抵抗阿霉素對(duì)Hela細(xì)胞的殺傷效應(yīng)。 三.轉(zhuǎn)染TRAF1可使線粒體發(fā)生聚集 以阿霉素未刺激/刺激的Hela細(xì)胞為靶細(xì)胞,分別設(shè)未轉(zhuǎn)染組,轉(zhuǎn)染空載組和轉(zhuǎn)染TRAF1組,1μM阿霉素刺激各組細(xì)胞24小時(shí),在熒光顯微鏡油鏡下觀察阿霉素刺激前后TRAF1轉(zhuǎn)染所導(dǎo)致的線粒體形態(tài)變化。阿霉素未刺激時(shí),3組細(xì)胞線粒體結(jié)構(gòu)均呈緊密的管網(wǎng)狀結(jié)構(gòu)細(xì)胞,而TRAF1轉(zhuǎn)染組與未轉(zhuǎn)染組和轉(zhuǎn)染空載組相比,表現(xiàn)為線粒體聚集。阿霉素刺激后,未轉(zhuǎn)染組與轉(zhuǎn)染空載組線粒體結(jié)構(gòu)松散紊亂,并有片段化的線粒體出現(xiàn),而轉(zhuǎn)染TRAF1組仍能保持聚集狀態(tài)。 四.轉(zhuǎn)染TRAF1可增強(qiáng)線粒體膜電位穩(wěn)定性 以阿霉素未刺激/刺激的Hela細(xì)胞為靶細(xì)胞,分別設(shè)未轉(zhuǎn)染組,轉(zhuǎn)染空載組和轉(zhuǎn)染TRAF1組,1μM阿霉素刺激各組細(xì)胞24小時(shí),觀察阿霉素刺激前后各組細(xì)胞線粒體膜電位的穩(wěn)定性。結(jié)果顯示:轉(zhuǎn)染TRAF1組在阿霉素刺激前后,膜電位的穩(wěn)定性均比未轉(zhuǎn)染組和轉(zhuǎn)染空載組增強(qiáng)。阿霉素刺激之前,TRAF1組凋亡細(xì)胞與對(duì)照組和空載組相比,分別下降了19.8%和21.8%;刺激后,TRAF1組凋亡細(xì)胞與對(duì)照組和空載組相比,分別下降了16.8%和14%。提示TRAF1能維持線粒體膜電位的穩(wěn)定性。 五.轉(zhuǎn)染TRAF1可減少caspase-9的活化 以阿霉素未刺激/刺激的Hela細(xì)胞為靶細(xì)胞,分別設(shè)未轉(zhuǎn)染組,轉(zhuǎn)染空載組和轉(zhuǎn)染TRAF1組,1μM阿霉素刺激各組細(xì)胞24小時(shí),通過(guò)Western blot觀察阿霉素刺激前后各組細(xì)胞胞漿蛋白中caspase-9的活化。無(wú)論阿霉素刺激前后,轉(zhuǎn)染TRAF1均可使caspase-9的活化明顯減少。結(jié)合上述膜電位的結(jié)果,提示TRAF1可能通過(guò)抑制線粒體凋亡途徑,提高線粒體膜電位的穩(wěn)定性,減少caspase-9的活化而發(fā)揮其抵抗殺傷的作用。 六.轉(zhuǎn)染TRAF1可增加IκB的降解 以阿霉素未刺激/刺激的Hela細(xì)胞為靶細(xì)胞,分別設(shè)未轉(zhuǎn)染組,轉(zhuǎn)染空載組和轉(zhuǎn)染TRAF1組,1μM阿霉素刺激各組細(xì)胞24小時(shí),觀察阿霉素刺激前后各組細(xì)胞胞漿蛋白中IκB的降解。轉(zhuǎn)染TRAF1后,IκB的降解增加,提示TRAF1可活化NF-κB,從而抵抗凋亡。 結(jié)論:TRAF1可能通過(guò)增加線粒體融合蛋白Mfn1的表達(dá)而引起線粒體聚集,此種聚集所導(dǎo)致的生物學(xué)效應(yīng)為抵抗細(xì)胞凋亡,表現(xiàn)為轉(zhuǎn)染TRAF1可抵抗化療藥物阿霉素對(duì)Hela細(xì)胞的殺傷。其抵抗殺傷(凋亡)的機(jī)制之一可能是通過(guò)抑制凋亡線粒體途徑:提高線粒體膜電位的穩(wěn)定性、減少caspase-9的活化。另一方面,也可能通過(guò)NF-κB的活化而促進(jìn)細(xì)胞生存。因此,TRAF1通過(guò)影響細(xì)胞的生存和死亡兩方面的共同作用,最終發(fā)揮抵抗細(xì)胞凋亡的效應(yīng)。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

【共引文獻(xiàn)】

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本文編號(hào)：2017386