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

【摘要】：TRAF1屬于TNF受體相關因子(TNFR-associated factor TRAF)家族。TRAF家族是一類胞內銜接蛋白,能介導TNF受體和IL-1受體超家族的信號轉導引起NF-?B和JNK等的活化,從而對細胞的生存與死亡產生影響。TRAF1不具有TRAF家族其他成員所共有的環指和與之相鄰的鋅指結構,其可募集一些TNF受體超家族成員,包括TNFR2,CD30,CD27,TRANCE-R等。據報道,TRAF1可抵抗/抑制細胞凋亡:在轉基因動物中過表達TRAF1,可抑制抗原誘導的CD8+細胞的凋亡;TRAF1可通過募集cIAP以執行其抗凋亡作用。但也有不同研究結果表明TRAF1可促進細胞凋亡。 本室前期工作首次證實高表達TRAF1能與線粒體共定位并引起線粒體聚集,這一現象具有普遍性。而且轉染TRAF1的HepG2細胞,其NF-κB活性增強,能抵抗TM-TNF-α的殺傷。轉染TRAF1能上調線粒體融合蛋白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。 本課題選取Hela細胞作為研究對象,借助Realtime-PCR觀察高表達TRAF1線粒體融合蛋白Mfn1轉錄水平的變化;以化療藥物阿霉素誘導Hela細胞凋亡為模型,觀察高表達TRAF1對于阿霉素殺傷作用的影響,并通過檢測線粒體膜電位的變化、caspase-9的活化以及I?B的降解,進一步深入探討TRAF1導致線粒體聚集的生物學效應及其分子機制。 一.轉染TRAF1使線粒體融合蛋白Mfn1的mRNA水平增高 以Realtime-PCR分別觀察Hela細胞各實驗組:未轉染組、轉染空載體pDsRed組和轉染pDsRed-TRAF1重組質粒組中線粒體融合蛋白Mfn1的mRNA水平的變化。轉染TRAF1后,Mfn1分子的mRNA水平明顯增高,為未轉染組的2.18倍(P0.01),為轉染空載體組的2.48倍(P0.01)。提示TRAF1可能通過上調Mfn1的表達而導致線粒體聚集。 二.轉染TRAF1可抵抗化療藥物阿霉素的殺傷效應 以Hela細胞為靶細胞,設未轉染組、轉染空載體組和轉染TRAF1組,以1μM阿霉素分別刺激各組細胞48小時,觀察TRAF1對阿霉素殺傷Hela細胞的影響。MTT結果顯示:轉染TRAF1組表現為對阿霉素殺傷作用的抵抗,其殺傷率與未轉染組和轉染空載組相比,分別下降了18%和17.21% (P0.05)。提示轉染TRAF1可抵抗阿霉素對Hela細胞的殺傷效應。 三.轉染TRAF1可使線粒體發生聚集 以阿霉素未刺激/刺激的Hela細胞為靶細胞,分別設未轉染組,轉染空載組和轉染TRAF1組,1μM阿霉素刺激各組細胞24小時,在熒光顯微鏡油鏡下觀察阿霉素刺激前后TRAF1轉染所導致的線粒體形態變化。阿霉素未刺激時,3組細胞線粒體結構均呈緊密的管網狀結構細胞,而TRAF1轉染組與未轉染組和轉染空載組相比,表現為線粒體聚集。阿霉素刺激后,未轉染組與轉染空載組線粒體結構松散紊亂,并有片段化的線粒體出現,而轉染TRAF1組仍能保持聚集狀態。 四.轉染TRAF1可增強線粒體膜電位穩定性 以阿霉素未刺激/刺激的Hela細胞為靶細胞,分別設未轉染組,轉染空載組和轉染TRAF1組,1μM阿霉素刺激各組細胞24小時,觀察阿霉素刺激前后各組細胞線粒體膜電位的穩定性。結果顯示:轉染TRAF1組在阿霉素刺激前后,膜電位的穩定性均比未轉染組和轉染空載組增強。阿霉素刺激之前,TRAF1組凋亡細胞與對照組和空載組相比,分別下降了19.8%和21.8%;刺激后,TRAF1組凋亡細胞與對照組和空載組相比,分別下降了16.8%和14%。提示TRAF1能維持線粒體膜電位的穩定性。 五.轉染TRAF1可減少caspase-9的活化 以阿霉素未刺激/刺激的Hela細胞為靶細胞,分別設未轉染組,轉染空載組和轉染TRAF1組,1μM阿霉素刺激各組細胞24小時,通過Western blot觀察阿霉素刺激前后各組細胞胞漿蛋白中caspase-9的活化。無論阿霉素刺激前后,轉染TRAF1均可使caspase-9的活化明顯減少。結合上述膜電位的結果,提示TRAF1可能通過抑制線粒體凋亡途徑,提高線粒體膜電位的穩定性,減少caspase-9的活化而發揮其抵抗殺傷的作用。 六.轉染TRAF1可增加IκB的降解 以阿霉素未刺激/刺激的Hela細胞為靶細胞,分別設未轉染組,轉染空載組和轉染TRAF1組,1μM阿霉素刺激各組細胞24小時,觀察阿霉素刺激前后各組細胞胞漿蛋白中IκB的降解。轉染TRAF1后,IκB的降解增加,提示TRAF1可活化NF-κB,從而抵抗凋亡。 結論:TRAF1可能通過增加線粒體融合蛋白Mfn1的表達而引起線粒體聚集,此種聚集所導致的生物學效應為抵抗細胞凋亡,表現為轉染TRAF1可抵抗化療藥物阿霉素對Hela細胞的殺傷。其抵抗殺傷(凋亡)的機制之一可能是通過抑制凋亡線粒體途徑:提高線粒體膜電位的穩定性、減少caspase-9的活化。另一方面,也可能通過NF-κB的活化而促進細胞生存。因此,TRAF1通過影響細胞的生存和死亡兩方面的共同作用,最終發揮抵抗細胞凋亡的效應。
[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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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本文編號：2017386