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  本文選題：tmTNF-α + TNFR1��； 參考：《華中科技大學(xué)》2012年博士論文

【摘要】：跨膜型TNF-a (transmembrane tumor necrosis factor-alpha, tmTNF-a)是分泌型TNF-a (secretory TNF-a, sTNF-a)的前體,它以穩(wěn)定的同源三聚體的形式表達(dá)于活化的巨噬細(xì)胞、淋巴細(xì)胞或其他類型細(xì)胞的表面。sTNF-a被TNF轉(zhuǎn)化酶(TNF-a converting enzyme,TACE)從tmTNF-a上剪切釋放到胞外,與其相應(yīng)的受體結(jié)合并發(fā)揮生物學(xué)功能。越來越多的證據(jù)顯示,不僅是sTNF-a, tmTNF-a亦可通過TNFR1和TNFR2介導(dǎo)多種生物學(xué)效應(yīng),包括細(xì)胞的凋亡與增殖、細(xì)胞因子的產(chǎn)生以及局部炎癥反應(yīng)的調(diào)節(jié)等。 tmTNF-α既可作為配體傳遞正向信號,也可作為受體來傳遞反向信號。本室前期工作證實(shí)tmTNF-α可通過TNFR1介導(dǎo)細(xì)胞凋亡；然而該分子如表達(dá)在腫瘤細(xì)胞則可通過反向信號激活NF-κB,導(dǎo)致抗凋亡。但其具體的分子機(jī)制尚不清楚。本研究主要探討tmTNF-a作為配體通過TNFR1轉(zhuǎn)導(dǎo)正向信號介導(dǎo)腫瘤細(xì)胞凋亡及其作為受體轉(zhuǎn)導(dǎo)反向信號保護(hù)腫瘤細(xì)胞抵抗凋亡的分子機(jī)制。其主要結(jié)果如下： 一、tmTNF-a通過TNFR1介導(dǎo)細(xì)胞凋亡的分子機(jī)制 1. tmTNF-a不能介導(dǎo)TNFR1的內(nèi)化：用流式與激光共聚焦顯微鏡證實(shí)sTNF-a導(dǎo)致細(xì)胞表面TNFR1表達(dá)降低,TNFR1胞漿移位明顯增加,用MDC預(yù)處理則可明顯抑制sTNF-a誘導(dǎo)的TNFR1向胞漿內(nèi)的內(nèi)化；而tmTNF-a則對細(xì)胞表面TNFR1的表達(dá)無明顯影響,且不誘導(dǎo)受體向胞漿內(nèi)移位。提示tmTNF-a不能誘導(dǎo)TNFR1內(nèi)化。 2. tmTNF-a的胞毒效應(yīng)不依賴TNFR1的內(nèi)化：首先用MTT證實(shí)用MDC抑制TNFR1的內(nèi)化,可明顯降低sTNF-a的胞毒效應(yīng),卻對tmTNF-a胞毒效應(yīng)無影響。進(jìn)一步用AnnexinV-PI凋亡檢測試劑盒和WB證實(shí)用MDC可明顯降低sTNF-a誘導(dǎo)的caspase-3的活化及細(xì)胞凋亡,但不影響tmTNF-a誘導(dǎo)的caspase-3的活化及細(xì)胞凋亡。上述結(jié)果提示,與sTNF-a胞毒效應(yīng)依賴TNFR1內(nèi)化的特征相反,tmTNF-a誘導(dǎo)靶細(xì)胞凋亡為TNFR1內(nèi)化非依賴性的。 3. tmTNF-a通過細(xì)胞膜上的TNFR1募集TRADD,FADD和caspase-8:分別提取細(xì)胞總蛋白,IP-western證實(shí)：tmTNF-a與sTNF-a均能通過TNFR1募集TRADD、FADD和caspase-8,形成DISC信號復(fù)合物。進(jìn)一步分離胞膜蛋白和胞漿蛋白,用IP證實(shí)tmTNF-a誘導(dǎo)TNFR1形成的DISC信號復(fù)合物位于膜蛋白中,且可用激光共聚焦顯微鏡觀察到tmTNF-a誘導(dǎo)DISC信號復(fù)合物的成員之一FADD從胞漿轉(zhuǎn)位到細(xì)胞膜；而sTNF-a誘導(dǎo)TNFR1形成的DISC信號復(fù)合物則位于胞漿蛋白中。提示兩型TNF-a盡管作用于同一受體,但二者分別在細(xì)胞不同部位誘導(dǎo)DISC信號復(fù)合物的形成。 4.tmTNF-a不能通過TNFR1在細(xì)胞膜上募集RIP-1,TRAF2和cIAPl:提細(xì)胞膜蛋白進(jìn)行IP證實(shí)：sTNF-a可通過TNFR1在細(xì)胞膜上募集RIP-1, TRAF2和cIAP1等激活NF-κB的信號復(fù)合物；而tmTNF-a則無明顯類似效應(yīng)。提示與sTNF-a不同,tmTNF-a不能通過TNFR1在細(xì)胞膜上募集抗凋亡信號分子。 5. tmTNF-a不依賴于TNFR1的死亡結(jié)構(gòu)域募集死亡信號分子：分別將野生型TNFR1和缺失DD的TNFR1突變體瞬時轉(zhuǎn)染HEK293細(xì)胞,兩型TNF刺激30分鐘后提取細(xì)胞膜蛋白進(jìn)行IP。結(jié)果發(fā)現(xiàn)tmTNF-a不但可通過野生型TNFR1,也能夠通過缺失DD結(jié)構(gòu)域的TNFR1募集TRADD, FADD和caspase-8等DISC信號復(fù)合物。提示與sTNF-a不同,tmTNF-a通過TNFR1募集DISC信號復(fù)合物不依賴于受體的死亡結(jié)構(gòu)域。 6. tmTNF-a通過TNFR1非DD結(jié)構(gòu)域在細(xì)胞膜上募集STAT1,形成DISC信號復(fù)合物：為探討tmTNF-a誘導(dǎo)TNFR1募集DISC復(fù)合物分子的機(jī)制,用胞膜蛋白進(jìn)行IP,證實(shí)：tmTNF-a可通過野生型和缺失DD的TNFR1募集STAT1,但sTNF-a則不能。提取細(xì)胞總蛋白進(jìn)行IP,進(jìn)一步證實(shí)sTNF-a誘導(dǎo)TNFR1形成的DISC復(fù)合物中有大量STAT1參與,但TNFR1缺失DD則不能被誘導(dǎo)形成DISC復(fù)合物,且無STAT1共沉淀,提示STAT1參與sTNF-a誘導(dǎo)的DISC信號復(fù)合物,且依賴TNFR1的DD結(jié)構(gòu)域；然而,tmTNF-a通過野生型和缺失DD的TNFR1形成的DISC復(fù)合物中均有明顯STAT1的共沉淀,提示STAT1可能通過與TNFR1非DD結(jié)構(gòu)域直接結(jié)合,參與tmTNF-a通過TNFR1募集DISC信號復(fù)合物。 二、tmTNF-α反向信號通路的研究 1. NIK, SODD參與tmTNF-α反向信號復(fù)合物：用tmTNF-a單抗對高表達(dá)tmTNF-a的Raji細(xì)胞進(jìn)行IP,證實(shí)tmTNF-a可與NIK、SODD發(fā)生免疫共沉淀。反之,我們用NIK或SODD的特異性抗體分別進(jìn)行反向IP,證實(shí)均有tmTNF-a共沉淀。提示除了前期工作發(fā)現(xiàn)的TRAF1,IKKa和NF-KBp52以外,NIK和SODD也是tmTNF-a反向信號復(fù)合物中的成員。 2. tmTNF-a通過胞漿段(TNF-LS)募集TRAF1:我們將TRAF1, IKKa和tmTNF-a野生型或突變體質(zhì)粒共轉(zhuǎn)染于293T細(xì)胞中,提取總蛋白,用tmTNF-a單抗進(jìn)行IP。證實(shí)：野生型tmTNF-a和缺失胞外段的TNF-LS均可募集TRAF1,而缺失胞漿段的ACS-tmTNF-a則不能募集TRAF1。提示tmTNF-a通過其胞漿段募集TRAF1。 3. tmTNF-a胞漿段(TNF-LS)不能直接與IKKa結(jié)合：本研究為了驗(yàn)證細(xì)胞內(nèi)TNF-LS和IKKa的相互作用,將二者共轉(zhuǎn)到293T細(xì)胞中,IP-Western顯示TNF-LS并未與IKKa共沉淀。提示TNF-LS不能直接結(jié)合IKKa,可能通過TRAF1間接募集該分子。 4. TRAF1是連接tmTNF-a與IKKa的接頭分子：使用siTRAF1下調(diào)Raji細(xì)胞表達(dá)TRAF1,導(dǎo)致與tmTNF-a免疫共沉淀的IKKa明顯減少。提示TRAF1作為銜接分子可將IKKa募集到tmTNF-a反向信號復(fù)合物中。 5.抑制tmTNF-α磷酸化可促進(jìn)其反向信號復(fù)合物TRAF1/IKKa/NF-KBp52的形成：tmTNF-a胞漿段含有CKI磷酸化酶的作用位點(diǎn),故我們用CKI的特異性抑制劑D4476抑制tmTNF-a的磷酸化,結(jié)果發(fā)現(xiàn)tmTNF-a反向信號復(fù)合物(TRAF1/IKKa/NF-KBp52)形成明顯增加。提示抑制tmTNF-a的磷酸化能促進(jìn)其反向信號復(fù)合物的形成。 綜上所述,本課題闡明了tmTNF-a通過TNFR1介導(dǎo)正向信號導(dǎo)致靶細(xì)胞凋亡的信號通路,證實(shí)tmTNF-a可能直接通過細(xì)胞膜上TNFR1的非DD結(jié)構(gòu)域與STAT1結(jié)合,進(jìn)而募集TRADD、FADD和caspase-8,形成DISC信號復(fù)合物,導(dǎo)致caspases的級聯(lián)活化,進(jìn)而誘導(dǎo)細(xì)胞凋亡。同時在前期工作的基礎(chǔ)上,我們進(jìn)一步闡明了tmTNF-a反向信號復(fù)合物的組成及其成員之間的相互關(guān)系,tmTNF-a一方面通過其胞漿段募集TRAF1、NIK和IKKa,進(jìn)而激活NF-KBp52(非經(jīng)典途徑),另一方面可能通過其胞漿段與TNFR1競爭SODD,參與激活NF-κB經(jīng)典途徑,導(dǎo)致tmTNF-a(+)的腫瘤細(xì)胞抵抗凋亡。 本研究深入探討了tmTNF-a通過TNFR1介導(dǎo)正向信號誘導(dǎo)靶細(xì)胞凋亡以及通過反向信號促進(jìn)tmTNF-a(+)的腫瘤細(xì)胞抵抗凋亡的分子機(jī)制,為臨床腫瘤治療及干預(yù)腫瘤耐藥提供新的思路和靶點(diǎn)。
[Abstract]:Tumor necrosis factor - alpha ( tmTNF - a ) is a precursor of secreted TNF - a ( sTNF - a ) , which is expressed in activated macrophages , lymphocytes or other types of cells in the form of stable homotrimer . sTNF - a is cleaved from tmTNF - a by TNF - a converting enzyme ( TACE ) to the extracellular domain , and plays a biological function . More and more evidence shows that not only sTNF - a , tmTNF - a can also mediate various biological effects through TNFR1 and TNFR2 , including apoptosis and proliferation of cells , production of cytokines and regulation of local inflammatory response .
tmTNF - 偽 can be used both as a ligand to deliver a positive signal , and can be used as a receptor to transmit a reverse signal . The previous work of the chamber proves that tmTNF - alpha can mediate apoptosis through TNFR1 ;
However , if the molecule is expressed in tumor cells , NF - 魏B can be activated by the reverse signal , which leads to anti - apoptosis . However , the specific molecular mechanism is not clear . The main results of this study are as follows :
One , tmTNF - a molecule mechanism of cell apoptosis mediated by TNFR1
1 . tmTNF - a did not mediate the internalization of TNFR1 : the expression of TNFR1 in the cell surface was decreased by the flow cytometry and the laser confocal microscope , and the expression of TNFR1 was significantly increased , and the amount of TNFR1 induced by sTNF - a was significantly inhibited by MDC pretreatment , and the internalization of TNFR1 induced by sTNF - a was inhibited significantly ;
However , tmTNF - a did not significantly affect the expression of TNFR1 in the cell surface , and did not induce the translocation of the receptor to the cytoplasm .
2 . The cytotoxic effect of tmTNF - a did not depend on the internalization of TNFR1 : firstly , MTT was used to confirm that MDC could inhibit the internalization of TNFR1 and reduce the cytotoxic effect of sTNF - a .
3 . tmTNF - a raised TRADD , FADD and caspase - 8 through TNFR1 on the cell membrane : the total protein of the cells was extracted , and IP - western confirmed that both tmTNF - a and sTNF - a could raise TRADD , FADD and caspase - 8 through TNFR1 to form DISC signal complexes .
The DISC signal complex induced TNFR1 induced by sTNF - a is located in the cytoplasm protein . It is suggested that the two types of TNF - a induce the formation of DISC signal complexes at different sites of the cell , although they act on the same receptor .
4 . tmTNF - a was unable to recruit RIP - 1 , TRAF2 and cIAPl on the cell membrane by TNFR1 : the membrane protein was confirmed by IP : sTNF - a could induce the activation of NF - 魏B signal complexes on the cell membrane via TNFR1 , TRAF2 and cIAP1 .
The results suggested that tmTNF - a could not recruit anti - apoptotic signal molecules on the cell membrane by TNFR1 , unlike sTNF - a .
5 . tmTNF - a did not rely on the death domain of TNFR1 to recruit death signal molecules : the wild type TNFR1 and the deleted DD TNFR1 mutant were transiently transfected into 293 cells and the two types of TNF stimulated 30 minutes to extract the membrane protein . The results showed that tmTNF - a not only can raise DISC signal complexes such as TRADD , FADD and caspase - 8 through TNFR1 with the DD domain .
6 . tmTNF - a raised STAT1 on the cell membrane via TNFR1 non - DD domain to form DISC signal complex . In order to investigate the mechanism of tmTNF - a - induced TNFR1 - raised DISC complex molecule , it was confirmed that tmTNF - a was able to raise STAT1 through wild - type and missing DD - induced TNFR1 , but sTNF - a could not be induced to form DISC complex , but sTNF - a could not be induced to form DISC complex , but without STAT1 co - precipitation , STAT1 was involved in sTNF - a - induced DISC signal complex and depended on the DD domain of TNFR1 ;
However , tmTNF - a is co - precipitated with STAT1 in DISC complexes formed by wild - type and missing DD TNFR1 , suggesting STAT1 may be directly bound to the TNFR1 non - DD domain and participate in tmTNF - a to raise DISC signal complexes through TNFR1 .
Study on the reverse signal pathway of di , tmTNF - 偽
1 . NIK and SODD were involved in tmTNF - a reverse signaling complexes : the presence of tmTNF - a monoclonal antibody against the high expression of tmTNF - a . The results showed that tmTNF - a could co - precipitate with NIK and SODD . On the contrary , we used NIK or SODD - specific antibodies to reverse IP respectively to confirm that tmTNF - a co - precipitated .
2 . tmTNF - a raised TRAF1 through the cytoplasmic segment ( TNF - LS ) : we co - transfected TRAF1 , IKKa and tmTNF - a wild - type or mutant plasmid into 293 cells , extracted the total protein and used tmTNF - a monoclonal antibody for IP . It was confirmed that both wild - type tmTNF - a and TNF - LS of the absent extracellular segment could raise TRAF1 , whereas the ACS - tmTNF - a in the missing cell segment could not raise TRAF1 . It was suggested that tmTNF - a raised TRAF1 through its cytoplasmic segment .
3 . The TNF - LS was not directly bound to IKKa . In order to verify the interaction between TNF - LS and IKKa in the cells , TNF - LS was not co - precipitated with IKKa . It was suggested that TNF - LS could not directly bind IKKa and could indirectly raise the molecule via TRAF1 .
4 . TRAF1 is a linker molecule linked to tmTNF - a and IKKa : Down - regulation of TRAF1 with siTRAF1 results in a significant decrease in IKKa co - precipitated with tmTNF - a . It is suggested that TRAF1 can be used as a linking molecule to raise IKKa to the tmTNF - a reverse signal complex .
5 . Inhibition of tmTNF - a phosphorylation can promote the formation of reverse signal complexes TRAF1 / IKKa / NF - KBp52 : the tmTNF - a cytoplasmic segment contains the action site of CKI phosphorylase , so we use the specificity inhibitor D4476 of CKI to inhibit the phosphorylation of tmTNF - a .
In conclusion , we have clarified that tmTNF - a can induce apoptosis of target cells through TNFR1 - mediated positive signals . It is confirmed that tmTNF - a may directly bind to STAT1 through the non - DD domain of TNFR1 in cell membrane , which leads to the cascade activation of caspases , thus inducing apoptosis . On the other hand , we can compete with TNFR1 to activate NF - KBp52 ( non - classical pathway ) . On the other hand , it is possible to activate NF - 魏B classical pathway through its cytoplasmic segment , which leads to the tumor cells of tmTNF - a ( + ) to resist apoptosis .
In this study , the molecular mechanism of tmTNF - a mediated by TNFR1 to induce apoptosis of target cells and to promote the apoptosis of the tumor cells of tmTNF - a ( + ) by reverse signal is discussed in detail , which provides new ideas and targets for clinical tumor therapy and intervention of tumor drug resistance .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 石文芳,李卓婭,龔非力,熊平,徐勇;跨膜型與分泌型TNF-α細(xì)胞毒效應(yīng)的比較[J];中華微生物學(xué)和免疫學(xué)雜志;1998年06期

2 石文芳,李卓婭,龔非力;跨膜型和分泌型TNF-α對TNF受體作用的比較[J];中國免疫學(xué)雜志;1998年04期

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本文編號：2030279