【作者】 刘涛；

【导师】 李卓娅；

【作者基本信息】 华中科技大学 ， 分子免疫学， 2009， 博士

【摘要】 第一部分酶切位点缺失的跨膜型TNF-α突变体的构建及其功能的研究跨膜型TNF-α(Transmembrane TNFα，tmTNF-α)是sTNF-α的前体，分子量为26kD，主要表达在活化的单核细胞和免疫细胞表面。tmTNF-α在N端比sTNF-α多76个氨基酸组成的引导肽，具有疏水区，故可跨膜成为膜分子。表达于细胞膜表面的tmTNF-α在TNF转化酶(TACE／ADAM17)的作用下，被剪切释放出sTNF-α。由于tmTNF-α可被剪切成sTNF-α，为了排除sTNF-α的影响，在研究tmTNF-α生物学功能的时候需要用其酶切位点缺失的突变体。常用的缺失1-12位的突变体虽然基本不被TACE剪切，但是其正向信号介导的胞毒效应下降，反向信号传递缺陷，故不是一个理想的研究tmTNF-α的突变体。有报道称△1-9，K11E突变体也不被TACE剪切，并且其胞毒效应和野生型tmTNF-α一致，但是这种突变是否影响tmTNF-α反向信号尚不清楚。本研究通过定点突变的方法，将tmTNF-α的酶切作用位点缺失，使得tmTNF-α不被剪切为sTNFα；并研究突变体对正反向信号的影响。主要结果如下：1利用重叠PCR技术，成功构建△1-12tmTNF-α、△1-9，K11E tmTNF-α突变体，经酶切鉴定，PCR鉴定及测序鉴定确认除预期突变外，无任何其它点突变，移码及缺失突变。2将tmTNF-α及其突变体转染Hela细胞后，经Western blot鉴定wt tmTNF-α在26kD处有特异性条带，突变体在25kD左右有特异性条带，符合预期分子量。3用野生型tmTNF-α及其突变体转染Hela细胞，证实转染野生型tmTNF-α的细胞能分泌大量的sTNF-α，而转染△1-12 tmTNF-α和△1-9，K11E tmTNF-α突变体的细胞上清均未检测到sTNF-α，提示两个缺失突变体都丧失被TACE酶解为sTNF-α的能力。4用生物活性检测证实wt tmTNF-α和△1-9，K11E tmTNF-α对靶细胞有明显的杀伤作用；但是△1-12 tmTNF-α的胞毒效应却减少了近一半，说明△1-9，K11E tmTNF-α保留了野生型tmTNF-α正向信号介导的胞毒效应。5用Western blot检测证实△1-9，K11E tmTNF-α与wt tm TNF-α一样能通过其反向信号有效降解IκB-α，激活NF-κB，而△1-12tmTNF-α则失去了这种能力。上述结果表明△1-9，K11E tmTNF-α突变体既丧失被酶解的能力，又能保留野生型tmTNF-α正向信号和反向信号传递能力，为进一步研究tmTNF-α的生物学功能提供了有力的工具。第二部分脂筏与tmTNF-α的正／反向信号脂筏是近年被发现的存在于细胞膜上微结构域，区别于经典的胞膜脂质双分子层，它富含胆固醇、磷脂酰肌醇。已知很多受体及其相关的信号分子均能分布在脂筏内。由于脂筏在质膜上分布比较分散，且能侧向漂移和聚集，故可形成信号转导平台，参与活化受体募集信号转导分子，向胞内传递信号。tmTNF-α不仅能和受体结合，向靶细胞传递正向信号，本身还能作为受体，用其胞内段募集信号分子，传递反向信号。有报道tmTNF-α的-47位半胱氨酸是棕榈酰化酰基化位点，故我们推测tmTNF-α可能定位在脂筏中，但是tmTNF-α与脂筏的关系尚不清楚。此外，TNFR也能定位在脂筏，脂筏内外的TNFR与tmTNF-α之间的关系也不清楚。为阐明tmTNF-α与脂筏的关系，我们利用蔗糖密度梯度离心法提取脂筏结构，明确tmTNF-α的脂筏定位；并从表达tmTNF-α的效应细胞和表达TNFR的靶细胞两方面入手，研究脂筏在tmTNF-α正反向信号传导中的作用。其主要结果如下：1脂筏与tmTNF-α反向信号1．1 tmTNF-α可定位脂筏内：利用蔗糖密度梯度离心法分离高表达tmTNF-α的Raji细胞的脂筏结构，用Western blot检测证实部分tmTNF-α定位在脂筏内。用10mMMCD破坏脂筏，所有tmTNF-α均分布到脂筏外。1．2脂筏内tmTNF-α导致sTNF-α耐受：高表达tmTNF-α的Raji细胞对sTNF-α耐受，用MCD破坏脂筏后对sTNF-α胞毒效应的敏感性明显增加；而低表达tmTNF-α的T24细胞对sTNF-α敏感，脂筏破坏后，sTNF-α的胞毒效应也明显增强。提示定位在脂筏内的tmTNF-α可导致sTNF-α耐受1．3破坏脂筏导致NF-κB活性下降：高表达tmTNF-α的Raji细胞经MCD处理后，IκB-α降解明显受到抑制；而低表达tmTNF-α的T24细胞经MCD处理后，IκB-α水平则无明显变化。提示破坏脂筏能使tmTNF-α反向信号减弱。1．4建立稳转完全定位在脂筏内或外的tmTNF-α突变体的细胞株：利用重组PCR成功构建cav-tmTNF-α和C-47A tmTNF-a突变体，连同野生型tmTNF-a稳转T24细胞。提取脂筏分析证实cav-tmTNF-α完全定位于脂筏内，而C-47A tmTNF-a则完全定位于脂筏外。1．5脂筏内tmTNF-α诱导对sTNF-α胞毒效应的耐受：用胞毒实验证实sTNF-α可有效杀伤定位在脂筏外的C-47A tmTNF-α表达细胞，而对部分定位脂筏内的wttmTNF-α表达细胞的胞毒效应则明显下降。提示只有脂筏内的tmTNF-α才可导致细胞抵抗sTNF-α的胞毒效应。1．6脂筏内tmTNF-α可组成性活化NF-κB：Western blot证实部分定位脂筏内的wttmTNF-α可组成性降解IκB-α，使NF-κB p65磷酸化；而仅在脂筏外的C-47AtmTNF-α则无此作用。1．7脂筏内tmTNF-α可上调抗凋亡分子cIAP1基因表达，下调促凋亡分子Bax基因表达：用Real time PCR检测证实部分定位在脂筏内的wt tmTNF-α诱导cIAP1基因表达明显增加，却明显抑制Bax的基因转录；但是定位在脂筏外的C-47AtmTNF-α则无明显影响。1．8 CKI抑制剂D4476可增强脂筏内tmTNF-α对sTNF-α的抵抗：用CKI的特异性抑制剂抑制tmTNF-α的磷酸化，增强反向信号，结果可增强转染wt tmTNF-α细胞对sTNF-α胞毒效应的抵抗，却对sTNF-α杀伤定位在脂筏外的C-47A tmTNFα表达细胞则无明显作用。提示tmTNFα诱导的sTNF-α耐受是由定位在脂筏内的tmTNF-α通过其反向信号介导的。1．9 tmTNF-α以脂筏为平台传递反向信号：用可溶性TNF受体刺激tmTNF-α反向信号或转染定位脂筏内、外的tmTNF-α及其突变体证明激活tmTNF-α可导致该分子本身向脂筏内聚集，且募集其反向信号分子TRAF1、IKK-α和NF-κBp52至脂筏内；而定位在脂筏外的C-47A tmTNF-α则对这些信号分子脂筏内外的分布无影响。提示tmTNF-α可能是以脂筏为平台传递反向信号的。2脂筏与tmTNF-α正向信号2．1效应细胞脂筏对tmTNF-α正向信号介导的生物学功能的影响2．1．1 tmTNF-α胞毒效应与其定位脂筏内外无关：用MCD破坏Raji细胞的脂筏，发现与TNF抗体封闭一样，几乎可以完全阻断tmTNF-α对T24细胞的胞毒效应。但是，转染野生型tmTNF-α、完全定位在脂筏外的C-47A tmTNF-a突变体或完全定位在脂筏内的cav-tmTNF-α突变体的效应细胞都具有相似的胞毒效应，三者间无明显差异，提示效应细胞脂筏内外的tmTNF-α均可有效杀伤靶细胞，即tmTNF-α杀伤靶细胞与其是否定位脂筏无关。2．1．2破坏ICAM-1脂筏定位抑制效靶细胞粘附，从而阻断tmTNF-α胞毒效应：用MCD破坏脂筏则所有ICAM-1分布至脂筏外，效／靶细胞粘附下降，tmTNF-α胞毒作用几乎被完全阻断，而ICAM-1中和抗体可部分抑制效／靶细胞之间的粘附，并部分阻断tmTNF-α胞毒作用。提示MCD阻断tmTNF-α胞毒效应是由于破坏粘附分子脂筏定位，进而抑制效／靶细胞粘附所致。2．1．3破坏脂筏导致sTNF-α分泌增加：用MCD破坏Raji细胞的脂筏，可导致部分本来分布在脂筏内的TACE转至脂筏外，sTNF-α产生明显增加。2．2靶细胞脂筏对tmTNF-α正向信号介导的生物学效应的影响2．2．1 tmTNF-α刺激诱导靶细胞TNFR1向脂筏内聚集：用固定的高表达tmTNF-α的Raji细胞作用于T24细胞，可见T24细胞大量TNFR1从脂筏外转移至脂筏内，此结果提示TNFR定位在脂筏可能影响tmTNF-α的正向信号。2．2．2 sTNF-α及脂筏内外的tmTNF-α对靶细胞脂筏内外TNFR1的作用：用MCD破坏靶细胞脂筏导致sTNF-α胞毒效应显著增加，却明显抑制wt tmTNF-α、C-47AtmTNF-α和cav-tmTNF-α的杀伤作用，并且三者间没有明显差异。提示靶细胞脂筏在sTNF-α和tmTNF-α的胞毒效应中所起作用不同，而效应细胞脂筏则与tmTNF-α正向信号无关。本研究证实tmTNF-α能定位在脂筏，且依赖于其-47位半胱氨酸。tmTNF-α的反向信号传递依赖其脂筏定位；而tmTNF-α正向信号传递则与效应细胞脂筏无关，却与靶细胞TNFR的脂筏定位相关。更多还原

【Abstract】 Part 1:Construction and Function Research of Transmembrane TNF-αMutants Deleted Catalytic SiteTransmembrane tumor necrosis factor-α(tmTNF-α) is the precursor of sTNF-αand themolecular weight is 26kD,which is mainly expressed in activated monocytes and immunecells.As a transmembrane protein tmTNF has a 76 amino signal peptide which composesof a hydrophobic domain compared to sTNF-α.The molecule expressed on the cell surfacecan be catalyzed by TNF-converting enzyme (TACE/ADAM17) to release sTNF-α.When we study the biological function of tmTNF-α,we get the integrated effects ofboth tmTNF-αand sTNF-αbecause it is often difficult to avoid the catalysis of tmTNF-α.Itis necessary to generate a mutant which can not release sTNF-αbecause the digested site ismutated.Although the mutant deleted the site of 1 to 12 lost the ability to be catalyzed,thecytotoxicity of which were also affected.Our lab found that the mutant also had defects inreverse signal transduction.So this mutant is not an ideal mutant for research.ThetmTNF-αmutant deleted the sites of 1 to 9 and mutated in the site of 11 from K to E caninhibit the function of TACE,and its cytotoxicity is same to wild-type tmTNF-α.It isunclear whether this mutant effects the reverse signal transduction.In this study,we aim tomutate the digestion site of TACE using site-directed mutagenesis method and to study theforward and reverse signal transduction of the mutants.1 We successfully constructed mutants lack catalysis site for TACE asΔ1-12 tmTNF-αandΔ1-9,K11E tmTNF-αby using overlap PCR technology.It was confirmed thatthere were absence of any other point mutations,frame-shift and deletion mutations butexpected mutations by restriction enzyme digestion,PCR and sequencingidentification.2 Hela cells were transfected with tmTNF-αand mutants and total proteins wereextracted.A specific band about 26kD was detected in wide type tmTNF-αgroup byWestern blot.A specific band about 25kD was detected in mutants groups. 3 The supernatant of Hela transfected cells with wild type tmTNF-αand mutants wascollected to detect the content of sTNF-α.We found that wt tmTNF-αgroup cansecrete large amount of tmTNF-αand there was not detected sTNF-αcompletely in themutants transfected grouPs.Both of the mutants can inhibitor the digestion of TACE.4 Hela cells tranfected with wt tmTNF-αand mutants were fixed with paraformaldehydeto kill the T24 cells.We found that groups both tranfected with wt tmTNF-αandΔ1-9,K11E tmTNF-αcan significantly induce cell death and the cytotoxicity ofΔ1-12tmTNF-αwas reduced nearly half of other groups.It was shown thatΔ1-9,K11EtmTNF-αmutant has retained the ability to induce cytotoxicity.5 Western blot was used to detect the IκB-αexpression of Hela cells transfected withtmTNF-αand its mutants.IκB-αdegraded in the cells transfected with wt tmTNF-αandΔ1-9,K11E tmTNF-α,meanwhileΔ1-12 tmTNF-αcan not degrade IκB-αtoactivate NF-κB.The results showed thatΔ1-9,K11E tmTNF-αnot only lost the ablity to releasesTNF-αbut also can retain the ability to transduce forward and reverse signals.It’s a betterand power tool to research the bio-function of tmTNF-αthanΔ1-12 tmTNF-α.Part 2:Lipid Rafts and the Bidirectional Signaling of tmTNF-αRecent years,Lipid rafts have been found as the existence of the micro-structure in themembrane.They are distinct from classical lipid bilayer membrane and rich of cholesteroland phosphatidylinositol.Many receptor molecules and their associated signalingmolecules can be located in lipid rafts.Because lipid rafts in the plasma membranedistributes scattered and can lateral drift to gather to form a platform for signal transductionwhich is involved in activation of receptor signal transduction and the intracellular signaltransmission.tmTNF-αnot only can bind to TNFR of target cells to induce signal transduction whichis termed forward signal,but also can act as a receptor itself to accept external signals andtransmit intracellular signals reversely.It has been reported that -47 site is palmitoylated.We speculated that tmTNF-αmay be partitionated to lipid rafts and the lipid rafts may be related to the location-related signal transduction.In addition it was reported that TNFR canlocalized to lipid rafts,but the relationship between tmTNF-αand the lipid raftslocalization of TNFR is not clear now.In order to clarify the relationship between tmTNF-αand the lipid rafts,we confirm thelipid rafts localization of tmTNF-αusing sucrose density gradient centrifugation.We studythe functions of lipid rafts in tmTNF-αforward and reverse signal transduction via twoways including effector cells expressed tmTNF-αand the target cells expressed TNFR.1 Lipid rafts and reverse signaling of tmTNF-α1.1 tmTNF-αcan localize in the lipid rafts.The lipid rafts of Raji cells which have highexpression of tmTNF-αwere separated using the mothod of sucrose density gradientcentrifugation and were detected by Western blot.A part of tmTNF was within the lipidrafts.Raji cells were treated with 10mM MCD for 45min to destroy lipid rafts,alltmTNF-αwere distributed outside of lipid rafts.1.2 Disruption of lipid rafts enhanced the cytotoxicity of sTNF-αsensitivity.The highexpression of tmTNF-αon Raji cells can induce sTNF-αtolerance.After lipid rafts weredisrupted,the cytotoxicity of sTNF-αincreased markedly.T24 ceils which have noneexpression of tmTNFs were sensitive to sTNF-αand sTNF-αcytotoxicity also enhancedafter lipid rafts were damaged,but the increasement was significantly lower than Rajicells1.3 Destruction of lipid rafts resulted in decreased activity of NF-κB.When Raji cellswere treated with MCD the level of IκB-αsignificantly become higher which indicatedthe inhibition of NF-κB signal pathway.But it was not change significantly for T24cells.It was shown that the destruction of lipid rafts can weaken reverse signaltransduction of tmTNF-α.1.4 Construction the mutants only in or outside of the lipid rafts and establish stablecell lines.We constructed a mutant named cav-tmTNF-c which can locate only in lipidrafts and another mutant C-47A tmTNF-αwhich only partitionate out of lipid rafts.T24cells were stably transfected with wild type tmTNF-αand the mutants and theirlipid rafts were extracted to confirm the location. 1.5 tmTNF-αwithin lipid rafts induced sTNF-αtolerance.We observed thecytotoxicity of stable cell lines induced by sTNF-α.Results showed that parental T24cells,the cells transfected with empty vector and C-47A tmTNF-αwere induced about50% of cell death by sTNF-α.The cytotoxicity of stable cells transfected with wttmTNF-αwas significantly decreased.The results showed that only tmTNF-αin lipidrafts may resist to sTNF-αinduced death death.1.6 NF-κB pathway can be constructively activated by tmTNF-αin the lipid rafts.Itwas found that the level of IκB-αwas degraded and the level of phosphorylated p65was increased constructively in cell lines stable transfected with wt tmTNF-α.Thelevel of IκB-αand p-p65 had no significant changes for the C-47A tmTNF-αcompared with the control groups.1.7 tmTNF-αin lipid rafts can up-regulate the expression of cIAP1,down-regulatethe expression of Bax.Realtime PCR was used to detect gene expressions.It wasfound that in wt tmTNF-αgroup anti-apoptosis genes cIAP1 expression increasedsignificantly and the pro-apoptotic gene Bax gene transcription were obviouslyinhibited compared with the control group.But these two genes of C-47A are nosignificant difference from that of the control group.1.8 CKI inhibitor D4476 is helpful for tmTNF-αin lipid rafts to resist tosTNF-αinduced cytotocity.CKI-specific inhibitor can inhibit the phosphorylation oftmTNF-αand enhance the reverse signal transduction.It was observed that wttmTNF-αtransfected cells can resist to sTNF-αinduced cytotoxicity.CKI inhibitorD4476 can enhance the role of the resistance,but it had no significant effect to C-47AtmTNF-αtransfected cell.1.9 Lipid rafts as a platform to transmit tmTNF-αreverse signals.The reverse signalof tmTNF-αcan be activated by sTNFR2 stimulation and transfection of tmTNF-αinthe lipid rafts,at the same time related signal molecules can be recruited to lipid rafts.The activation of reverse signal can induced the aggregation of tmTNF-αTRAF1IKK-αand p52 to lipid rafts.The molecules translocation from outside to inside of lipid rafts can not been seen for the C-47A tmTNF-αtransfected cells.It is suggestedthat Lipid rafts as a platform to transmit tmTNF-αreverse signals.2 Lipid rafts and forward signal of tmTNF-α2.1 Effect of lipid rafts to biological functions tmTNF-αmediated by forward signal.2.1.1 Lipid rafts location of tmTNF-αhas no relationship to its cytotoxicity.ThetmTNF-αcytotoxicity to T24 cells was almost completely blocked as inhibited withTNF-αAb when Raji cells were treated with MCD to destroy lipid rafts.In order torule out the possibility of non-specific effects of MCD,we use Hela cells transientlytransfected with mutants which only locate in or out of lipid rafts to kill the T24 cells.The results showed that the effector cells transfected with wild-type and mutanttmTNF-αhave similar cytotoxicity and there’s no significant difference among thethree mutants.It was shown that tmTNF-αwithin and outside lipid rafts both hadcell-killing ability,the tmTNF-αcytotoxicity has no relation to lipid rafts location.2.1.2 Destruction of ICAM-1 location in lipid rafts led to decline of adhesionbetween target and effetor cells and blocked the cytotoxicity of tmTNF-α.Becausethe cytotoxicity of tmTNF-αis dependent on cell-cell contact which is often mediatedby adhesion molecules.We further observed the relationship between lipid rafts,ICAM-1 and anti-tumor effect of tmTNF-α.The results showed that some of ICAM-1distributes in lipid rafts in resting state and all ICAM-1 target to non-rafts when lipidrafts were destroyed by MCD.Also the cytotoxicity to t24 ceils was almost completelyobstructed and the adhesion between target and effector cells was decrease.ICAM-1antibodies only partially inhibited the adhesion between the target and effector cellsand partially blocked the cytotoxicity of tmTNF-α.2.1.3 Destruction of lipid rafts resulted in increased secretion of sTNF-α.We furtherobserved the effect of lipid rafts destruction on the generation of sTNF-α.lipid raftsdamagement of Raji cells with MCD can lead to TACE,some of which have beenfound in lipid rafts,to the out of lipid rafts,and sTNF-αincreased significantly.2.2 Lipid rafts affected the bio-function of tmTNF-αmediated by forward signals2.2.1 tmTNF-αcaused TNFR1 of target cells recruit to lipid rafts.It was found that a small part of TNFR1 of T24 cells located in lipid rafts in resting state.A large numberof TNFR1 from outside to lipid rafts when we engaged fixed Raji cells to T24 cells.This result suggested that TNFR recruitment to lipid rafts may affect the forward signalof tmTNF-α.2.2.2 sTNF-αand tmTNF-αinside or outside of lipid rafts have the different effectto TNFR1 inside or outside of lipid rafts.Cytotoxicity of sTNF-αincreasedsignificantly when lipid rafts were destroyed by MCD,but that of tmTNF-αsignificantly decreased and there was no significant difference among the three mutants.These results suggest that lipid rafts played the different roles in sTNF-αand tmTNF-αinduced cytotoxicityOur study revealed that tmTNF-αcan target to lipid rafts which rely on cysteine on thesite of -47.Reverse signal tranduction of tmTNF-αdepends on its lipid raft localization.For effector cells,tmTNF in or out of lipid rafts have no difference in forward signaltransduction.But lipid rafts of target cells play an important role in forward signaltransduction of tmTNF-α.更多还原