【作者】 师伟；

【导师】 张嘉宁；

【作者基本信息】 大连医科大学 ， 生物化学与分子生物学， 2009， 硕士

【摘要】 背景与目的:Caveolae(胞膜窖)是细胞质膜向内凹陷的一种结构,于1953年由G.E.Palade首次通过电子显微镜在毛细血管内皮中发现。胞膜窖能够介导胞吞、胞饮、胆固醇转运和信号转导。胞膜窖的标志性蛋白是窖蛋白家族(Caveolins),包括Caveolin-1(α,β),Caveolin-2 (α,β,γ),Caveolin-3。其中Caveolin-1广泛的存在于组织细胞中,Caveolin-2与Caveolin-1的分布基本相同,Caveolin3主要位于肌细胞中。研究表明Caveolin-1是细胞内许多信号分子相互作用的枢纽,它可通过影响多种信号通路进而影响细胞的增殖、转化、迁移和转移、细胞生存与死亡等细胞行为。目前发现Cav-1具有磷酸化位点,棕榈酰化位点[6],跨膜区,脚手架区(CSD)等功能域,它们在细胞生物学行为中发挥着各自不同的作用。例如:Cav-1可通过CSD区域与相关蛋白作用,进而影响相关蛋白的活性。Cav-1的表达对caveolae形成形态学上的结构起着必要的作用,且在caveolin-1存在的条件下,caveolae通过调节细胞对信号分子的摄取及信号的持续时间进一步影响其下游分子[11]。CD147分子是一种糖基化的(glucosylated)免疫球蛋白超家族(immunoglobulin Superfamily,IGSF)成员,是具有单次跨膜结构的糖蛋白。CD147为N-连接糖蛋白,其糖基化形式主要两种,低糖基化(LG-CD147)和高糖基化(HG-CD147,~32kDa),糖基化形式与肿瘤恶性程度及淋巴道转移潜能有关。CD147又称为基质金属蛋白酶诱导因子(extracellular matrix metalloproteinase inducer,EMMPRIN),能够诱导基质金属蛋白酶(MMPs)在间质和肿瘤细胞内表达并分泌出来。大部分MMPs几乎能够降解细胞外基质和基底膜的全部成分,在肿瘤侵袭和肿瘤转移中发挥着重要的促进作用[12,13]。目前认为HG-CD147在促进MMPs分泌和表达方面比LG-CD147具有更积极的作用。本课题组研究结果表明,在小鼠肝癌细胞HCa-F(高淋巴道转移)中Cav-1表达量高于HCa-P(低淋巴道转移),而在Hepa 1-6(无淋巴道转移)中无Cav-1的表达,提示Cav-1在小鼠肝癌细胞淋巴道转移中可能发挥作用。进一步通过RNA干扰下调Hca-F细胞中Cav-1的表达时,与对照相比,HG-CD147表达减少,LG-CD147表达增多;同时,MMPs-11表达下调,细胞体外迁移及侵袭能力下降。本文旨构建Cav-1的不同功能域突变体重组真核表达载体,研究其在Hepa1-6细胞中表达、细胞内的定位及对CD147糖基化的影响。方法:1.突变Cav-1的14位磷酸化位点(记为Y14F),133,143和156位棕榈酰化位点(记为C133A,C143A,C156A),删除133-178位氨基酸(记为△133-178),删除跨膜区102-134位氨基酸(记为△TMD),并将突变体分别与pMD18-T simple vector进行连接,记为T/Cav-1(Y14F), T/Cav-1(C133A), T/Cav-1(C143A), T/Cav-1(C156A), T/Cav-1(△133-178), T/Cav-1(△TMD)。再经转化、抗生素初步筛选、限制性核酸内切酶酶切和DNA测序获得初步重组质粒,再经过限制性核酸内切酶酶切、琼脂糖凝胶电泳、DNA纯化,将纯化的DNA产物与真核表达载体pcDNA3.1连接,获得Caveolin-1突变体重组质粒分别记为pcDNA3.1/Cav-1(Y14F), pcDNA3.1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178),pcDNA3.1/Cav-1(△TMD),进行DNA序列分析。2.利用脂质体转染的方法,将以上Caveolin-1突变体重组真核表达载体分别转染至小鼠肝癌细胞Hepa1-6中,记为Hepa1-6/Cav-1(Y14F), Hepa1-6/Cav-1(C133A) Hepa1-6/Cav-1(C143A), Hepa1-6/Cav-1(C156A), Hepa1-6/Cav-1(△TMD) , Hepa1-6/Cav-1(△133-178),以转染空载体pcDNA3.1的Hepa1-6细胞(记为Hepa1-6/mock)与Hepa1-6细胞作对照。3.Western blotting检测瞬时转染Caveolin-1及其突变体后, Hepa1-6细胞内CD147糖基化的改变。结果:1.经XhoLI/EcoRI双酶切和测序鉴定证实,成功构建并获得6种Caveolin-1突变体重组真核表达载体分别记为pcDNA3.1/Cav-1(Y14F), pcDNA3.1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178),pcDNA3.1/Cav-1(△TMD)。2.经免疫细胞化学和Western blotting的方法证明,以上Caveolin-1突变体重组真核表达载体均能在Hepa1-6细胞内瞬时表达,并富集于胞浆内,但Cav-1(Y14F)表达水平较低。3.与Hepa1-6/mock相比, Hepa1-6/Cav-1(wt)及Hepa1-6/Cav-1(Y14F)细胞中, LG-CD147基本消失;而转染其余突变体的细胞,LG-CD147和HG-CD147表达均未见显著变化。4. Cav-1(△TMD)蛋白表达在Hepa1-6细胞内36小时左右达高峰,但随后蛋白表达量逐渐减少,至96小时完全消失;但是蛋白酶体抑制剂MG132处理使Cav-1(△TMD)蛋白表达增加。结论:1.构建的6种小鼠Caveolin-1突变体重组真核表达载体pcDNA3.1/Cav-1(Y14F),pcDNA3.1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178),pcDNA3.1/Cav-1(△TMD),在小鼠肝癌细胞Hepa1-6中实现瞬时表达,其Caveolin-1突变体蛋白主要定位于细胞浆中。2.Caveolin-1第14位酪氨酸磷酸化修饰没有影响CD147的糖基化,但133,143,156位半胱氨酸的棕榈酰化修饰、跨膜区以及133-178位胞内羧基端可能促进胞内LG-CD147向HG-CD147的转变,而133-178位胞内羧基端对CD147糖基化的影响可能是通过三个棕榈酰化修饰位点来实现的。3.首次发现Caveolin-1跨膜区可以保护Caveolin-1不被泛素—蛋白酶体途径降解,可能对Caveolin-1在胞内的稳定性有一定贡献。更多还原

【Abstract】 Objective:Caveolaes are invaginations of the plasma membrane that were firstly described in 1953 in the Capillary Endothelium by G.E. Palade. Caveolae mediates endocytosis, pinocytosis, cholesterol homeostasis and signal transduction. There are three identified caveolin proteins, Caveolin-1(α,β), Caveolin-2(α,β,γ) and Caveolin-3, which serve as markers for identification of caveolae and are critically involved in caveolae function. Caveolin-1 expresses in most cell types. Caveolin-2 co-localizes with Caveolin-1 and is found in the same cells as Caveolin-1. Caveolin-3 is a muscle specific caveolin and is found in muscle cells.Many results show that Caveolin-1 is a nodal point of many signal molecules. Caveolin-1 plays important roles in cell proliferation,transfor- mation,apoptosis and metastasis. There are some modified sites on Cave- olin-1, for instance, phosphorylation of Tyr14, palmitoylation of Cys133, Cys143 and Cys156. There are some other domains such as transmembrane domain and Caveolin scaffolding domain(CSD) which play different actions in cell biological behaviours. For example, Caveolin-1, through its CSD, is able to bind to upstream and downstream components of cell signaling pathways and then change activities of some proteins. It is now known that Caveolin-1 expression is sufficient and necessary to drive the formation of morphologically identifiable caveolae. Caveolae affects downstream molecules through modulating the internalization of signal molecules and the duration of signal.CD147 belongs to the highly glycosylated immunoglobulin super- family transmembrane protein, enriched on the surface of many malignant tumor cells. As a result of heterogeneous N-glycosylation, CD147 exists in both a highly glycosylated form, HG-CD147 and lowly glycosylated form, LG-CD147. CD147 mediates the interaction of tumor cells and matrix, and is responsible for inducing the expression of fibroblasts and tumor cells themselves MMPs, accelerating angiogenesis which promote tumor metastasis.Our previous study showedCaveolin-1 expression levels were higher in Hca-F cells than that in Hca-P and Hepa1-6 cells which have character of high lymphatic metastatic potential, low lymphatic metastatic potential and no lymphatic metastasis potential respectively. And the expression of caveolin-1 was undetectable in Hepa1-6 cells. These results indicate that caveolin-1 may play a role in Hepatocacinoma cell lines lymphatic metastasis. When caveolin-1 expression was down-regulated specifically in Hca-F cells using RNAi technology, Western blot analysis showed that the Caveolin-1 levels were significantly reduced in Hca-F/RNAi cells. HG- CD147 expression was obviously decreased while LG-CD147 expression showed a marked increase.The main aim of this study was to construct Cav-1 mutants expression vectors, to express Cav-1(wt) and mutants in Hepa1-6 cells and observe their location and their effect on CD147 glycosylation.Methods:Cav-1 mutants of phosphorylation site mutant (Y14F), palmitoylation site mutant (C133A, C143A, C156A), 133-178 residues at C-terminal cyto- solic domain deletion (△133-178) and 102-134 residues of transmembrane domain deletion(△TMD) were constructed. The above mutants were subcloned into pMD18-T simple vector, and then transformed into E.coli. DH5a. The positive clones were selected by PCR, restriction enzyme ana- lysis and DNA sequencing. The positive plasmid DNA was subcloned into expression vector pcDNA3.1/Myc-His(-)B and the recombinant plasmids were identified by restriction enzymes, PCR and DNA sequencing.Recombinant plasmids,pcDNA3.1/Cav-1(Y14F), pcDNA3.1/Cav-1 (C 133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178), pcDNA3.1/Cav-1(△TMD) were transfected into Hepa1-6 cells by liposome, respectively. Untransfected cells and trasfected pc DNA3.1/Cav-1(wt) or empty vectors cells were used as controls.The Cav-1 expression and CD147 glycosylation in Cav-1 mutants were revealed by immunocytochemistry and Western blot analysis.Results:1. The recombinant plasmids of pcDNA3.1/Cav-1(Y14F), pcDNA3. 1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178), pcDNA3.1/Cav-1(△TMD) were constructed successfully. The temporarily expressed Cav-1 mutants proteins were found in the cytosol of Hepa1-6 cells, but the expression level of Cav-1(Y14F) was lower.2. The expression of LG-CD147 protein was undetectable. But this changes of CD147 glycosylation in Hepa1-6/Cav-1(wt) cells were not also found among Hepa1-6 cells transfected pcDNA3.1/Cav-1(C133A), pc DNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178).3. The expression of Cav-1(△TMD) protein reached top level at 36h after transfection and then the protein level of Cav-1(△TMD) decreased and disappeared at 96h. The reduction of Cav-1(△TMD) protein was inhibited by treatment Heap1-6/ Cav-1(△TMD) with 0.2μM of proteosome inhibitor MG132.Conclusions:1. 6 recombinant Cav-1 mutant plasmids of pcDNA3.1/Cav-1(Y14F), pcDNA3.1/Cav-1(C133A),pcDNA3.1/Cav-1(C143A),pcDNA3.1/Cav-1(C156A),pcDNA3.1/Cav-1(△133-178), pcDNA3.1/Cav-1(△TMD) expressed in Hepa1-6 cells and the proteins of those Cav-1 mutants mainly localized in the cytosol of Hepa1-6 cells.2. The palmitoylation rather than phosphorylation of Cav-1 may contri- bute to CD147 glycosylation in Hepa1-6 cells.3. Transmembrane domain of Cav-1 protects Cav-1 protein from prote- olysis by ubiquitin-proteosome pathway.更多还原