【作者】 闫媛；

【导师】 杨渝珍；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 目的构建含有多对靶向TACE的小发夹结构RNA(shRNA)的真核表达载体，观察干扰TACE表达对人子宫颈癌HeLa细胞增值、凋亡、粘附和侵袭的影响。方法设计针对TACE基因的4个shRNA序列，构建分别含有单对shRNA真核表达载体pG1-T1，pG1-T2，pG1-T3和pG1-T4和同时含有这四对shRNA的干扰载体pG4-T。通过酶切和测序等方法进行鉴定。转染HeLa细胞后，用RealtimePCR和WesternBlot的方法检测其对HeLa细胞TACE基因表达的影响；用ELISA检测细胞分泌的sTGF-α和Western Blot检测EGFR磷酸化水平。利用MTT方法检测细胞增值活性，流式细胞术检测细胞凋亡，最后通过细胞粘附和侵袭实验观察细胞粘附和侵袭功能的变化。结果构建出的针对TACE基因的三个有效的特异性真核表达载体pG1-T1，pG1-T2，pG1-T3，均在不同程度上抑制了HeLa细胞TACE基因的表达。同时含有四对shRNAs的载体pG4-T表现更强的干扰率。同时发现干扰TACE基因后，HeLa细胞的生长受到抑制，同时凋亡率升高，粘附和侵袭能力均降低，与对照组相比有显著性差异(P＜0．05)。结论多个shRNA的同时干扰一个或多个基因，在预防和治疗肿瘤上是一个强有力的策略。同时，针对TACE的siRNA干扰载体在肿瘤治疗中有可能带来治疗性的效果。目的为了解人TACE的解整合索域对肿瘤细胞粘附和侵袭的影响以及其独立于其他结构域发挥作用的能力。方法(1)重组TACE解整合素域、酶催化域和胞外功能结构域原核表达重组体的构建、表达、纯化和鉴定：从单核细胞系THP1中提取RNA，RT-PCR扩增出TACE全长基因，构建了PMD18T-TACE载体。对PMD18T-TACE质粒进行PCR，分别扩增T300(300bp，编码解整合素域)、T800(800bp，编码酶催化域)及T1300(1300bp，编码胞外域)三个片段，将其分别亚克隆至表达载体pET-28a(+)。将pET-28a-T300、pET-28a-T800和pET-28a-T1300转化大肠杆菌BL21(DE3)，经IPTG诱导表达出带有His-tag的目的蛋白(T300、T800和T1300)，三者均为包涵体。经过溶解包涵体、BBST NTA树脂柱亲和层析并透析复性，获得高纯度的三种活性蛋白。(2)重组蛋白的功能研究：细胞粘附实验、Transwell小室侵袭实验分别检测重组蛋白对A549细胞粘附、侵袭功能的作用。(3)系列突变重组体及稳定转染HeLa细胞株的建立及生物活性研究：以PMD18T-FL-TACE为模版，应用重叠延伸PCR扩增出缺失解整合素区的TACE片段，利用DNA重组技术将其分别定向插入真核表达在pIRES2．0-EGFP中，同时TACE全长也从PMD18T-TACE上双酶切下来构建入pIRES2．0-EGFP。这两种种真核表达载体经酶切和测序鉴定后，用脂质体法转染HeLa细胞，经过G41 8筛选，获得稳定转染的HeLa细胞系，采用Western blot及荧光法检测表达。将稳定转染细胞分别进行MTT，细胞粘附和侵袭实验检测，观察其增殖、粘附和侵袭能力的变化。结果(1)成功构建了TACE解整合素结构域、酶结构域和胞外结构域的原核表达载体pET-28a-T300、pET-28a-T800和pET-28a-T1300。镍柱亲和层析后得到重组蛋白T300、T800和T1300。(2)重组蛋白的体外功能研究：细胞粘附实验、Transwell小室侵袭实验分别显示TACE解整合素域、胞外域蛋白均能明显以剂量依赖性的方式抑制A549细胞与纤维粘连蛋白的粘附，并能抑制Transwell小室中A549细胞对Matrigel模拟的天然基底膜的侵袭，而酶结构域蛋白则无相应的抑制能力。(3)成功构建了pIRES-EGFP／FL-TACE、pIRES-EGFP／disΔ-TACE真核表达载体，并建立了稳定转染的HeLa细胞系，成功地表达了目的基因。实验证明稳定转染的细胞的增殖、粘附和侵袭能力增强，其中转染了pIRES-EGFP／FL-TACE的细胞的改变更显著。结论表明重组的TACE解整合素域蛋白可影响肿瘤细胞的粘附和侵袭，为深入研究该区域的作用及TACE在肿瘤发病中的机制提供了新的认识。稳定表达TACE和缺失解整合素TACE片段的HeLa细胞的建立，为进一步研究TACE的功能提供了细胞模型，同时也证明了解整合素域在TACE分子在肿瘤细胞中发挥更有效的作用中是必不可少的部分。目的探讨RNAi抑制小鼠ADAM10和PC7基因表达对H22细胞小鼠体内成瘤能力的影响。方法将含有两对针对ADAM10和两对针对PC7的干扰载体PG4-ADAM10／PC7转染小鼠肝癌细胞H22，RT-PCR检测转染前后ADAM10和PC7的mRNA水平的改变。观察PG4-ADAM10／PC7对小鼠的负瘤局部注射后负瘤的生长变化。免疫组化检测瘤组织中ADAM10和PC7蛋白表达的变化。结果RT-PCR检测转染组ADAM10和PC7基因mRNA的表达水平下调(P＜0．05)。注射PG4-ADAM10／PC7的小鼠负瘤生长缓慢，与对照组瘤体积比较差异有显著性(P＜0．05)；采用免疫组化法检测注射PG4-ADAM10／PC7组，ADAM10和PC7蛋白的表达下调(P＜0．05)。结论PG4-ADAM10／PC7可抑制ADAM10／PC7基因表达．对负瘤的生长有抑制作用。更多还原

【Abstract】 Objective To construct the shRNA eukaryotic expression vectors of TACE geneand to investigate their inhibiting in cellular functions related to tumor malignancy.Methods A single vector containing four shRNAs that target four regions of theTACE gene was constructed.After transfection with our multiple shRNAs vector,Realtime quantitative PCR and Western blot analysis were used to measure the TACEmRNA and protein expression.Four shRNA vectors each containing one shRNA werealso constructed and compared their ability to silence TACE gene expression with thatof the multiple shRNAs vector.The level of proliferation in HeLa cells transfectedwith different shRNA vectors were compared using the MTT assay.The FlowCytometry was used to examine apoptosis.The adhesive and invasive ability weretested by plated adhesion model and Transwell assay.At last,ELISA kit and Westernblot analysis respectively evaluated sTGF—αand the activation of EGFRs aftertransfection of TACE shRNA vectors.Results We found that in HeLa cells our multiple shRNAs vector produced a higherlevel of TACE knockdown than any single shRNA vector containing only one TACEshRNA.Silencing TACE expression in HeLa cells decreased their malignancy bydecreasing the proliferation,adhesion and migration,as well as inducing apoptosis inthese cells.Furthermore,our data suggest that the effects of TACE on the malignancyof HeLa cells may be mediated via activation of the EGFR（epidermal growth factorreceptor）signaling pathway.Conclusion Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy fortumors. Objective:To explore effects ofdisintegrin domain of TACE on the proliferation,adhesion and invasion potential of tumor cells in vitroMethods:（1）The construction,expression and purification of prokaryoticexpression plasmids of TACE:The total RNA wasisolated from THP1 cell.TACEcDNA was amplified by RT-PCR and subcloned into PMD18-T vector to constructPMD18T—TACE vector.Primers were designed and synthesized according to thepublished cDNA sequence of TACE.The amplified PCR products（T1300,T591）were ligated into the pET-28a（+）prokaryotic expression vector digested with the samerestriction enzymes to create pET-28a-T300,pET-28a-T800 and pET-28a（+）-T1300,which were transformed into E.coli BL21（DE3）.After induced by IPTG,therecombinant proteins were purified using a Ni²⁺-chelating resin affinity column.Thepurified productions were analyzed by SDS-PAGE and Western blot.（2）Thebiological activites of the proteins in vitro.The adhesive and invasive ability wereexamined by plated adhesion model and Transwell assay.（3）The full-length TACEcDNA fragment was amplified by PCR from the human THP1 cell cDNA andsubcloned into pMD18T vector to construct PMD18T-TACE vector.The cDNAfragment of dis△-TACE and met△-TACE were amplified from plasmidPMD18T-TACE by using the overlap extension PCR and cloned into pIRES2.0-EGFPto construct the expression vector pIRES2.0-EGFP/FL-TACE andpIRES2.0EGFP/dis△-TACE.The full-length of TACE was obtained by doubledigesting the plasmid pMD18T-TACE and then inserted into pIRES2.0-EGFP.Thesethree eukaryotic expressing vectors were transfected into HeLa cells by Lipofectamine 2000 after identification of digestion and sequencing.The stabletransfected HeLa cell lines were then established by screening culture withG418.And the transcription and expression of TACE and dis△-TACE wereidentified by Western blot and immunofl uorescence.The proliferation,adhesion andmigration ability of stable transfected HeLa cells were detected by MTT,adhesionand migration test.Results:（1）SDS-PAGE and Western blotting analysis revealed that there proteinsof about 18KD,37KD and 55KD were expressed and recognized by anti-His Abrespectively,and the purity of the proteins purified by Ni²⁺-NTA resin was more than90%.（2）The protein pET28a（+）-T300 and pET28a（+）-T1300 can reduce the adhesionand invasion ability of the human lung carcinoma cell A549 in vitro,but otherwisethe pmtein pET-28a（+）-T800 had not shown the inhibitive function.（3）Theeukaryotic expression vector pIRES2.0-EGFP/FL-TACE andpIRES2.0-EGFP/dis△-TACE were constructed successfully,stable transfected HeLaeelllines were established,and the aim proteins were expressed successfully.Conclusion:The disintegrin domain of TACE have the similar biological function toother disintegrins,which can be used for further research on function of TACE ininflammation and tumor. Objective To investigate the influence on growth of transplanted tumor byshRNA expression vector PG4-ADAM10/PC7 inhibiting expression of ADAM10 andPC7 gene in vitro.Methods The vector was transfected into H22 and the mRNA expression ofADAM10 and PC7 of H22 before and after transfection was detected byRT-PCR.The tumor—beating mice were treated by PG4-ADAM10/PC7.Results The RNA expressions of ADAM 10 and PC7 gene were down-regulated in thetransfected group of RT-PCR assay.The transplanted tumors were grown slowly intransfected PG4-ADAM10/PC7 mice and comparing with the volume of controlgroup,there was significant difference（P＜0.05）.Using immuno-histochemistrymethod to detect injected PG4-ADAM10/PC7 goup,PG4-ADAM10/PC7 expressionwere down-regulated（P＜0.05）.Conclusions PG4-ADAM10/PC7 can inhibitory action for the growth of transplantedtumors.更多还原