中华临床实验室管理电子杂志
中華臨床實驗室管理電子雜誌
중화림상실험실관리전자잡지
2014年
1期
38-44
,共7页
方旭前%刘湘帆%姚玲%顾志冬%陈长强%倪培华%郑新民%樊绮诗
方旭前%劉湘帆%姚玲%顧誌鼕%陳長彊%倪培華%鄭新民%樊綺詩
방욱전%류상범%요령%고지동%진장강%예배화%정신민%번기시
剪接突变体%酪氨酸激酶%黏着斑激酶%肿瘤
剪接突變體%酪氨痠激酶%黏著斑激酶%腫瘤
전접돌변체%락안산격매%점착반격매%종류
Splicing mutant%Protein tyrosine kinase%Focal adhesion kinase%Tumor
目的:探讨外显子33缺失型黏着斑激酶(focal adhesion kinase,FAK)突变体(FAK-Del33)的亚细胞定位及生物学特性。方法采用基础实验研究设计。将野生型FAK以及突变型FAK-Del33分别构建到pEGFP表达载体,并转染乳腺癌细胞株,以研究分析其细胞表达和定位情况。另构建过表达FAK或FAK-Del33的MDA-MB-468乳腺癌稳转细胞株,研究评价其细胞表达和体外克隆形成能力。结果构建FAK-GFP融合蛋白表达载体转染MDA-MB-468后的细胞定位显示,野生型FAK在胞浆中弥散分布,而FA K突变体蛋白在胞浆中呈点状不均匀分布,并聚集成簇。经限制性内切酶酶切分析与测序分析,成功构建出过表达FAK的慢病毒载体,以293T细胞包装的重组慢病毒能够高效感染乳腺癌细胞;经抗生素puromycin筛选和荧光定量PCR鉴定,成功筛选到稳定表达FAK的细胞株。在软琼脂克隆形成实验中,FAK野生型的克隆形成数为(335±48)/1000个,FAK突变体为(735±91)/1000个,后者对MDA-MB-468乳腺癌稳转细胞株的增殖能力高于前者(t=9.437, P<0.01)。结论初步研究表明外显子33缺失可改变FAK的亚细胞定位;并能增强肿瘤细胞株的体外克隆形成能力,这种FAK突变体可能参与肿瘤的发生发展。
目的:探討外顯子33缺失型黏著斑激酶(focal adhesion kinase,FAK)突變體(FAK-Del33)的亞細胞定位及生物學特性。方法採用基礎實驗研究設計。將野生型FAK以及突變型FAK-Del33分彆構建到pEGFP錶達載體,併轉染乳腺癌細胞株,以研究分析其細胞錶達和定位情況。另構建過錶達FAK或FAK-Del33的MDA-MB-468乳腺癌穩轉細胞株,研究評價其細胞錶達和體外剋隆形成能力。結果構建FAK-GFP融閤蛋白錶達載體轉染MDA-MB-468後的細胞定位顯示,野生型FAK在胞漿中瀰散分佈,而FA K突變體蛋白在胞漿中呈點狀不均勻分佈,併聚集成簇。經限製性內切酶酶切分析與測序分析,成功構建齣過錶達FAK的慢病毒載體,以293T細胞包裝的重組慢病毒能夠高效感染乳腺癌細胞;經抗生素puromycin篩選和熒光定量PCR鑒定,成功篩選到穩定錶達FAK的細胞株。在軟瓊脂剋隆形成實驗中,FAK野生型的剋隆形成數為(335±48)/1000箇,FAK突變體為(735±91)/1000箇,後者對MDA-MB-468乳腺癌穩轉細胞株的增殖能力高于前者(t=9.437, P<0.01)。結論初步研究錶明外顯子33缺失可改變FAK的亞細胞定位;併能增彊腫瘤細胞株的體外剋隆形成能力,這種FAK突變體可能參與腫瘤的髮生髮展。
목적:탐토외현자33결실형점착반격매(focal adhesion kinase,FAK)돌변체(FAK-Del33)적아세포정위급생물학특성。방법채용기출실험연구설계。장야생형FAK이급돌변형FAK-Del33분별구건도pEGFP표체재체,병전염유선암세포주,이연구분석기세포표체화정위정황。령구건과표체FAK혹FAK-Del33적MDA-MB-468유선암은전세포주,연구평개기세포표체화체외극륭형성능력。결과구건FAK-GFP융합단백표체재체전염MDA-MB-468후적세포정위현시,야생형FAK재포장중미산분포,이FA K돌변체단백재포장중정점상불균균분포,병취집성족。경한제성내절매매절분석여측서분석,성공구건출과표체FAK적만병독재체,이293T세포포장적중조만병독능구고효감염유선암세포;경항생소puromycin사선화형광정량PCR감정,성공사선도은정표체FAK적세포주。재연경지극륭형성실험중,FAK야생형적극륭형성수위(335±48)/1000개,FAK돌변체위(735±91)/1000개,후자대MDA-MB-468유선암은전세포주적증식능력고우전자(t=9.437, P<0.01)。결론초보연구표명외현자33결실가개변FAK적아세포정위;병능증강종류세포주적체외극륭형성능력,저충FAK돌변체가능삼여종류적발생발전。
ObjectiveTo investigate the subcellular localization and the biological characteristics of the novel FAK splicing mutant (FAK-Del33).MethodsBasic experimental research applied. FAK wide-type and FAK-Del33 cDNA were cloned into pEGFP expression vector, and then transfected into breast cancer cell lines. The expression and subcellular localization of FAK wild-type and FAK-Del33 cDNA were analyzed. FAK or FAK-De133 overexpressing MDA-MB-468 were constructed. Its expression and in vitro colony forming ability was evaluated.ResultsFAK-GFP fusion protein expression plasmids were constructed and then transfected into MDA-MB-468. FAK-WT dispersed evenly in the cytoplasma while FAK-Del33 signal enriched into strong signal points at different subcellular sites; restriction enzyme analysis and sequencing confirmed the correct construction of lentivirus plasmids. The stable cell lines with expression of FAK were generated by lentivirus infection, puromycin screening and lfuorescence quantitative PCR identiifcation. Soft agar colony formation test showed that clone number from mutant FAK had signiifcantly higher levels than FAK wide-type [(735±91)/1000 vs. (335±48)/1000,t=9.437,P<0.01).ConclusionThis work clearly indicates that the exon 33 deletion change the subcellular localization of FAK and can enhance tumor cell line′ s in vitro colony-forming ability and the FAK mutant may participate in the occurrence and development of tumor.