RUNX3基因真核表达载体的构建及其在乳腺癌T47D细胞中的表达

目的：构建真核表达载体pcDNA3.1（＋）-RUNX3,并在乳腺癌T47D细胞株中表达。方法：应用基因重组技术和限制性内切酶EcoRI和XhoI酶切,构建并鉴定pcDNA3.1（＋）-RUNX3真核表达载体,经脂质体Lipofectamine2000介导质粒转染T47D细胞后应用逆转录-聚合酶链反应（RT-PCR）和Western blot实验,检测RUNX3在T47D细胞中的表达。结果：重组真核表达载体pcDNA3.1（＋）-RUNX3经限制性内切酶EcoRI和XhoI酶切,电泳后显示1.3kb的RUNX3目的片段和5.4kb的pcDNA3.1（＋）载体片段。测序证实酶切片段与gene bank中登记的RUNX3序列相同,证实pcDNA3.1（＋）-RUNX3真核表达载体构建成功。经RT-PCR和Western blot检测,表明转染pcDNA3.1（＋）-RUNX3的T47D细胞RUNX3阳性表达。结论：重组真核表达载体构建正确,并建立稳定表达RUNX3的T47D细胞系,从而为后续研究提供有用的细胞研究模型。     

VEGFR-3胞外区基因真核表达载体的构建-与鉴定

目的构建VEGFR-3胞外区基因真核表达载体pcDNA3.1-VR-3,并在体外进行表达和鉴定。方法采用 RT-PCR技术,扩增C57BL/6小鼠胚胎VEGFR-3胞外区cDNA片段,通过基因重组技术将其插入到真核表达载体pcDNA3.1,构建重组质粒pcDNA3.1-VR-3。经限制性酶切鉴定和DNA 序列测定结果证实后,将重组质粒经脂质体法转染COS-7细胞, Western blotting检测其蛋白表达。结果克隆了C57BL/6小鼠胚胎VEGFR-3胞外区cDNA片段,并构建了真核表达载体pcDNA3.1-VR-3,Western blot 证实目的基因可在COS-7细胞中表达。结论构建的真核表达载体pcDNA3.1-VR-3,可在真核细胞内正确表达,这为进一步的动物实验奠定了基础。     

Fascin真核表达载体的构建及其在肺癌A549细胞中的表达

目的:构建fascin基因的pcDNA3.1(+)表达载体pcDNA3.1(+)-fascin,将其转染肺癌A549细胞,观察其表达,并检测转染前后细胞内F-肌动蛋白(F-actin)的变化。方法:应用RT-PCR从Hela细胞中扩增fascin基因,酶切后和真核表达载体pcDNA3.1(+)重组,获得重组表达载体pcDNA3.1(+)-fascin。经酶切、电泳,DNA测序鉴定后,将其及对照空载体转染肺癌A549细胞,采用Western blotting检测fascin在A549细胞中的表达情况;免疫荧光染色后采用激光共聚焦显微镜观察fascin对F-actin的影响。结果:成功克隆fascin基因片段,并将其重组到pcDNA3.1(+)载体中,经酶切及DNA测序鉴定正确。pcDNA3.1(+)-fascin转染A549细胞,Western blotting鉴定转染后fascin在A549细胞中高表达。激光共聚焦显示fascin基因能够在A549细胞内促进F-actin形成丝状突出。结论:成功构建了pcDNA3.1(+)-fascin真核表达载体,并将其转染肺癌A549细胞;激光共聚焦观察显示fascin能够促进A549细胞内F-actin形成丝状突出,可能是促进A549细胞转移的结构基础之一。     

分泌型核心蛋白聚糖真核表达载体的构建及在HepG2细胞中的表达

目的构建分泌型核心蛋白聚糖(decorin)真核表达载体,并在肝癌细胞HepG2中表达,为研究核心蛋白聚糖的抗肿瘤作用奠定基础。方法利用特异性引物,应用PCR技术扩增核心蛋白聚糖全长基因cDNA片段,与pcDNA3.1载体进行连接,并转化到大肠杆菌JM109中扩增以获得重组载体,应用双酶切、PCR以及测序鉴定此重组载体;脂质体介导重组载体转染HepG2,经G418筛选建立稳定转染细胞株,分别采用RT-PCR、免疫组化法和westernblot检测其表达。结果获得了约1080bp大小的特异性DNA片段;PCR产物与真核表达载体进行连接,经过双酶切、PCR以及测序鉴定证实分泌型核心蛋白聚糖cDNA片段正确插入真核表达载体pcDNA3.1中。RT-PCR方法可见转染组mRNA表达明显增多,免疫组化和westernblot方法可见转染组细胞decorin蛋白表达明显增高。结论成功构建了真核表达载体pcDNA3.1-decorin,建立了稳定转染decorin的HepG2细胞株。     

Nucleostemin基因荧光真核表达载体构建及表达特性

目的:构建Nucleostemin(NS)基因真核表达载体,并在COS-7细胞中表达.方法: 设计引物在肺癌细胞株(LTEP-a-2)中扩增NS全长cDNA片段,插入pMD-18T 质粒中得pMD-18T-NS载体,并测序;再将pMD-18T-NS质粒经酶切后导入真核表达载体pcDNA3.1(+)-GFP质粒中,构建pcDNA3.1(+)-GFP-NS载体,并检测其真核表达情况.结果: 经RT-PCR扩增得到1 650 bp的全长NS cDNA片段,酶切及测序后鉴定证明pMD-18T-NS构建成功.pcDNA3.1(+)-G-FP-NS载体转染COS-7细胞经蛋白印迹及激光共聚焦显微镜检测显示,该GFP-N-S融合基因在COS-7细胞中获得较好表达.转染后见该基因定位于核仁,细胞逐渐失去黏附贴壁的特性,细胞的增殖受阻;稳定表达(4～20周)后,细胞形态发生改变,体积增大,核增多,细胞成瘤细胞样改变.结论: 成功构建pcDNA3.1(+)-GF-P-NS真核表达载体,并在COS-7细胞中有效表达;NS基因在COS-7发生瘤样改变过程中发挥着重要作用,可能与肿瘤发生密切相关.     

Fascin真核表达载体的构建及其在肺癌A549细胞中的表达

目的：构建fascin基因的pcDNA3.1（＋）表达载体pcDNA3.1（＋）-fascin,将其转染肺癌A549细胞,观察其表达,并检测转染前后细胞内F-肌动蛋白（F-actin）的变化。方法：应用RT-PCR从Hela细胞中扩增fascin基因,酶切后和真核表达载体pcDNA3.1（＋）重组,获得重组表达载体pcDNA3.1（＋）-fascin。经酶切、电泳,DNA测序鉴定后,将其及对照空载体转染肺癌A549细胞,采用Western blotting检测fascin在A549细胞中的表达情况;免疫荧光染色后采用激光共聚焦显微镜观察fascin对F-actin的影响。结果：成功克隆fascin基因片段,并将其重组到pcDNA3.1（＋）载体中,经酶切及DNA测序鉴定正确。pcDNA3.1（＋）-fascin转染A549细胞,Western blotting鉴定转染后fascin在A549细胞中高表达。激光共聚焦显示fascin基因能够在A549细胞内促进F-actin形成丝状突出。结论：成功构建了pcDNA3.1（＋）-fascin真核表达载体,并将其转染肺癌A549细胞;激光共聚焦观察显示fascin能够促进A549细胞内F-actin形成丝状突出,可能是促进A549细胞转移的结构基础之一。     

人RASSF2基因的真核表达载体的构建及其表达

 目的　构建人RASSF2基因的真核表达载体 ,为研究RASSF2在肿瘤细胞凋亡、细胞周期阻滞等方面的作用奠定基础。方法　采用RT PCR自正常人外周血单个核细胞RNA中扩增出人RASSF2基因的全长cDNA ,利用DNA重组技术将其插入到真核表达载体 pcDNA3.1/HisC中获得重组质粒。用脂质体将重组质粒转染人胰腺癌细胞株PANC 1,RT PCR检测其表达。结果　酶切图谱分析及基因测序证明人RASSF2表达片段已被完整、正确的插入到 pcDNA3.1/HisC中 ,RT PCR结果显示转染细胞RASSF2表达水平上调。结论　成功构建了RASSF2基因的真核表达载体 pcDNA3.1RASSF2     

人白细胞介素-2真核表达质粒的构建及鉴定

目的构建高效表达人IL－2的真核表达质粒,为IL－2的肿瘤基因治疗提供重要的工具。方法运用PCR扩增出人IL－2基因,经BamHI及EcoRI双酶切后,回收片段与BamHI及EcoRI处理的表达载体pcDNA3在T4DNA连接酶的作用下连接,并用多种限制性内切酶对重组质位进行酶切鉴定。结果PCR扩增片段的长度约为480bp,重组质粒经酶切显示,构建的质粒中含480bp的IL－2插人片段,将此重组质粒命名为pcDNA3－IL－2。结论本研究结果为进一步研究表达IL－2质粒的肿瘤基因治疗及免疫调节作用打下了基础。     

pcDNA3.1/DLC-1 重组质粒的构建及其在HT-29细胞中的表达

 目的 构建和表达DLC-1(deleted in liver cancer)基因重组质粒。 方法 用PCR法得到DLC-1基因片段,此基因片段带有Xba Ⅰ和BamH Ⅰ两个酶切位点,然后将此片段 和真核表达载体pcDNA3.1连接,转化大肠杆菌DH5a,利用脂质体介导将pcDNA3.1/DLC-1重组质 粒转染到结肠癌HT-29细胞中,再用RT-PCR法检测重组质粒的表达。 结果 重组质粒经Xba Ⅰ和BamH Ⅰ双酶切和测序与DLC-1基因序列一致;利用脂质体介导将 pcDNA3.1/DLC-1重组质粒导入HT-29细胞,获得了DLC-1基因的表达。 结论 成功构建pcDNA3.1/DLC-1重组质粒并在HT-29细胞内表达。     

miR-181a对人食管癌TE11细胞增殖、迁移和侵袭能力的影响

目的:通过构建miR-181a的真核表达载体,研究其对人食管癌TE11细胞增殖、迁移和侵袭能力的影响。方法:以95C细胞基因组DNA为模板,扩增得到miR-181a前体序列,插入表达载体pcDNA3.1(+)克隆获得pcDNA3.1(+)-miR-181a。将真核表达载体pcDNA3.1(+)-miR-181a转染到TE11细胞中,并采用实时荧光定量-PCR(real-time fluorogentic quantitative-PCR,RFQ-PCR)对其表达情况进行验证。分别采用MTT法、细胞划痕法和Boyden小室法检测转染重组质粒pcDNA3.1(+)-miR-181a对TE11细胞增殖能力、迁移能力和侵袭能力的影响。结果:成功构建了插入miR-181a基因片段的真核表达载体pcDNA3.1(+)-miR-181a;RFQ-PCR检测结果表明,转染pcDNA3.1(+)-miR-181a的TE11细胞能够过表达成熟的miR-181a(P<0.05)。过表达miR-181a的TE11细胞增殖能力、迁移能力和细胞侵袭能力均明显增强。结论:miR-181a在TE11细胞中过表达能够增加细胞的增殖、迁移和侵袭能力,为进一步深入研究miR-181a在肿瘤中的作用机制提供了实验依据。     

The role of the tumor suppressor RUNX3 in giant cell tumor of the bone

RUNX3 is a tumor suppressor gene localized in 1p36. In various human tumors, the region is frequently inactivated through hypermethylation, histone modulation and other processes. Recent studies have suggested that loss of RUNX3 expression is involved in stomach, colon and breast cancer. However, the relationship between RUNX3 expression and giant cell tumor of the bone (GCTB) remains elusive. The aim of our study was to elucidate the roles of RUNX3 expression in carcinogenesis and progression of giant cell tumor of the bone. The levels of RUNX3 mRNA and protein were evaluated in human GCTB specimens and cell lines. To assess RUNX3 methylation we employed methylation-specific polymerase chain reaction using GCTB specimens and cell lines. In addition, to examine the roles of RUNX3 in giant cell tumor of the bone, GCTB cells were transfected with pcDNA3.1-RUNX3 (RUNX3 was cloned into the pcDNA3.1 plasmid). Flow cytometry (FCM) was used to analyze the apoptosis and cell cycle. The mobility of cells was tested by transwell migration assay. The expression rates of RUNX3 in patients with GCTB were significanly lower than normal bone tissues. Thirty of 47 human cancer specimens exhibited suppression (P<0.05). Down-regulation of RUNX3 mRNA in the same GCTB cell lines was associated with RUNX3 DNA methylation. In in?vitro experiments, exogenous expression of RUNX3 strongly inhibited cell growth in GCTB by MTT (P<0.05), induced apoptosis as evidenced by Annexin?V-FITC and increased G1 phase ratio by PI (P<0.05). Transwell migration assay showed that less RUNX3 positive cells migrated to the lower side of the membrane than negative ones (P<0.05). These results show that RUNX3 is a tumor suppressor in GCTB. RUNX3 DNA methylation may be the molecular basis for its lower expression. These data may be applied in GCTB for diagnostics and therapeutics.     

人miR-34a真核表达载体的构建及其表达

目的:构建人miR-34a的真核表达载体,并使其在骨肉瘤SOSP-9607细胞中表达.方法:从人骨肉瘤SOSP-9607细胞基因组DNA中用PCR法扩增出miR-34a的前体序列,并引入酶切位点和保护碱基,双酶切后定向插入真核表达载体pcD-NA3.1(+)中,构建miR-34a真核表达载体pcD-NA-miR34a,然后进行菌落PCR、酶切和测序鉴定.同时用构建好的载体pcDNA-miR34a转染骨肉瘤细胞SOSP-9607细胞,筛选miR-34a稳定表达细胞系,用Northern blot及real time RT-RCR法鉴定pcD-NA-miR34a可于真核细胞中过表达miR-34a的生物学特性.结果:成功构建了人miR-34a真核表达载体pcDNA-miR34a.pcDNA-miR34a可于骨肉瘤SOSp-9607细胞中上调miR-34a的表达,获得了miR-34a高表达骨肉瘤细胞系.结论:miR-34a的真核表达载体可在骨肉瘤细胞SOSP-9607细胞中有效表达,为进一步研究miR-34a在骨肉瘤中的功能及基因调控机制奠定了实验基础.     

Restoration of RUNX3 enhances transforming growth factor-beta-dependent p21 expression in a biliary tract cancer cell line

RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region commonly inactivated by deletion and methylation in various human tumors. To elucidate the role of RUNX3 in transforming growth factor (TGF)-beta signaling in biliary tract cancer, we transfected Mz-ChA-2 cells, which do not express RUNX3 but have intact TGF-beta type II receptor and SMAD4 genes, with the RUNX3 expression plasmid pcDNA3.1/RUNX3 or with the vector pcDNA3.1 as a control. Four Mz-ChA-2/RUNX3 clones and one control clone were obtained. Although TGF-beta1 only slightly inhibited growth of the control cells, growth inhibition and TGF-beta-dependent G(1) arrest were significantly enhanced in the RUNX3-transfected clones. None of the clones, however, exhibited apoptosis. The slightly increased TGF-beta1-induced p21 expression in the control clone was strongly enhanced in the RUNX3-transfected clones, and was accompanied by augmented decreases in the expression of cyclins D1 and E. When RUNX3 small interfering RNA was added, TGF-beta-dependent induction of p21 was reduced in the RUNX3-transfected clones. Xenografts of the clones in nude mice demonstrated that tumorigenicity was significantly decreased in the RUNX3-transfected clones in inverse proportion to the expression levels of RUNX3. Based on these results, RUNX3 is involved in TGF-beta-induced expression of p21 and the resulting induction of TGF-beta-dependent G(1) arrest.     

人类α型叶酸受体基因的克隆及其真核表达载体的构建

目的 探讨如何以人类α型叶酸受体(FOLR-1)基因为基础构建核酸疫苗。方法 应用RT-PCR技术，从人类卵巢癌细胞系-SKOV3细胞中扩增FOLR-1基因，插入克隆载体pGEM-T Easy，经DNA自动测序仪测序证实后，以亚克隆法构建于真核表达载体pcDNA3．1( )，并使用限制性内切酶酶切鉴定。结果 从卵巢癌细胞系SKOV3中成功扩增出FOLR-1基因，并克隆人pcDNA3．1( )载体。结论 成功构建FOLR-1的重组克隆及真核表达载体，为今后利用FOLR-1进行卵巢上皮性肿瘤的免疫及导向治疗研究打下了基础。