利用CRISPR/Cas9基因编辑技术构建CDH1基因敲除的人乳腺癌MCF-7稳定细胞系

目的:利用CRISPR/Cas9基因编辑技术构建CDH1基因缺失的人乳腺癌MCF-7稳定细胞系。方法:根据CRISPR/Cas9靶点设计原则,设计能特异性针对CDH1基因的sgRNA,以lentiCRISPR v2质粒为骨架构建能表达此sgRNA和Cas9蛋白的重组质粒。测序鉴定后,将重组质粒与逆转录病毒包装质粒VSVG、PAX2在氯化钙介导下共同转入HEK293T细胞进行病毒包装,转染48 h后收集病毒上清,直接感染人乳腺癌MCF-7细胞。采用嘌呤霉素筛选CDH1缺失的乳腺癌MCF-7细胞,通过DNA测序、Western印迹及免疫荧光染色实验验证获得的MCF-7细胞。结果:构建了靶向CDH1的CRISPR/Cas9质粒;DNA测序和Western印迹实验结果表明获得了稳定敲除CDH1的人乳腺癌MCF-7细胞。免疫荧光染色结果显示,相比对照组,稳定敲除CDH1的MCF-7细胞中已无法明显观察到E-钙黏蛋白的表达分布。结论:通过CRISPR/Cas9基因编辑技术构建了CDH1基因缺失的MCF7细胞系,为进一步研究CDH1在肿瘤免疫治疗中的作用提供了基础。     

Pmscv-Ubc9逆转录病毒表达载体的构建及产毒细胞系的建立

目的：构建含Ubc9的逆转录病毒表达载体,筛选建立携带该基因的高滴度产毒细胞系,深入研究SUMO化修饰的作用。方法：聚合酶链反应（PCR）扩增获取目的基因Ubc9,定向插入逆转录病毒表达载体pMSCVneo,形成重组质粒pMSCV-Ubc9;脂质体法将pMSCV-Ubc9转染逆转录病毒包装细胞PT67;G418筛选产毒细胞克隆,扩大培养产毒细胞克隆,收获病毒感染NIH3T3细胞。结果：限制性酶切和测序鉴定证实Ubc9正确插入逆转录病毒表达载体。G418筛选获得稳定产毒的抗性细胞克隆,收获病毒能有效感染NIH3T3细胞。结论：携带Ubc9基因的重组逆转录病毒表达载体pMSCV-Ubc9构建成功,转染PT67细胞后包装出重组逆转录病毒,进而筛选获得了能转录表达Ubc9的产毒细胞系PT67-Ubc9。     

iRhom2及其突变基因重组慢病毒的包装和稳定表达细胞系的建立

目的通过重组慢病毒感染,建立iRhom2及其突变基因的Vero细胞稳定细胞表达系,用于iRhom2的功能研究。方法将iRhom2及其突变基因克隆到慢病毒载体Lenti-OE-Flag,构建重组慢病毒载体Lenti-OEiRhom2和Lenti-OE-iRhom2mut,将重组质粒瞬时转染HEK-293T包装细胞,获得重组慢病毒。将重组病毒感染Vero细胞,利用puromycin进行加压筛选,获得二者的重组表达细胞系。结果构建了iRhom2及其突变基因的逆转录病毒载体,并获得了重组逆转录病毒,并利用该病毒获得了二者的稳定表达细胞系。Western-blot实验证实,二者能够在Vero细胞内稳定表达。结论利用重组逆转录病毒感染,成功获得了iRhom2及其突变系的Vero细胞稳定表达株,为进一步研究iRhom2的生物学功能及其机制奠定了良好的基础。     

Mo MLV gag-pol基因在NIH3T3细胞中的表达和鉴定

目的 构建含MoMLV gag-pol基因的重组表达载体,实现其在NIH3T3细胞中稳定表达。方法 应用RT-PCR方法反转录并扩增gag-pol基因,克隆入真核表达载体pcDNA4/HisMaxA上,构建重组表达载体pcDNA4/HisMaxA-gag-pol,用脂质体法转染NIH3T3细胞,Zeocin筛选稳定表达细胞株,通过SDS-PAGE分析检测表明, gag-pol基因在NIH3T3细胞实现了表达,产物相对分子质量(kD)为194.78×103。然后,将逆转录病毒载体导入此细胞系,包装逆转录病毒。用PCR与标记基因补救分析法检测野生型辅助病毒。结果 酶切鉴定的片段大小分别为5.2-kb,与预期大小一致,经Zeocin筛选后获得稳定表达细胞株,SDS-PAGE实验表明产物融合蛋白相对分子质量(kD)为194.78×103,与预期相符。脂质体转染包装细胞,嘌呤霉素加压筛选出高病毒滴度(4.0×106CFU/ml)的细胞克隆,且未检测到辅助病毒。结论 本工作构建的融合表达载体pcDNA4/HisMaxA-gag-pol及其在NIH3T3细胞中的表达,构建成功具有靶向性的逆转录病毒包装细胞系,该细胞系能够包装出高滴度的逆转录病毒,为肝细胞的基因治疗提供了一种新的基因转移系统。     

Pmscv-Ubc9 逆转录病毒表达载体的构建及产毒细胞系的建立

目的:构建含Ubc9的逆转录病毒表达载体,筛选建立携带该基因的高滴度产毒细胞系,深入研究SUMO化修饰的作用。方法:聚合酶链反应(PCR)扩增获取目的基因Ubc9,定向插入逆转录病毒表达载体pMSCVneo,形成重组质粒pMSCV-Ubc9;脂质体法将pMSCV-Ubc9转染逆转录病毒包装细胞PT67;G418筛选产毒细胞克隆,扩大培养产毒细胞克隆,收获病毒感染NIH3T3细胞。结果:限制性酶切和测序鉴定证实Ubc9正确插入逆转录病毒表达载体。G418筛选获得稳定产毒的抗性细胞克隆,收获病毒能有效感染NIH3T3细胞。结论:携带Ubc9基因的重组逆转录病毒表达载体pMSCV-Ubc9构建成功,转染PT67细胞后包装出重组逆转录病毒,进而筛选获得了能转录表达Ubc9的产毒细胞系PT67-Ubc9。     

逆转录病毒表达系统及其在外源蛋白高效表达中的应用

逆转录病毒表达系统是一种新的重组蛋白高效表达系统,它由逆转录病毒载体,包膜蛋白载体和包装细胞系构成,在基因治疗和生物制药领域都有很大的应用潜力。逆转录病毒和宿主细胞基因组的重组倾向于发生在转录活性区;口炎疱疹病毒-G蛋白 (vesicular stomatitis virus G, VSV-G)能有效扩大逆转录病毒感染宿主细胞的范围、提高逆转录病毒的感染效率;用高滴度的重组病毒感染细胞,经过简单的筛选即可获得高表达细胞株。本文对逆转录病毒表达系统的组成、感染的特点和机制及其应用前景作了概述。     

改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

小鼠T-bet逆转录病毒载体的构建与功能验证

为在小鼠细胞中表达并研究T-bet功能,首先构建了含有报告基因Thy1.1的小鼠T-bet逆转录病毒载体,并将构建的载体质粒转染病毒包装细胞系包装成重组病毒,再利用重组病毒分别感染NIH-3T3和D9细胞系检测其感染能力。之后,使用该重组病毒感染T-bet敲除小鼠的CD4+T淋巴细胞,流式细胞术检测T-bet及其下游靶基因Ifng的表达情况。经验证,重组的逆转录病毒感染T-bet敲除小鼠T淋巴细胞后可以在细胞中表达T-bet,并进一步引起下游靶基因Ifng的表达上调,证明本研究中外源表达的转录因子T-bet具有正常功能。综上所述,本实验成功构建了含有报告基因的小鼠T-bet重组逆转录病毒载体,为进一步在小鼠细胞中研究T-bet功能奠定了基础。     

携带靶向性可调控自杀基因的重组相关腺病毒载体的构建及其活性检测

目的:构建携带受Tet-On以及VEGF启动子双调控自杀基因HSVtk的重组腺相关病毒载体,研究其在乳腺癌细胞MCF-7中的可调控表达.方法:用PCR方法扩增VEGF启动予,将其插入pAAV/TRE/HSVtk/Tet-On中形成重组载体pAAV/VEGF/TRE/HSVtk/Tet-On质粒.进行病毒包装后得到了rAAV/VEGF/TRE/HSVtk/Tet-On重组腺相关病毒.用重组腺相关病毒感染乳腺癌细胞株MCF-7和正常乳腺HBL-100细胞,用MTT法及RT-PCR检测在Dox诱导下,GCV对rAAV感染的MCF-7细胞和HBL-100细胞的杀伤作用以及HSVtk基因在MCF-7细胞内的表达情况.结果:在rAAV+Dox+GCV组,GCV对rAAV感染的MCF-7细胞的杀伤作用明显高于rAAV+Dox组,rAAV+Dox组以及HBL-100组,并且RT-PCR结果显示经Dox诱导HSVtk表达较明显.结论:成功构建了携带双调控自杀基因的重组腺相关病毒载体,该病毒载体能有效的感染乳腺癌细胞MCF-7,并能联合GCV治疗,抑制肿瘤细胞生长,而且具有靶向性.     

骨形成蛋白7基因诱导人肝癌细胞的凋亡

目的 构建表达重组人骨形成蛋白7 （bone morphogenic protein 7, BMP7）基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并探讨其作用机制。方法 克隆BMP7基因,以loxP同源重组法构成逆转录病毒载体pLP-LNCX-BMP7（pLLBMP7）,转染包装细胞PT67进行病毒包装并测定病毒滴度;将逆转录病毒感染人成骨细胞,MTT法检测细胞生长变化,琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;Western blotting检测BMP7,caspase-3和bcl-2蛋白表达。结果 重组逆转录病毒载体pLLBMP7经鉴定连接正确,转染PT67细胞后上清液中可得到病毒,滴度达1×109pfu;MTT检测见pLLBMP7病毒组48和72h细胞抑制率高于对照组（35.1% vs. 5.3%,68.5% vs.18.3%,均p<0.05）,48h可见BMP7蛋白高表达。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达14.42%;BMP7蛋白高表达时caspase-3蛋白的表达亦有显著升高,但bcl-2蛋白未见表达差异。结论 构建了BMP7逆转录病毒,在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能是通过激活caspase-3而发生作用。     

受强力霉素调节的自杀基因表达载体系统的构建和在乳腺癌细胞株(MCF-7)的表达

自杀基因治疗是恶性肿瘤基因治疗中最常用的途径之一,以递转录病毒载体-pRevTRE为基础进行载体构建,首先使用PCR技术对单纯疱疹病毒胸苷激酶基因（HSVtk)进行扩增,将HSVtk基因插入到pRe-vTRE,形成重组载体pRevTRE/HSVtk,用磷酸钙共沉淀法,经过两轮转染,分别将pRevTRE/HSVtk和pRevTet-On质粒导入乳腺癌细胞株（MCF-7）经过潮霉素B（HygromycinB)和G418筛选,建立了一株稳定的受四环素衍生物-强力霉素（Doxycycline,Dox)调控,表达HSVtk基因产物的人乳腺癌细胞株MCF/TRE/tk/Tet-On,HSVtk基因表达产物可以将无毒性的药物前体Ganciclovir(GCV)转变成一种有毒的代谢产物,从而杀死乳腺癌细胞株（MCF-7）,达到基因治疗的目的。     

乳腺癌细胞株MCF-7-PC-1-46的建立

以人前列腺癌C4-2细胞基因组DNA为模板,扩增出PC-1基因N端编码46个氨基酸残基及其上游非编码区共599bp的DNA序列,将其正向克隆到真核表达载体pIRES2中,并在脂质体介导下,转染人乳腺癌细胞MCF-7,经G418筛选获得阳性单克隆,细胞扩大培养后,进行PCR和RT-PCR分析,检测外源PC-1基因在靶细胞中的整合与转录,PCR和RT-PCR结果表明,稳定转梁细胞株MCF-7-PC-1-46具有外源目的基因的整合和相应mRNA的高表达,说明成功建立了稳定表达外源PC-1基因N端46个氨基酸的人乳腺癌细胞株,为进一步研究PC-1基因的生物学功能提供了实验材料。     

Connective tissue growth factor induces apoptosis in human breast cancer cell line MCF-7

Connective tissue growth factor (CTGF) is a member of an emerging CCN gene family that is implicated in various diseases associated with fibro-proliferative disorder including scleroderma and atherosclerosis. The function of CTGF in human cancer is largely unknown. We now show that CTGF induces apoptosis in the human breast cancer cell line MCF-7. CTGF mRNA was completely absent in MCF-7 but strongly induced by treatment with transforming growth factor beta (TGF-beta). TGF-beta by itself induced apoptosis in MCF-7, and this effect was reversed by co-treatment with CTGF antisense oligonucleotide. Overexpression of CTGF gene in transiently transfected MCF-7 cells significantly augmented apoptosis. Moreover, recombinant CTGF protein significantly enhanced apoptosis in MCF-7 cells as evaluated by DNA fragmentation, Tdt-mediated dUTP biotin nick end-labeling staining, flow cytometry analysis, and nuclear staining using Hoechst 33258. Finally, recombinant CTGF showed no effect on Bax protein expression but significantly reduced Bcl2 protein expression. Taken together, these results suggest that CTGF is a major inducer of apoptosis in the human breast cancer cell line MCF-7 and that TGF-beta-induced apoptosis in MCF-7 cells is mediated, in part, by CTGF.     

Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling

In the present study we evaluated the anti-tumor potential of samsum ant venom (SAV) from Pachycondyla sennaarensis on the human breast carcinoma cell line MCF-7. We found that SAV induced growth arrest of MCF-7 cells without affecting the viability of MCF-10 (non-tumorigenic normal breast epithelial cells) and normal PBMCs. We then analyzed its impact on IGF-1-mediated MCF-7 cell proliferation and its effect on the underlying IGF-1 signaling pathways. Using flow cytometry analysis, we showed that the percentage of apoptotic cells was fourfold higher in SAV-treated cells as compared to untreated cells. More importantly, treatment with SAV induced a marked reduction in actin polymerization and a subsequent marked reduction in IGF-1-mediated cell proliferation. In addition to growth-inhibitory and pro-apoptotic effects, significant reductions were also observed in the phosphorylation of AKT and ERK, but not p38MAPK, in SAV-treated cells as compared to untreated cells. Our data reveal unique anti-tumor effects of samsum ant venom.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

Cell-cycle arrest and apoptosis induction in human breast carcinoma MCF-7 cells by carboxymethylated β-glucan from the mushroom sclerotia of Pleurotus tuber-regium

The mechanism for the anti-tumor activity of a water-soluble carboxymethylated β-glucan (CMPTR), partially synthesized from an insoluble native glucan isolated from the sclerotia of Pleurotus tuber-regium, was studied using human breast carcinoma MCF-7 breast cancer cells in vitro. CMPTR-induced anti-proliferative activity dose-dependently, with an IC₅₀ of 204 μg/ml. CMPTR inhibited the cell proliferation of MCF-7 by arresting the G₁ phase of its cell cycle after 48 h of incubation as shown by flow cytometry. Such G₁ phase arrest was associated with the down-regulation of cyclin D1 and cyclin E expressions in the breast cancer cells. In addition, the CMPTR-treated MCF-7 cancer cells were associated with decreased expression of anti-apoptotic Bcl-2 protein and increased expression of Bax/Bcl-2 ratio. This study shows that CMPTR can inhibit the proliferation of MCF-7 by cell-cycle arrest and apoptosis induction. The potential development of this mushroom polysaccharide as a water-soluble anti-tumor agent requires further investigation.     

siRNA-cyclin D1对乳腺癌MCF-7细胞增殖的抑制作用

研究小干扰RNA(small interfering RNA,siRNA)对乳腺癌MCF-7细胞株cyclin D1表达的抑制及对细胞增殖的影响。化学合成针对cyclin D1基因的siRNA,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin D1 mRNA和蛋白的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。在实验中,10、50、100 nmol/L siRNA-cyclin D1分别使MCF-7细胞cyclin D1 mRNA表达降低了57．85%、63．22%和68．02%,蛋白表达降低了51．13%、62．09%、77．68%。转染siRNA-cyclin D1后,细胞增殖受到抑制,细胞周期阻滞于G1期,软琼脂克隆形成率降低。结果提示siRNA可以有效抑制MCF-7细胞株中cyclin D1的表达,使细胞周期阻滞于G1期,从而抑制细胞增殖。     

CCAAT/Enhancer binding protein delta (c/EBPdelta) regulation and expression in human mammary epithelial cells: I. "Loss of function" alterations in the c/EBPdelta growth inhibitory pathway in breast cancer cell lines

"Loss of function" alterations in growth inhibitory signal transduction pathways are common in cancer cells. In this study, we show that growth arrest (GA) treatments--serum and growth factor withdrawal and growth inhibitory IL-6 family cytokines (Interleukin-6 and Oncostatin M (OSM))--increase STAT3 phosphorylation (pSTAT3), increase CCAAT enhancer binding protein delta (C/EBPdelta) gene expression and induce GA of primary, finite-lifespan human mammary epithelial cells (HMECs), and immortalized breast cell lines (MCF-10A and MCF-12A). In contrast, serum and growth factor withdrawal from human breast cancer cell lines (MCF-7, SK-BR-3, T-47D, and MDA-MB-231) for up to 48 h induced a relatively modest increase in pSTAT3 levels and C/EBPdelta gene expression and resulted in varying levels of GA. In most breast cancer cell lines, IL-6 family cytokine treatment increased pSTAT3 levels and C/EBPdelta gene expression, however, growth inhibition was cell line dependent. In addition to "loss of function" alterations in growth inhibitory pathways, breast cancer cell lines also exhibit "gain of function" alterations in growth signaling pathways. The Akt growth/ survival pathway is constitutively activated in T-47D and MCF-7 breast cancer cells. The Akt inhibitor LY 294,002 significantly enhanced T-47D growth inhibition by serum and growth factor withdrawal or IL-6 family cytokine treatment. Finally, we show that activation of the pSTAT3/C/EBPdelta growth control pathway is independent of estrogen receptor status. These results demonstrate that "loss of function" alterations in the pSTAT3/C/EBPdelta growth inhibitory signal transduction pathway are relatively common in human breast cancer cell lines. Defective activation of the pSTAT3/ C/EBPdelta growth inhibitory signal transduction pathway, in conjunction with constitutive activation of the Akt growth stimulatory pathway, may play a synergistic role in the etiology or progression of breast cancer.     

Epigenetic regulation of bone morphogenetic protein-6 gene expression in breast cancer cells

Bone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Our previous research found that BMP-6 gene expression can be activated dose-dependently by estrogen in estrogen receptor positive (ER⁺) breast cancer cell line MCF-7, but not in ER negative (ER⁻) cell line MDA-MB-231. This experiment is designed to investigate the epigenetic regulatory mechanism of the BMP-6 gene expression in breast cancer cell lines MDA-MB-231, MCF-7 and T47D with regard to the methylation status in the 5′ flanking region of the human BMP-6 gene. The endogenous level of BMP-6 mRNA in ER⁻ cell line MDA-MB-231 was relatively lower than that in ER⁺ MCF-7 and T47D cell lines. After the treatment with 5-aza-2′-deoxycytidine (5-aza-dC, especially in the concentration of 10 μM), the BMP-6 mRNA expression in MDA-MB-231 was obviously up-regulated. However, 5-aza-dC treatment failed to regulate the expression of BMP-6 in MCF-7 and T47D cells. Using enzyme restriction PCR (MSRE-PCR), as well as bisulfite sequencing (BSG), methylation of human BMP-6 gene promoter was detected in MDA-MB-231; while in MCF-7 and T47D, BMP-6 gene promoter remained demethylated status. In 33 breast tumor specimens, promoter methylation of BMP-6 was detected by methylation-specific PCR, hypermethylation of BMP-6 was observed in ER negative cases (16 of 16 cases (100%)), while obviously lower methylation frequency were observed in ER positive cases (3 of 17 cases (18%)), indicating that BMP-6 promoter methylation status is correlated with ER status in breast cancer.