反义细胞周期蛋白E真核表达载体的构建及其对体外人乳腺癌细胞的抑制作用

 为了探讨细胞周期蛋白 E(cyclin E)与人乳腺癌细胞恶性特征间的相关性 ,利用反义 RNA抑制基因表达的技术 ,构建了细胞周期蛋白 E反义 RNA的真核表达载体并转入人乳腺癌细胞中 .通过 G41 8筛选出阳性克隆 ,经 PCR和 Western印迹检测 ,确定细胞中含有重组质粒 ,并且细胞周期蛋白 E蛋白的水平明显降低 ,由此获得了反义 RNA表达载体导致的细胞周期蛋白 E表达受抑制的细胞 .细胞模型建立后 ,观察分析了细胞形态 ,细胞生长的血清依赖性以及软琼脂成集落能力 ,与对照细胞相比所发生的变化 .结果显示 ,细胞周期蛋白 E受抑制后 ,乳腺癌细胞体积变大 ,细胞生长对血清依赖性增加 ,低血清培养到第 6d时 ,细胞密度约为对照细胞的五分之一 ,细胞成集落能力也显著下降 ,软琼脂中克隆形成率下降 57% .这些变化都表明乳腺癌细胞恶性程度由于细胞周期蛋白 E表达受抑制而减弱 ,可以推测 cyclin E与乳腺癌细胞的恶性增殖及非锚定依赖性生长有着明显的关系 .     

尿激酶受体反义RNA抑制人乳腺癌细胞的侵袭作用

将尿激酶受体 u PAR反义 RNA表达质粒 p URAS以脂质体法转染高侵袭性人乳腺癌细胞株 MDA- MB- 2 31 ,G41 8筛选抗性克隆 .Northern印迹法检测 u PAR反义 RNA的表达 ,RT- PCR法检测 u PAR的表达 ,牛奶板法测定细胞培养上清中纤溶活性 .改良 Boyden小室模型和裸小鼠乳房脂肪垫接种试验分别检测肿瘤细胞体外和体内侵袭能力 .反义克隆细胞能表达 u PAR反义RNA,其 u PAR表达水平及培养上清中纤溶活性明显降低 .反义细胞克隆体外侵袭能力比原代细胞 MDA- MB- 2 31和转染载体细胞克隆显著降低 .裸小鼠体内侵袭实验表明 ,反义细胞克隆的成瘤性、生长性和侵袭性均显著受到抑制 .u PAR至少在一部分恶性乳腺癌侵袭行为中发挥重要作用 ,反义 RNA可望成为抗肿瘤侵袭治疗的一种有效手段 .     

人U6基因POI Ⅲ启动子表达反义VEGF cDNA抑制SMMC-7721肝癌细胞VEGF表达的观察

 由 RNA聚合酶启动子在细胞内转录高浓度反义 RNA是抑制靶蛋白的一种有效手段 ,有报道 POI 启动子转录小分子 RNA存在效率不高 ,带有较多的非特异性序列等缺点 ,为了克服这些问题 ,表达反义 VEGF RNA的人 U6基因表达盒对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制作用进行了研究 .首先 PCR扩增 2 0 0 bp VEGF c DNA以正、反向插入人 U6 sn RNA.通过测序证实反向插入的正确性 .采用细胞原位杂交 ,RNA酶保护分析 ,Northern印迹来证实反义 RNA表达的情况 ,利用 RT- PCR方法研究了其对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制效果 .细胞原位杂交结果显示 U6启动子转录产物主要分布于细胞核内 ,细胞浆内亦有表达 ,RNA酶保护分析显示 U6基因 POI 启动子能高表达所需大小反义 RNA,Northern印迹结果显示脂质体 lipo-fectamine介导含 U6基因 POI 启动子的质粒转染人肝癌细胞株 SMMC- 772 1后 1 2 h即有表达且可持续表达 6 d. RT- PCR证实 U6基因 POI 启动子转录的反义 VEGF RNA能有效抑制SMMC- 772 1细胞 VEGF的 m RNA表达 .已有的研究结果揭示 POI 启动子是反义基因治疗中一种好的选择     

核苷酸切除修复基因XPA反义RNA表达载体的构建及其抑瘤功能

采用RT PCR方法扩增出 4 2 6bp着色性干皮病A(xerodermapigmentosumgroupA ,XPA)cDNA片段 (2～ 4 2 7bp) ,反向插入pcDNA3 1质粒构建XPA反义RNA表达载体 .经测序证实 ,该片段序列与XPAmRNA对应片段完全互补 .通过脂质体Lipofectamine 2 0 0 0将重组质粒转染肺癌A5 4 9细胞 ,RT PCR检测表明转染XPA反义RNA重组质粒能够抑制肺癌细胞XPAmRNA表达 ;MTT实验表明转染XPA反义RNA的肺癌细胞对顺铂敏感性增强 .本研究为深入探讨NER途径基因功能及临床克服肿瘤耐药提出了一个新的思路     

用ribozyme抑制增殖细胞核抗原的表达对HaLa细胞增殖的影响

增殖细胞核抗原(PCNA)是DNA聚合酶δ的辅助蛋白,它是细胞染色体DNA复制所必需的。人工设计的ribozyme具有可特异地切割PCNA mRNA的性质,将此ribozyme的自修剪体内表达质粒导入HeLa细胞,从细胞总RNA中分离相应部分能在体外切割靶RNA片段,证明此表达质粒在细胞内能表达出有活性的ribozyme分子。与对照相比,导入ribo-zyme表达质粒的HeLa细胞进入S期的时间从12 h推迟到20 h,而突变ribozyme的对照表明反义抑制对细胞进入S期的影响较小(推迟到15 h)。证明该ribozyme能有效抑制He-La细胞DNA复制,同时亦证明PCNA对于细胞DNA复制及细胞周期进程的重要性。     

应用载体介导的RNAi技术抑制HCMV的UL49基因表达

为了研究RNA干涉抑制HCMV UL49基因的作用,以pLXSN(U6启动子)为模板通过两步PCR的方法扩增含U6启动子的siRNA表达片段,并通过TA克隆将siRNA表达片段克隆到pMD18-T载体构建成siRNA表达质粒,同时以人巨细胞病毒AD169病毒株基因组为模板PCR扩增UL49基因,将其克隆到pEGFP-N1构建融合质粒pEGFP-UL49。通过脂质体介导将siRNA表达质粒和pEGFP-UL49质粒共转染人宫颈癌细胞系HeLa,在荧光显微镜下观察RNA干涉结果。通过这种方法得到具有介导RNA干涉的siRNA片段,为UL49基因沉默研究提供技术基础。     

人尿激酶原cDNA在中国仓鼠卵巢细胞(CH0)中高效表达研究

通过构建高效表达载体,改进转染方法,与二氢叶酸还原酶(dhfr)基因共扩增等手段在CH0细胞内高效表达了人尿激酶原(Pr0-UK)cDNA。首先将pro—UK cDNA插入到sR α启动子的下游,构建成表达质粒pMGl0102,在cos－7细胞内进行暂时性表达,结果表明此启动子的表达水平比SV40早期启动子高约5倍。然后将质粒pMG10102和pSV2－dhfr线性化后用磅酸钙共沉淀法转染CH0－dhfr-细胞,经一系列筛选后获得20个能表达pro—UK的细胞克隆,纤维蛋白溶解平板法(FAPA)测定表达水平为12．5—100IU／10⁶cells／d．再经MTX加压共扩增,得到9株高表达细胞系,其中最高的表达水平达到400—500Iu／10‘cells／d。2—3个月连续传代,表达水平未下降,表明细胞株是稳定的。Western Blot分析证明细胞分泌的重组pro－UK具有与天然pro—UK相同的分子量,而且培养液中不加蛋白酶抑制剂时,分泌的重组UK大部分为单链(60％以上)。     

SOX7基因启动子甲基化水平对乳腺癌MDA-MB-231细胞株体外迁移和侵袭的影响

目的:探讨乳腺癌MDA-MB-231细胞中,Y性别决定区基因7(SOX7)基因启动子甲基化水平对细胞的体外迁移和侵袭的影响。方法:脂质体转染pcDNA3.0-DNA甲基转移酶3a(DNMT3a)质粒至MDA-MB-231细胞中,并于24h、48h及72h后,采用蛋白质免疫印迹实验(WB)检测细胞内DNMT3a蛋白表达水平;甲基化特异性定量PCR(Q-MSP)检测DNMT3a处理组、5-aza-C处理组及对照(Control)组MDA-MB-231细胞中的SOX7基因启动子DNA甲基化水平;实时荧光定量PCR(qRT-PCR)及WB实验检测各组MDA-MB-231细胞中的SOX7 m RNA和蛋白表达水平;细胞划痕实验及细胞侵袭实验检测各组MDA-MB-231细胞的迁移和侵袭能力。结果:pcDNA3.0-DNMT3a质粒转染MDA-MB-231细胞24h时,细胞内的DNMT3a蛋白表达水平最高。DNMT3a能够显著提高SOX7基因启动子DNA甲基化水平,而5-aza-C则抑制了SOX7基因启动子DNA甲基化水平(P0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞中,SOX7的m RNA及蛋白表达水平均明显下降,而5-aza-C处理组SOX7的m RNA及蛋白表达水平均明显增加(P0.05)。与Control组相比,DNMT3a处理组的MDA-MB-231细胞的迁移和侵袭能力均显著增强(P0.05),而5-aza-C处理组的MDA-MB-231细胞的迁移和侵袭能力变化不大(P0.05)。结论:在恶性肿瘤中,SOX7低表达表受其基因启动子高甲基化调节,且乳腺癌MDA-MB-231细胞中低表达的SOX7能够影响细胞的外迁移和侵袭能力。     

利用脊髓灰质炎病毒5''''NCR和RNA聚合酶基因可提高外源基因表达

将含脊髓灰质炎病毒 (PV)RNA聚合酶的不同长度基因片段克隆到载体 pSG5质粒上 ,分别构建了 4个表达RNA聚合酶的质粒。体外转录实验证明 ,pSG5 POL1 99和 pSG5 POL2 0 3质粒转染细胞的提取物促进了特异的RNA转录 ,表明两质粒可表达RNA聚合酶。将PV的 5'NCR序列插在载体 pGREENLANTERN 1的CMV启动子下游 ,构建了 pGREENLANTERN 1 5'NCR质粒 ;用 LacZ基因替换GFP基因分别插入到PGREENLANTERN 1和pGREENLANTERN 1 5'NCR质粒上 ,构建成 pLacZLANTERN 1和 pLacZLANTERN 1 5'NCR质粒。表达RNA聚合酶的质粒与 pLacZ 5'NCR调控表达报告基因的质粒共转染 ,明显提高了报告基因的表达水平 ,表明PV的表达调控元件和RNA聚合酶基因可用于构建外源基因高效表达载体。     

反义RNA抑制猪传染性胃肠炎病毒复制的研究

根据反义RNA作用原理,设计一条互补猪传染性胃肠炎病毒基因(26888—27184)区的反义RNA序列。将该序列与逆转录病毒表达载体构建成质粒PLXSN—N5’,并与质脂体共转染PA317细胞,经G418(500μg／ml)筛选出稳定的产毒细胞克隆。取其上清液感染小鼠成纤维细胞NIH3T3,测定细胞克隆产生的假病毒滴度,用高滴度假病毒感染IBRS2细胞。提取被感染的IBRS2细胞总DNA和RNA,通过PCR和RT_PCR证明PL,XSN—N5’整合到IBRS2细胞基因组。病毒感染细胞病变表明,反义RNA有明显抑制TGEV复制的作用。     

人乳头瘤病毒16E6基因通过增加HMGB1表达调节口腔癌CAL27细胞侵袭的实验研究

人乳头瘤病毒16型(Human papillomavirus type 16,HPV16)感染与口腔癌、宫颈癌的发病有关,HPV16 E6基因编码的蛋白是重要的癌蛋白,已经被证实能够通过增加高迁移率族蛋白B1(High mobility group box-B1,HMGB1)表达来促进宫颈癌细胞的侵袭,但是否能调控口腔癌细胞的侵袭仍未明确。为研究HPV16 E6基因通过增加HMGB1表达调节口腔癌CAL27细胞侵袭的作用,口腔癌CAL27细胞被分为对照组、空白质粒组、HPV16 E6质粒组、NC-si RNA组(短片断干扰RNA阴性对照组)、NC-si RNA+HPV16 E6质粒组、HMGB1-si RNA+HPV16E6质粒组,检测细胞中HPV16 E6及HMGB1的表达、细胞的侵袭数目、培养基中HMGB1的含量。结果显示,HPV16 E6质粒组细胞中HPV16 E6及HMGB1的表达量、培养基中HMBG1的含量、细胞的侵袭数目均高于对照组及空白质粒组(P<0.05);HMGB1-si RNA组细胞中HMGB1的表达量明显低于对照组及NC-si RNA组(P<0.05);NC-si RNA+HPV16 E6质粒组的细胞侵袭数目均明显高于NC-si RNA组(P<0.05),HMGB1-si RNA+HPV16 E6质粒组的细胞侵袭数目均明显低于NC-si RNA+HPV16 E6质粒组(P<0.05)。本研究提示,HPV16 E6基因能够促进口腔癌CAL27细胞的侵袭且这一作用与增加HMGB1表达有关。     

Reversal MDR in breast carcinoma cells by transfection of ribozyme designed according the secondary structure of mdr1 mRNA

Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. The present study aims to investigate whether the ribozyme could reverse MDR in breast carcinoma cells. In this study, two GUC sites (GUC106 and GUC135) on the surface of mdr1 mRNA were selected according to the secondary structure of the 5'-region of mdrl mRNA. The ribozyme gene RZ106 and RZ135 complementary to two sides bases of the target GUC were synthesized and cloned into the plasmid pEGFP -C1 which has EGFP (Enhanced Green Fluorescence Protein) as report gene and Kan/Neo as selection gene. After transfection with the recombinant plasmid and selected by G418, the stable cell clones were produced and used for detection. The alteration of mdr1 mRNA and P-gp in the treated cells was detected by RT-PCR, flow cytometry and Rh123 retention. The reversal efficiency of the drug resistance for adriamycin was determined by MTT assay. The results showed that after transfection with RZ106 and RZ135, the amount of the mdr1 mRNA and P-gp decreased significantly and the efflux function of P-gp was inhibited accordingly. Nine-fold and 16-fold reduction of resistance for adriamycin was observed in the two groups of treated cells. These results suggested that both ribozymes can reverse the MDR phenotype by inhibiting the expression of mdr1 mRNA and P-gp, and the RZ135 showed the better cleavage efficiency. The ribozyme strategy designed according the secondary structure of the target RNA could be a useful therapy for reversal of MDR.     

Transient gene expression control: effects of transfected DNA stability and trans-activation by viral early proteins.

The effects of trans-acting factors and transfected DNA stability on promoter activity were examined with chloramphenicol acetyl transferase (CAT) transient expression analysis. With cotransfection into CV-1P and HeLa cells, simian virus 40 T antigen, adenovirus E1a, and herpes-virus IE proteins were compared for their ability to trans-activate a variety of eucaryotic promoters constructed into CAT plasmids. T antigen and the IE protein were promiscuous activators of all the promoters tested [the simian virus 40 late promoter, the adenovirus E3 promoter, the alpha 2(I) collagen promoter, and the promoter of the Rous sarcoma virus long terminal repeat]. Conversely the E1a protein was specific, activating only the adenovirus E3 promoter and suppressing the basal activity of the other promoters. This specificity of activation by E1a contrasted with the high activity generated by all of the promoter-CAT plasmids when transfected into 293 cells, which endogenously produce E1a protein. Examination of transfected 293 cells determined that they stabilized much greater amounts of plasmid DNA than any other cells tested (CV-1P, COS, NIH-3T3, KB). Thus the high activity of nonadenovirus promoter-CAT plasmids in 293 cells results from the cumulative effect of basal promoter activity from a very large number of gene copies, not from E1a activation. This conclusion was supported by similar transfection analysis of KB cell lines which endogenously produce E1a protein. These cells stabilize plasmid DNA at a level comparable to that of CV-1P cells and, in agreement with the CV-1P cotransfection results, did not activate a nonadenovirus promoter-CAT plasmid. These results indicate that the stability of plasmid DNA must be considered when transient gene expression is being compared between cell lines. The use of relative plasmid copy numbers for the standardization of transient expression results is discussed.     

人乳头瘤病毒16型 E7蛋白在子宫颈癌细胞内的定位

应用重组质粒在大肠杆菌中表达人乳头瘤病毒(HPV)16 型 E7 基因.以所产生的 E7 融合蛋白为抗原免疫家兔,制得抗 E7 蛋白抗血清.在子宫颈癌组织切片中用此抗血清作免疫组化染色(胶体金标记染色法).在光学显微镜下可观察到癌细胞中存在 E7 抗原黑色颗粒,位于细胞核内.主要附着于核膜,可证明 E7 基因在 HPV16 感染的子宫颈癌细胞中有强烈表达;提示 E7 基因可能即为 HPV16的癌基因.     

Splicing of a cap-proximal human Papillomavirus 16 E6E7 intron promotes E7 expression, but can be restrained by distance of the intron from its RNA 5' cap

Human papillomavirus 16 (HPV16) E6E7 pre-mRNA is bicistronic and has an intron in the E6 coding region with one 5' splice site and two alternative 3' splice sites, which produce E6(*)I and E6(*)II, respectively. If this intron remains unspliced, the resulting E6E7 mRNA expresses oncogenic E6. We found for the first time that the E6E7 pre-mRNA was efficiently spliced in vitro only when capped and that cellular cap-binding factors were involved in the splicing. The cap-dependent splicing of the E6E7 pre-mRNA was extremely efficient in cervical cancer-derived cells, producing mostly E6(*)I, but inefficient in cells transfected with a common retrovirus expression vector, pLXSN16E6E7, due to the large size of this vector's exon 1. Further studies showed that efficient splicing of the E6E7 pre-mRNA depends on the distance of the cap-proximal intron from the RNA 5' cap, with an optimal distance of less than 307nt in order to facilitate better association of U1 small nuclear RNA with the intron 5' splice site. The same was true for splicing of human beta-globin RNA. Splicing of the E6E7 RNA provided more E7 RNA templates and promoted E7 translation, whereas a lack of RNA splicing produced a low level of E7 translation. Together, our data indicate that the distance between the RNA 5' cap and cap-proximal intron is rate limiting for RNA splicing. HPV16 E6E7 pre-mRNA takes advantage of its small cap-proximal exon to confer efficient splicing for better E7 expression.     

Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors.

Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant. Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase. When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein. Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature. Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.