逆转录病毒载体介导IL 6基因转导成纤维细胞的表达

目的将ＩＬ６基因转导至成纤维细胞ＮＩＨ３Ｔ３，并使转染株有效地表达ＩＬ６，为ＩＬ６转基因治疗奠定基础．方法利用重组载体构建技术将质粒ｐＵＣＩＬ６ｃＤＮＡ的目的片段连接于逆转录病毒载体上，并以脂质体介导的方法将重组载体转染包装细胞ＰＡ３１７，以Ｇ４１８筛选克隆细胞，浓缩克隆细胞上清以制备重组病毒液，继之感染ＮＩＨ３Ｔ３细胞后，进行Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ分析，检测目的基因在靶细胞的整合与转录水平．结果成功地构建了重组载体ｐＺＩＰＩＬ６ｃＤＮＡ，筛选出抗生较强的克隆细胞，制备了高滴度的重组病毒液．杂交结果表明转导株３Ｔ３ＩＬ６具有ＩＬ６基因的整合和相应ｍＲＮＡ的高表达．结论ＩＬ６基因能稳定整合至靶细胞并进行有效的转录表达，为ＩＬ６基因治疗的应用奠定了可靠的基础     

PRS载体介导的表达CTGF-SiRNA逆转录病毒载体的构建

【目的】梅建表达人结缔组织生长因子（CTGF）小分子干扰RNA（small interfering RNA,SiR—NA）的PRS-CTGF-SiRNA逆转录病毒载体。【方法】根据SiRNA靶序列设计要求及PRS逆转录病毒载体特点分别设计含64bpDNA序列的4对寡核苷酸,并在体外合成。PRS载体用BgⅡ、HindⅢ双酶切完全后,分别与退火的4对寡核苷酸进行连接,连接后去载体自连,构建成表达人CTGF小分子干扰RNA的PRS-CTGF-SiRNA逆转录病毒载体。【结果】经酶切、连接后构建成的质粒称之为PRS-CTGF—SiRNA1～4,经酶切与测序证实构建成功,无任何碱基突变。【结论】成功构建了能表达人CTGF-SiRNA的PRS-CTGF—SiRNA逆转录病毒载体,为腹膜透析腹膜纤维化防治的体内、外研究提供一种可能有效的方法。     

Inhibitory effect of retroviral vector containing anti-sense Smad4 gene on Ito cell line, LI90

Background Transforming growth factor-β1 (TGF-β1) exerts strong fibrogenic potential in culture-activated HSCs. Smad4 is a key intracellular mediator for the transforming growth factor-β (TGF-β) superfamily of growth factors. The aim of this study was to assess the effects of the antisense Smad4 gene on Ito cell line, LI90.Methods The recombinant retroviral vector pLXSN-Smad4 was constructed by cloning the rat antisense Smad4 cDNA into the retroviral vector pLXSN. Retroviruses with or without the antisense gene were obtained by transfecting pLXSN-Smad4 and pLXSN vectors into PA317 cells. Human hepatic stellate cells (HSCs) LI90 were infected with these retroviruses followed by selection with G418. The expression of Smad4 was detected by Northern and Western blots. Cell biological characteristics, including cell growth curve, 3H-TdR and 3H-proline uptake by HSCs and the production of extracellular matrix were assessed.Results mRNA and protein expressions of Smad4 in LI90 cells transfected with retrovirus containing the antisense Smad4 gene were much lower than those in LI90 cells transfected with empty vector or parental LI90 cells. Cells hypoexpressing the Smad4 gene exhibited a slower rate of growth, a lower uptake of 3H-TdR and 3H-proline (P<0.01), and smaller production of th extracellular matrix, compared with parental LI90 cells and cells transfected with empty retrovirus.Conclusions The antisense Smad4 gene can suppress the expression of the Smad4 gene, reduce endogenous production of Smad4 mRNA and protein, block TGF-β1 signaling pathway, inhibit activation of Ito cells, obstruct the growth of Ito cells, decrease the production of the extracellular matrix （ECM）. Our results may provide a basis for the development of antifibrotic gene therapy.     

用双拷贝逆转录病毒载体转导人G－CSF cDNA的实验研究

为克服逆转录病毒中内部启动子对插入外源基因表达的干扰，将人Ｇ－ＣＳＦ（粒细胞集落刺激因子）ｃＤＮＡ插入双拷贝载体Ｎ２Ａ的３＇ＬＴＲＵ３区上的多克隆位点，酶切鉴定后将重组质粒用电击转导法转入Ｐｓｉ－２嗜单性包装细胞系，经Ｇ－４１８选择压力，两周后挑出阳性克隆，测定感染滴度后转染ＮＩＨ３Ｔ３细胞，选择挑出抗性克隆，测定Ｇ－ＣＳＦ表达量，最高达５３．３Ｕ／１０６细胞。Ｓｏｕｔｈｅｒｎ印迹和Ｎｏｒｔｈｅｒｎ印迹分析证明嵌合的目的基因在感染细胞中通过基因复制而转染到５＇ＬＴＲ的转录单位之外并且在染色体ＤＮＡ中稳定整合，复制产生两个拷贝，在３＇ＬＴＲ和５＇ＬＴＲ中各有一个基因。     

NDRG2基因反转录病毒载体的构建及表达

背景：NDRG2(N-Myc Downstream Regulated Gene 2)是一个新的抑癌基因,既可以增强经典抑癌通路的抗肿瘤效应,又可以对正常细胞的癌变起到监控。有关NDRG2在骨髓瘤发生中的功能和作用至今还未见报道。 目的：构建NDRG2基因反转录病毒表达载体,利用包装的病毒感染人骨髓瘤细胞系U266,检测NDRG2的表达情况。 方法：设计与合成引物,提取U266细胞的RNA,反转录和PCR扩增,经BamHⅠ和TaqⅠ双酶切,琼脂糖凝胶电泳,切胶回收进行连接转化,并再次酶切鉴定;并将构建的载体包装为反转录病毒,感染U266细胞,筛选出稳定表达NDRG2的U266细胞(U266-NDRG2)克隆扩大培养,利用Western blot实验检测筛选到的U266细胞中NDRG2的表达。 结果与结论：成功构建了携带NDRG2基因表达的重组载体pBaba-puro-NDRG2,并包装为反转录病毒,筛选到的U266细胞(U266-NDRG2)中NDRG2蛋白表达明显高于U266-cherry细胞和U266细胞。结果可见利用反转录病毒载体基因重组技术成功构建出携带相应基因的反转录病毒,为研究NDRG2在人骨髓瘤中的作用奠定了实验基础。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

hG－CSF cDNA重组逆转录病毒的构建

通过ＤＮＡ重组技术，将不含非编码区的人粒细胞集落刺激因子（ｈＧ－ＣＳＦ）ｃＤＮＡ重组入逆转录病毒质粒ｐＬＸＳＮ中。重组质粒转染ＰＡ３１７细胞后，经Ｇ４１８筛选，抗性克隆细胞培养上清液能成功地感染ＮＩＨ３Ｔ３细胞，使之在筛选培养基中形成典型的Ｇ４１８抗性克隆。该克隆细胞染色体中成功地整合了ｈＧ－ＣＳＦｃＤＮＡ，并且表达了有生物学活性的ｈＧ－ＣＳＦ产物。     

Transduction of human hepatocellular carcinoma cells with human alpha-interferon gene via retroviral vector

AIM:To investigate the therapeutic potential of gamma interferon (IFN-alpha) genemodified human hepatocellular carcinoma (HCC) cells.METHODS:The IFN-alpha gene was introduced retrovirally into four HCC cell lines.Secreted IFN-alpha activity was assessed using bioassay. The expression of MHC molecules was detected by FACS.Tumorigenicity was analysed by tumor formation in nude mice.RESULTS:Four IFN-alpha gene transduced HCC cell lines secreted different amounts of IFN-alpha, as in the same case of five clones derived from one HCC cell line. Transduction with IFN-alpha caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class I was increased by 2-3 times in terms of mean fluorescence intensities, while for class II expression, the percentage of positive cells augmented from < 10% to &lg 50%. When equal amount of tumor cells were injected into nude mice, the tumor igenicity some transduced cells decreased dramantically.CONCLUSION:IFN-alpha gene transduction can convert weakly imunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.     

逆转录肿瘤病毒在细胞增殖和凋亡中的双向机制

目的 :研究逆转录肿瘤病毒在L6 15小鼠T细胞白血病增殖和凋亡中的机理。方法 :6 15近交系小鼠 2 0只 ,随机分为病毒感染组和正常对照组。感染组动物皮下接种携带小鼠逆转录病毒的L6 15白血病肿瘤株 ,检测外周血白细胞总数 ,观察肿瘤细胞凋亡形态特征及DNA电泳图谱 ,并检测肿瘤细胞中c myc和ras基因的表达。结果 :感染组在接种肿瘤细胞株后第 8天外周血中的肿瘤细胞急剧增高 [(0 2 199± 0 10 99)× 10 9 L],与正常对照组比较差异有极显著性 (P <0 0 1)。肿瘤细胞中c myc和ras基因的表达率分别为 (76 0 0± 3 2 318) %和 (5 6± 2 0 138) %。在肿瘤细胞中还能观察到典型的细胞凋亡小体 ,凋亡率为 (4 6 0± 0 97) % ,外周血淋巴细胞DNA电泳也显示典型的梯形电泳带。结论 :L6 15小鼠逆转录肿瘤病毒在引发T细胞白血病过程中 ,可导致肿瘤细胞c myc和ras基因异常表达 ,同时还激活宿主对肿瘤细胞的凋亡路径。