微小RNA-27 a对黑色素瘤WM239细胞增殖和凋亡的影响

目的：探讨微小RNA-27a（ miR-27a）模拟物和抑制物转染黑色素瘤WM239细胞后对细胞增殖和凋亡的影响。方法将miR-27a模拟物、抑制物及其阴性对照转染WM239细胞,荧光显微镜观察转染效率,实时荧光定量PCR检测相应的微小RNA,四甲基偶氮唑盐（ MTT）法检测细胞增殖,流式细胞仪检测细胞凋亡和细胞周期。结果细胞转染效率为80%~90%。转染miR-27a模拟物后,细胞内miR-27a表达量明显上升（2-△△CT值为26.98±0.01）,与正常对照组相比差异有统计学意义（ t＝－1123.67,P＝0.00）;转染miR-27a抑制物后,细胞中miR-27a的表达量下降（2-△△CT值为0.96±0.02）,与正常对照组相比差异无统计学意义（t＝4.04,P＝0.06）。转染miR-27a模拟物后,细胞增殖受到明显抑制,与正常对照组相比差异具有统计学意义[72 h吸光度（0.45±0.02）∶（0.72±0.01）,F＝129.56,P﹤0.05]。miR-27a模拟物组G0-G1期的细胞比例升高[（74.83±1.46）∶（63.73±1.25）,F＝30.33,P﹤0.05],S期和G2-M期细胞比例减少[（21.33±1.75）∶（27.50±1.25）,F＝14.98,P﹤0.05;（3.90±1.31）∶（8.80±2.10）,F＝3.66,P﹤0.05];模拟物组细胞凋亡率与正常对照组相比明显增加[（29.67±0.91）%∶（1.44±0.85）%, F＝530.90,P﹤0.01];而抑制物组对细胞周期和凋亡无明显作用。结论 miR-27a抑制黑色素瘤细胞增殖,具有抑瘤作用,这与其促进细胞凋亡,阻滞细胞周期于G0-G1期相关。     

In vitro cultivation of rat bone marrow mesenchymal stem cells and establishment of pEGFP/Ang-1 transfection method

ObjectiveTo obtain the bone marrow mesenchymal stem cells (BMSCs), complete phenotypic identification and successfully transfect rat BMSCs by recombinant plasmid pEGFP/Ang-1.MethodsBMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method, and purified. Then the recombinant plasmid pEGFP/Ang-1 was used to transfect BMSCs and the positive clones were obtained by the screen of G418 and observed under light microscopy inversely. Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.ResultsBMSCs cell lines were obtained successfully by adherence screening method and density gradient centrifugation. Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs, which can express Ang-1 for 3 d and decreased after 7 d.ConclusionsAdherence screening method and density gradient centrifugation can be effective methods to obtain BMSCs with high purity and rapid proliferation. Besides, the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.     

Bone Sialoprotein Promotes Tumor Cell Migration in Both In Vitro and In Vivo Models

The present study was conducted to determine the effects of bone sialoprotein (BSP) in promoting vascular invasion of tumor cells in metastasis. We used a Matrigel system and the MDA-231 human breast cancer cells transfected with human BSP cDNA (MDA-231/BSP). Quantative analysis indicated an average of 1.7-fold increase in cell numbers that migrated through the endothelial cells in MDA-231/BSP cells compared with empty vector-transfected MDA-231 cells (MDA-231/EV). In an in vivo assay, the MDA-231 cells were incubated with or without BSP antibodies and were then inoculated onto the upper chorioallantoic membrane (CAM) of chicken embryos, in which the only route for the tumor cells to reach the lower CAM was to migrate through the embryonic vasculature. PCR amplification using human Alu primers and genomic DNA from harvested lower CAM showed an average reduction of 67% in the samples treated with BSP antibodies. These preliminary data suggest that, in metastasis, BSP may enhance the penetrating ability of tumor cells through endothelial cells and basement membrane into blood vessels. BSP antibodies can specifically hinder this effect in an in vivo system.     

Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: Studies on formulation,biodistribution and vector clearance

We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT-18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vectors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immunization of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in significantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are mediated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications.     

菊花总黄酮对转染RSV的A549细胞RANTES及MCP-1表达的影响

【目的】探讨菊花总黄酮对呼吸道合胞病毒(Respiratory Syncytial Virus,RSV)转染 A549细胞诱导激活正常 T 细胞表达和分泌因子(regulated upon activation ,normal Tcell expressed and secreted ,RANTES)及单核细胞趋化蛋白-1(monocyte chemotactic protein 1,MCP-1)释放作用影响。【方法】实验分为正常对照组,病毒对照组,菊花总黄酮组和巴韦林组。在人喉癌上皮细胞(Hep-2细胞)和气道上皮细胞(A549)分别加入菊花总黄酮和巴韦林的含药维持液,测定上述两种药物的最大无毒浓度；RSV 转染 Hep-2细胞,观察药物对 RSV 的病毒抑制作用；RSV 转染 A549细胞,ELISA 法测细胞趋化因子 RANTES 及 MCP-1含量。【结果】菊花总黄酮组50%有效率优于巴韦林组,差异有统计学意义(P <0.05)；菊花总黄酮组 RANTES、MCP-1明显降低,但高于正常细胞组,优于巴韦林组,差异有统计学意义(P <0.05)。【结论】菊花总黄酮能够抑制 RSV 活性,明显降低 A549细胞释放 RANTES、MCP-1,缓解患儿的呼吸道症状。     

pcDNA3.1-CD81载体在人肝癌细胞中的表达及意义

目的研究人白细胞分化抗原CD81真核表达载体pcDNA3．1-CD81在人肝癌细胞系HepG2中的表达及意义。方法由人Molt-4细胞提取细胞总RNA，根据已公布的序列设计引物，运用RT-PCR及PCR方法扩增出人CD81基因，应用TA克隆插入PMD-18T载体；定向克隆入真核表达载体pcDNA3．1( )；通过脂质体介导转染人肝癌细胞系HepG2，用免疫组织化学方法(ABC法)及流式细胞技术检测目的蛋白的表达。结果由RT-PCR及PCR方法扩增出入CD81编码基因与Genebank公布的序列完全一致。重组真核表达载体pcDNA3．1-CD81经酶切和PCR鉴定分析正确。免疫组织化学方法(ABC法)及流式细胞技术证明转染的HepG2细胞表面能有效地表达CD81蛋白。结论所构建的pcDNA3．1-CD81真核表达载体在HepG2细胞有良好的表达，该转染细胞可作为研究CD81在HCV感染中的作用提供细胞模型，为研究丙型肝炎病毒(HCV)与CD81相互作用奠定基础。     

KAI1 is a potential target for anti-metastasis in pancreatic cancer cells

AIM： To investigate whether KAI1, as a metastasis suppressor gene, is associated with invasive and metastatic ability of pancreatic cancer cells.METHODS： KAI1 gene was transfected into pancreatic cancer cell line MiaPaCa Ⅱ by liposomes selected with G418. Expression of transfected cells was measured by Western blotting, immunofluorescence and immunocytochemistry. Tumor cell invasion and metastatic ability were detected through gelatinase activity and reconstituted basement membrane （Matrigel） assay. pCMV-KAI1 was directly injected into the heterotopic human pancreatic adenocarcinoma successfully established in the groin of BALB/C nude mice, by subcutaneous injection of MiaPaCa Ⅱ pancreatic cancer cells. The statistical analysis between groups was determined by Student＇s two tailed t test.RESULTS： By Western blotting, MiaPaCa Ⅱ cells transfected by KAI1 gene indicated KAI1 expression at approximately 29.1 kDa. Cytoplasm staining was positive and uniformly spread in transfected cancer cells, using immunohistochemistry and immunofluorescence. The most obvious difference was present after 30 h （MiaPaca Ⅱ 43.6 ± 9.42, pCMV-MiaPaca Ⅱ 44.8 ± 8.56, pCMV-KAI1-MiaPaca Ⅱ 22.0 ± 4.69, P 〈 0.05）. Gelatinolysis revealed a wider and clearer band of gelatinolytic activity in non-transfected than in transfected cells （MiaPaCa Ⅱ cells 30.8 ± 0.57, transfected cells 28.1 ± 0.65, P 〈 0.05）. In vivo tumor growth rates of KAI1 transfectants with KAI1-Lipofectamine 1.22 ± 0.31 in A group were lower than control 4.61 ± 1.98 and pCMV-KAI 11.67 ± 0.81. Analyses of metastases with and without KAI1 transfection in mice were different in liver and lung between controls 1.62 ± 0.39, 0.45 ± 0.09, pCMV-KAI 1.01 ± 0.27, 0.33 ± 0.09 and KAI1-Lipofectamine 0.99 ± 0.21, 0.30 ± 0.09 respectively （P 〈 0.05）.CONCLUSION： High expression of KAI1 gene was found in transfected MiaPaCa Ⅱ human pancreatic cancer cells with lower metastatic ability. KAI1 gene plays an important role in inhibiting     

反义肝素酶基因对胰腺癌细胞体外增殖和侵袭的抑制作用

目的:研究反义肝素酶基因对人胰腺癌SW1990细胞体外增殖和侵袭能力的抑制作用．方法:反义肝素酶基因转染胰腺癌sW1990细胞,并设空载体对照组和空白对照组,以流式细胞仪检测细胞周期;免疫组化、Western blot及RT-PCR检测肝素酶蛋白和mRNA表达;平板克隆形成实验检测细胞增殖活性,Transwell侵袭小室模型检测细胞体外侵袭能力．结果:与空白组和空载组比较,反义组细胞周期中S期比例明显减少(18.8％±2．5％vs 36．3％±2．2％,33．2％±2．1％,P<0．01),G1期细胞比例明显升高(66．0％±2．7％vs30．7％±3．2％,39．8％±4．9％,P<0．01);肝素酶蛋白及mRNA表达分别降低34．3％和37．8％:细胞克隆形成数目减少(12．2±2．8 vs 30．8±4．4,28．3±2．7,P<0．01);Transwell侵袭小室中24 h穿膜细胞数减少(13．0±3．5 vs 34．8±5．8,29．4±5．6．P<0．01)．结论:反义肝素酶基因抑制人胰腺癌SW1990细胞体外增殖及侵袭能力．     

旋毛虫DNA疫苗在中国仓鼠卵巢细胞中的表达

目的　观察编码旋毛虫相对分子质量(Mr)31000抗原的DNA疫苗(重组真核表达质粒pcDNA3 TspE1)在中国仓鼠卵巢(CHO)细胞中的体外表达 ,并分析其表达产物的抗原性。　方法　通过用阳离子脂质体Lipofectamine2000将重组质粒pcDNA3 TspE1转染CHO细胞,G418筛选阳性克隆,用逆转录聚合酶链反应(RT PCR)、间接荧光抗体试验(IFAT)、十二烷基硫酸钠 聚丙烯酰胺凝胶电泳(SDSPAGE)和蛋白质印迹法(Westernblotting)对表达产物进行鉴定。　结果　RT PCR结果显示,pcDNA3 TspE1转染的CHO细胞在876bp处有一条带,而用空质粒pcDNA3转染的CHO细胞未出现条带,表明pcDNA3 TspE1转染细胞中有TspE1基因转录。IFAT结果显示,pcDNA3 TspE1转染的CHO细胞与重组融合蛋白免疫小鼠血清反应呈现亮绿色荧光 ,而pcDNA3转染的CHO细胞及未转染细胞呈现橘黄色。Westernblotting显示在pcDNA3 TspE1转染的CHO细胞培养液中存在有一Mr约31000的蛋白带 ,且该条带能被重组融合蛋白免疫小鼠血清、旋毛虫肌幼虫可溶性抗原免疫兔血清、感染旋毛虫的小鼠及旋毛虫病患者血清识别。　结论　重组质粒pcDNA3 TspE1可转染CHO细胞,旋毛虫TspE1基因可在转染的CHO细胞中表达,表达蛋白能分泌到细胞培养上清中且具有旋毛虫     

Gene delivery of PAMAM dendrimer conjugated with the nuclear localization signal peptide originated from fibroblast growth factor 3

Polyamidoamine (PAMAM) is one of the widely employed non-viral vectors in gene therapy research, and shows excellent biocompatibility and relatively low cytotoxicity. However, it has poor transfection efficiency compared with that of polyethylenimine (PEI, 25 kDa). To enhance the gene expression efficiency, we introduced the RRRK peptide from mouse fibroblast growth factor 3 (FGF3) to PAMAM, which is a known nuclear localization signal (NLS). We synthesized PAMAM-KRRR and PAMAM-RRRK to verify the difference of the induced functional status from reversal of the N-terminus. PAMAM containing the FGF3 peptide showed a transfection efficiency corresponding to that of PEI in HEK293, and HeLa cells, and showed much higher gene expression capacity than that of PEI in NIH3T3 cells with relatively decreased cytotoxicity. These results imply that introduction of the FGF 3 peptide has the potential to provide a novel PAMAM-based vector by enhancing its gene expression efficiency.