质粒体

Methods Eukaryotic expression plasmid pCMVCD was constructed , and identified by re2 striction endoenzyme digestion. CD gene was transfected into NSCs from new2born Wistar rats using Lipofec2tamine2000. Positive clones (named NSCs/ CD cells) were screened by G418 presence. 52Fluorocytosine (52FC) ad2ministration of different concentrations were incubated with NSCs/ CD cells. NSCs/ CD cells viability ratios were mea2 sured by MTT assay.

通过构建真核表达质粒pCMVCD ，限制性内切酶消化鉴定后，采用Lipofectamine 2000 脂质体介导法转染新生大鼠室管膜下区神经干细胞(Neural stemcells ， NSCs)，G418 筛选阳性克隆，加入不同浓度的52氟胞嘧啶(52Flourocytosine ， 52FC)，MTT比色法测定NSCs的生存率。

Interval observations under thefluorescent microscop were performed with excition waves of 470—490 nm.The results displayed that GFP genes were expressed effectively in E.octocarinatus, and GFP was distributed unifimly around macronuclearand diffused to the entire cell gradually. The chromosomes can be kept inthe macronucleus for 90h.

用优化的脂质体转化方法将含有人工染色体的重组质粒pBTub-tel2转化到处于有性生殖分裂间期阶段的游仆虫细胞中，结果表明，GFP基因在游仆虫细胞中得以高效表达，36-48小时绿色荧光均匀分布在细胞核周围，逐渐扩散到整个细胞，染色体能够在大核中保持约90小时。

RESULTS A eukaryotic expression system for high expression humanmutantCD59 were successfully set up : The recombinant PALTER-MAX plasmid containing human mutantCD59 cDNA and PCDNA plasmid were co-transfected into CHO cell by cation lipoid mediating method ;and the cells were grown in F12 medium containing 400ug/ml G418 for 14 days, positive clones were grown in RPMI1640 medium to get stable expressing cell lines . Highly expressing clones were selected by flow cytometry ,and were named PALTER-CD59-CHO1PALTER-CD59-CHO2 . Flow cytometry indicated that expression rates of PALTER-CD59-CHO1 and PALTER-CD59-CHO2 were 53.7%and 54.5%. Further more, Stable highly expressing CHO cell lines were more detected by immunocytochemistry and immunofluorescence technology . PALTER-CD59 -CHO1 and PALTER-CD59-CHO2 were grown in RPMI1640 to get a large of cells . CD59 protein were obtained by spalling PALTER- CD59- CHO1 and PALTER - CD59 - CHO2 cells . Stable highly expressing cells were further validated by SDS-PAGE, immunoblot analysis and solid enzyme immunoassay . PALTER - CD59 - CHO1 and PALTER - CD59 - CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER-CD59-CHO1 or PALTER-CD59-CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59 -CHOI or PALTER-CD59-CHO2 was higher than unglycated ones . PALTER -CD59-CHO1 and PALTER -CD59 -CHO2 were glycated in RPMI1640 of 50mM ribose for 72 hours .BCECF releasing test indicted that the releasing rate of PALTER - CD59 - CHOI or PALTER - CD59 - CHO2 was less high than PALTER-CHO ,and the releasing rate of glycated PALTER-CD59-CHO1 or PALTER - CD5 9-CHO2 was higher than unglycated ones .

结果 成功构建突变人CD59的真核细胞表达系统：运用阳离子脂质体介导法将含有突变人CD59的PALTER—MAX重组质粒与PCDNA共转染入CHO细胞：用含有400ug／mlG418的F12培养基培养14天，筛选出稳定阳性表达克隆，RPMI1640培养基扩增获得稳定表达细胞株，并用流式细胞术进一步筛选出高效表达细胞株分别命名为PALTER—CD59—CH01、PALTER—CD59—CH02，表达率分别为53.7％、54.5％；应用免疫组化方法、免疫荧光技术进一步鉴定阳性细胞株；RPMI1640培养基大量扩增PALTER—CD59—CH01、PALTER—CD59—CH02细胞株，裂解细胞得到CD59蛋白质；通过SDS—PAGE凝胶电泳技术、免疫印迹技术、固相酶联免疫吸附试验验证了这两中文摘要个阳性细胞株CO59蛋白的高效表达；50mM核糖培养72小时，获得突变人CD59糖化细胞株，BCECF染料释放试验结果显示，PALTER一CD59一CHOI、pALTER一CD59一CHOZ细胞较PALTER一CHO细胞染料释放率低，未糖化PALTER一CD59一CHOI、PALTER一CD59一CHOZ细胞比较糖化后细胞染料释放率低。

Fluorescence in situ hybridization was carried out to confirm the integration of HBV DNA into male pronucleus and its replication with cell division in embryonic development.(Reverse transcriptase polymerase chain reaction,RT-PCR) and immunofluoresence assay were performed to observe the expression of the HBV gene in two-cell stage.

材料与方法：成熟雄鼠麻醉后双侧睾丸注射经脂质体DOSPER包裹的HBV质粒，手术后雄鼠与超排雌鼠合笼交配，用荧光原位杂变(Fluorescence in situ hybridization，FISH)分别检测单细胞胚和二细胞胚间期核中HBV DNA的存在与复制，用逆转录聚含酶链反应(Reverse transcriptase-polymerase chain reaction，RT-PCR)和免疫荧光方法检测HBV基因在二细胞胚胎中的表达。

METHODS: MyoD cDNA fragments were extracted from plasmids pEMSV-MyoD with polymerase chain reaction, and PCR was used to clone the whole-length gene of MyoD. After adding CACC sequence at 5' end, MyoD gene was cloned by orient topology into transfer ventor, pENTR/D-TOPO. Objective gene was transferred into adenoviral expression vector DNA via pENTR/D-TOPO vector. The recombinant adenoviral vectors transfected into HEK293A cells by using lipofectamine were packaged and amplified.

从pEMSV-MyoD质粒上用聚合酶链反应法扩增出MyoD cDNA片段，再通过聚合酶链反应使MyoD基因加上CACC序列接头，经过定向拓扑克隆使目的基因连接到转移载体上，再通过LR酶促反应，将目的基因转移到腺病毒表达载体DNA上，获得MyoD基因重组的腺病毒DNA，用脂质体转染法转染HEK293A细胞，包装扩增出MyoD基因重组的腺病毒。

These transgenic cells were able to secrete hEGF protein to certain extent, and have the biological activities to enhance the growth rate of Hacat cells in vitro. Conclusion: Adult epidermal keratinocytes in vitro transfected with exogenetic hEGF cDNA can express and secrete active hEGF.

质粒pcDNA3.1-hEGF在脂质体介导下成功转染成人皮肤角质形成细胞，转基因细胞能分泌有生物活性的EGF；体外培养的成人皮肤角质形成细胞可见少量EGF分泌。

Methods Femoral arteries isolated from the rabbits which had been fed with a high cholesterol diet and locally perfused with MM-LDL within the artery beforehand, were used as the models. Antisense MCP-1cDNA was transferred into the arterial wall by injecting recombinant LNCX-anti-MCP-1/liposomal complex in the femoral sheath and the periarterial tissue.

以实验性高脂血及轻度氧化LDL局部保留灌注的家兔股动脉为模型，采用DOTAP阳离子脂质体包裹LNCX-anti-MCP-1质粒，将反义MCP-1 cDNA定位导入家兔股动脉鞘及周围组织，以原位杂交、 Northern杂交及狭缝杂交法观察反义MCP-1基因的表达，应用扫描电镜观察单核细胞在动脉内膜粘附的情况。

By using liposome-mediated gene transfection technology, plasmid pCMVSV40T/PUR containing SV40Tag was transfected in primary embryonic precartilaginous stem cells, while non-transfected cells served as negative controls.

利用脂质体介导基因转染技术将含有SV40Tag的质粒pCMVSV40T/PUR转染原代胚胎前软骨干细胞，以未转染细胞作阴性对照。

METHODS Human IL 6 gene was reconstructed in retrovirus vector and transferred into incasing cells PA317 by lipofectamine mediated method. The clones of the cells transferred with hIL 6 were selected by G418. Targeted NIH3T3 cells were infected with the virus granules secreted from PA317 and also selected by G418. The insertion and expression of hIL 6 gene in NIH3T3 cells were analysed with Southern blot and Northern blot. RESULTS Human IL 6 retrovirus vector (pZIPIL-6) was successfully reconstructed.

利用重组载体构建技术将质粒pUCIL 6 cDNA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Southern blot和Northern blot分析，检测目的基因在靶细胞的整合与转录水平。

Results: it was efficient to transfect chicken embryonic fibroblast cells by the method of lipofectamine mediation. the expression levels of protein were significantly different.

结果 脂质体介导的方法能有效转染鸡胚胎成纤维细胞，实验中egfp蛋白相对表达量随质粒量的增加而明显升高（p.01）。

She gently rebuff ed him, but agreed that they could be friends

她婉言拒绝了，但同意作为朋友相处。

If in the penal farm, you were sure to be criticized.

要是在劳改农场，你等着挨绳子吧！