脂质体

Methods:Marrow stromal cells were obtained from the dual ilium of three adult dogs.

从狗的髂骨穿刺骨髓分离骨髓基质细胞，应用脂质体转染VEGF165基因，以RT-PCR检测转染的基因表达。

Rat glioma cell C6 was infected by the deficiency recombinant retrovirus (named C6TK). The sensitivity of C6TK cells to GCV or ACV increased 450 or 10 times than that of C6 cells respectively. Rat C6 glioma model was successfully built, and SD rats inoculated intracerebrally C6TK cells had normal gliomas. The average survival periods of the rats were about 15 days. However, after treated with ACV, the growth of C6TK tumors obviously regressed, and the survival periods extended to 57.8±8.1 days, especially in rats injected with mixed PA317TK and C6 cells or in situ PA317TK cells, which the tumors nearly disappeared after ACV administration and the survival periods extended to over 120 or 71.4±36.1 days respectively, P＜0.001.

结果：构建了带有HSV-tk基因的反转录病毒载体GINaTK，应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317，成为产病毒细胞PA317TK，用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞，命名为C6TK细胞，对GCV和ACV的敏感性分别高于亲本450倍和10倍；成功地建立了大鼠脑胶质瘤模型，并证实转染细胞C6TK的成瘤效应未改变，存活期约为15天；而经ACV治疗后，含有C6TK细胞的肿瘤生长明显被抑制，大鼠生存期延长为57.8±8.07天，尤其是采用PA317TK细胞混和治疗组和原位治疗组，肿瘤基本消失，大鼠生存期显著延长，混和治疗组存活120天以上，原位治疗组存活至71.4±36.1天。t检验，P值均小于0.001。

Results: GINaTK retroviral vector containing HSV-tk was constructed and transduced into PA317 retrovirus packaging cell by lipofectin (named PA317TK). Rat glioma cell C6 was infected by the deficiency recombinant retrovirus (named C6TK). The sensitivity of C6TK cells to GCV or ACV increased 450 or 10 times than that of C6 cells respectively. Rat C6 glioma model was successfully built, and SD rats inoculated intracerebrally C6TK cells had normal gliomas. The average survival periods of the rats were about 15 days. However, after treated with ACV, the growth of C6TK tumors obviously regressed, and the survival periods extended to 57.8±8.1 days, especially in rats injected with mixed PA317TK and C6 cells or in situ PA317TK cells, which the tumors nearly disappeared after ACV administration and the survival periods extended to over 120 or 71.4±36.1 days respectively, P ＜0.001.

结果：构建了带有HSV-tk基因的反转录病毒载体GINaTK，应用脂质体转移方法将GINaTK导入反转录病毒包装细胞PA317，成为产病毒细胞PA317TK，用带有HSV-tk基因的复制缺陷型反转录病毒感染C6细胞，命名为C6TK细胞，对GCV和ACV的敏感性分别高于亲本450倍和10倍；成功地建立了大鼠脑胶质瘤模型，并证实转染细胞C6TK的成瘤效应未改变，存活期约为15天；而经ACV治疗后，含有C6TK细胞的肿瘤生长明显被抑制，大鼠生存期延长为57.8±8.07天，尤其是采用PA317TK细胞混和治疗组和原位治疗组，肿瘤基本消失，大鼠生存期显著延长，混和治疗组存活120天以上，原位治疗组存活至71.4±36.1天。t检验，P值均小于0.001。

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。

The therapy groups displayed significant improvements in neuromotor functions and survivals of neuron and oligodendrocyte compared with control groups. These results demonstrated the neuroprotect roles of GGF2, and cationic liposome-mediated GGF2 transgene therapy has potential possibility.

结果：1、大鼠LFP致伤后3—7天，GGF2 mRNA在海马CAl区、CA2区、CA3区、DG及皮层表达明显增加。2、阳离子脂质体介导GGF2转基因治疗可促进大鼠伤后行为学的恢复，可促进损伤区神经元及少突胶质细胞的存活。3、原核表达方法得到GGF2重组蛋白。

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。

The labia have now been sutured together almost completely.The drains and the Foley catheter come out at the top.

此刻阴唇已经几乎完全的缝在一起了，排除多余淤血体液的管子和Foley导管从顶端冒出来。

To get the business done, I suggest we split the difference in price.

为了做成这笔生意，我建议我们在价格上大家各让一半。