腺病毒

Because different cell lines may show various levels of susceptibility to adenovirus infection and virus production, non-selectively replicating wtAd5 was used to be a comparison with the tested conditionally replicating adenovirus CNHK500.ONYX-015, the first replication-competent adenovirus entered into clinical trials was also used to be a comparison. We used virus proliferation assay, cell viability assay and western blot to evaluate the proliferation and cytolysis selectivity of CNHK500. Results demonstrated that the CNHK500 virus proliferation multiples in breast cancer cell lines after 48 hours is similar to that of wtAd5, ONYX-015 virus proliferation ability is less than that of CNHK500 in cancer cells.

然后，通过与ONYX-015（E1B 55 KD a蛋白缺失的2型和5型嵌和型腺病毒，美国ONYX生化制药公司研制）、wtAd5进行对比，利用病毒增殖实验和细胞生长抑制实验，验证了CNHK500选择性增殖能力和肿瘤特异性杀伤作用；利用Western Blot检测CNHK500腺病毒E1A和E1B在乳腺癌细胞和正常成纤维细胞中的表达差异，揭示了腺病毒肿瘤靶向性的机制；利用携带绿色荧光蛋白的CNHK500-EGFP感染乳腺癌细胞株和正常成纤维细胞株，直观地观察其感染乳腺癌细胞的能力和在乳腺癌细胞中的增殖过程。

Recombinant adenovirus particles were produced after 293 cells transfected with recombinant Ad genomic plasmid DNA digested with PacI, then they were further amplified in 293 cells.

纯化所得腺病毒滴度约为8×1012pfu/L，当MOI=100时，腺病毒感染HepG1细胞的效率＞75％，Western Blot证实在感染重组体腺病毒的细胞中有相应基因产物的表达。

The adenovirus plasmid was identified by PacI digestion and transfected into 293A cells to package a recombinant adenovirus which expressed the Fhit protein. Furthermore, the adenovirus rAd-Fhit was infected into colon cancer cells,and the expression of the ectogenic protein was detected by Western blotting. Finally, the proliferation of colon cancer cells was observed in adenovirus-infected cells by the MTT assay.

经PacI酶切鉴定正确后，将重组腺病毒质粒转染293A细胞获得表达Fhit蛋白的重组腺病毒rAd-Fhit，将获得的重组腺病毒感染结肠癌细胞，采用蛋白印迹法检测外源Fhit蛋白的表达，并进一步观察其对细胞增殖能力的影响。

Methods In this study,a plasmid vector,rAd5,and rAAV2 were used to transfect rat cochlear marginal cells of stria vascularis by injection into the perilymph through the round window membrane,and the transfection efficiency, target tissue accessibility, cell/ tissue toxicity, time course of expression, and effect on hearing were evaluated in vivo and in vitro.

结果三种载体对原代培养的大鼠耳边缘细胞的转染研究发现，腺病毒的转染效率最高，而腺相关病毒转染对细胞活性影响最小。rAAV2和rAd5对大鼠耳蜗组织的感染实验发现，腺病毒和腺相关病毒携带的增强型绿色荧光蛋白可在多种耳蜗组织中及转染对侧耳蜗组织表达，且并不引起明显的耳蜗组织细胞凋亡。rAAV2携带的EGFP基因在第90天时仍可检测到大量表达。rAd5携带的EGFP基因在第30天时表达明显减弱。

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。

Methods The promoter of hTERT and Cox-2 was subcloned from human genomic DNA, then ligated to shuttle vector pd306 which contains the Ela and Elb gene of adenovirus to control the expression of Ela and Elb,respectively.

将hTERT和Cox-2启动子从人类白细胞基因组中亚克隆出来，并将启动子分别插入到腺病毒穿梭载体pd306上的E1A和E1B基因前，使hTERT和Cox-2启动子分别调控腺病毒必须基因E1A和E1B的表达，再将构建后的pd306和腺病毒的骨架质粒BHGE3在Ad293细胞内进行同源重组，并用重组后的病毒感染Hela细胞检测病毒对肿瘤细胞的杀伤力。

Was simultaneously injected into cisterna magna at the second injection blood in the presence of subarachnoid blood. At 2 to 4 days, GFP was expressed in leptomeninges over the brain stem, cortex and cerebral arteries, smooth muscle cells of small vessels were occasionlly transduced. GFP was expressed in adventitia of spastic basilar artery on days 2 (day 5 after first injection blood) after injection 〓, but was undetectable by days 4, transgene was not expressed in medial or intimal layers. The prensence of subarachnoid blood can not prevent access of adenovirus to vessels and transgene expression.

注入腺病毒载体后2天（初次注血第5天）行荧光显微镜、免疫组织化学和RT-PCR检测，结果在颅内大血管如基底动脉的外膜可见外源基因的表达，而外源基因不能转移至血管中膜和内膜，4天时（初次注血第7天）基底动脉的外膜中外源基因表达消失；注入腺病毒载体后2～4天，外源基因可以有效转移至颅底软脑膜细胞，小血管外膜和平滑肌层也可见外源基因的表达，表明蛛网膜下腔内的血凝块不能阻止腺病毒载体介导外源基因转移至颅内血管。

The late phase expression cassette suited for adenovirus vectors was also constructed. The HBV S gene, HBV preS2+S gene and the late phase expression cassette +HBV S genes were respectively cloned into the Ad5 vector downstream of the E3 promoter. Semiconfluent monolayers of 293 cells were cotransfected with the above constructed plasmids and Ad5 DNA EcoRI A fragment. The progeny adenoviruses named rAd5S, rAd5MS, rAd5S2S were harvested.

通过共转染优化实验，建立了脂质体转染腺病毒的有效方法，并构建了适合腺病毒载体的晚期表达盒，进一步将乙型肝炎表面抗原基因、HBV preS2+S、腺病毒晚期表达盒+HBV S基因插入E3区，与EcoRI消化的Ad5 DNA A片段共转染细胞，并获得重组腺病毒，相应命名为：rAd5S、rAd5MS、rAd5S2S。

Viral vector mainly contain adenovirus, retrovirus,adeno-associated virus and so on, but it has potential danger of safety; it isrepeled by immune system when it is injected to organism for a greatimmunogenicity. An injection with adenovirus vector with highconcentration, it leads to serious inflammatory reaction of liver. Viralvector such as liposome and polycation are commonly used lately. But,liposome and polycation have low specificness and targetness of genetransfer tissue, have lower transfection efficiency and short period ofgene expression, for they can be phagocytized by endothelial system.

许多的载体，病毒载体和非病毒载体以前已广泛应用，病毒载体主要包括腺病毒，逆转录病毒，腺相关病毒等；但病毒载体在安全性方面存在潜在的危险；免疫原性比较强，注射到机体后很快会被机体的免疫系统排斥，当静脉注射高浓度的腺病毒载体会使肝脏发生严重的炎症反应；非病毒载体目前常用的有脂质体及多聚阳离子聚合物；但脂质体和阳离子聚合物介导基因转移缺乏组织的特异性和靶向性，转染效率较低且易被网状内皮系统吞噬，基因表达时间短；因此研制新型的非病毒载体已成为研究的热点，纳米颗粒具有小尺寸效应，表面效应，随着颗粒直径变小，比表面积将会显著增大，故具有很高的化学活性，因而纳米成为了最有应用前景的非病毒载体。

Objective: To investigate the relationship between adenovirus type 5 infection efficiency and expression of Coxsackie virus adenovirus receptor and integrin in different tumor cell lines, providing a basis for further study of adenovirus gene therapy.

目的：探讨不同肿瘤细胞系柯萨奇病毒－腺病毒受体和整合素的表达水平与5型腺病毒感染效率的关系，为腺病毒基因治疗研究奠定基础。

I didn't watch TV last night, because it .

昨晚我没有看电视，因为电视机坏了。

Since this year, in a lot of villages of Beijing, TV of elevator liquid crystal was removed.

今年以来，在北京的很多小区里，电梯液晶电视被撤了下来。