CellProfiler分析重组杆状病毒转导OL细胞的最佳条件

[目的]定量分析重组杆状病毒在少突神经胶质细胞(oligodendrocyte,OL)中的表达水平,为探索重组杆状病毒最佳转导条件。[方法]首先构建重组杆状病毒,将重组杆状病毒转导至OL细胞,通过荧光显微镜观察细胞形态及EGFP表达情况,并采集细胞的数字图像。利用CellProfiler软件对重组杆状病毒在OL细胞中转导后的表达水平进行数字图像分析,得到各个优化条件的EGFP阳性细胞比率及表达强度,绘制图表并分析。[结果]40MOI重组杆状病毒转导OL细胞并添加10 mmol/L丁酸钠,转导时间为12 h,培养时间为72 h时病毒转导效率最高;培养时间为48 h,其它条件相同情况下EGFP表达强度最高。[结论]定量分析优化后,重组杆状病毒转导OL细胞的表达效率及EGFP的表达水平得到显著上调。     

辅助病毒依赖型腺病毒载体转基因的体外表达效率

构建可表达增强型绿色荧光蛋白 (Enhanced green fluorescent protein,EGFP) 的辅助病毒依赖型腺病毒载体 (Helper-dependent adenoviral vector,HDAd),并完成大量制备、纯化和体外表达鉴定。荧光显微镜证实HDAd/EGFP可表达,电镜下观察到经CsCl纯化后的腺病毒的典型形态。分光光度计法测定病毒的浓度为4.0×1012 颗粒数 (Virus particle,vp) /mL。与可表达EGFP的第一代腺病毒载体 (First generation adenoviral vector,FGAd) FGAd/EGFP进行了体外感染和转基因表达效率的比较研究,分别用约2 000 vp/细胞的HDAd/EGFP和FGAd/EGFP感染A549细胞,流式细胞仪检测EGFP的表达情况。通过相同时间点流式细胞仪分析EGFP的表达情况,可见HDAd/EGFP感染早期的A549细胞较FGAd/EGFP有更高的荧光表达率及更高的表达强度,显示HDAd载体具有转基因瞬时高表达的特性,是一种更有价值的疫苗载体。     

增龄引起复制性衰老细胞胞内钙信号转导的变化

为了观察血管紧张素Ⅱ (AngⅡ )引起复制性衰老细胞胞内游离钙的变化以及衰老对其的影响 ,初步阐明衰老引起胞内游离钙变化的机制 .选用人胚肺二倍体成纤维细胞WI 38细胞株 ,利用逆转录PCR(RT PCR)技术及Northern杂交技术检测衰老细胞血管紧张素Ⅱ 1型受体 (AT1R)、血管紧张素Ⅱ 2型受体 (AT2R)mRNA水平的表达 ;利用激光共聚焦显微成像技术 (LSCM )观察WI 38细胞在AngⅡ刺激 ,Valsartan阻断条件下细胞内钙离子荧光强度的改变 .AngⅡ通过AT1受体介导增加WI 38细胞内游离钙的水平 ,并随着WI 38细胞传代增加至衰老状态 ,AT2受体高表达 ,AT1受体介导的钙离子信号转导的活性逐渐降低 .提示WI 38细胞衰老过程中钙离子信号的转导活性降低 ,并且AT1R和AT2R在血管紧张素Ⅱ介导的细胞内钙信号活性中具有不同的作用与机制 .为探讨WI 38衰老细胞内钙信号变化机制提供了实验依据     

细胞因子基因转导对人乳腺癌细胞MHC抗原及细胞膜糖蛋白表达的影响

为探讨细胞因子基因(人IL-2、IL-6)转导对于肿瘤细胞膜MHC抗原及细胞膜糖蛋白表达调控的影响,本文利用脂质体介导的方法,将含人IL-6、IL-2基因的逆转录病毒载体分别导入人乳腺癌细胞系MCF-7细胞中,采用间接免疫荧光染色流式细胞仪测定法,对基因转导的瘤细胞细胞膜糖蛋白及MHC抗原表达进行测定。结果表明经两种基因修饰的MCF-7细胞MHCⅠ型抗原表达均获得增强,此外,基因转导细胞可程度不同地表现出细胞膜多种糖蛋白表达的变化。提示肿瘤细胞膜抗原及糖蛋白表达的改变可能是细胞因子基因转导影响肿瘤细胞免疫原性的重要结构基础。     

EGFP—pBABE逆转录病毒载体的构建及鉴定

目的构建表达增强绿色荧光蛋白（enhancedgreenfluorescentprotein,EGFP）的EGFP—pBABE逆转录病毒载体并对其表达进行鉴定。方法从pEGFP—N3质粒上切下EGFP片段,连接到pBABE载体,构建EGFP—pBABE重组质粒;将该重组质粒转染到Fr67细胞后,荧光显微镜下观察EGFP的表达;收集逆转录病毒感染宫颈癌SiHa细胞后荧光显微镜下观察EGFP的表达。结果成功构建EGFP—pBABE重组质粒。该质粒转染PT67细胞24h后,荧光显微镜下可观察到EGFP的表达;用该重组质粒包装的逆转录病毒感染宫颈癌SiHa细胞36h后,在荧光显微镜下可观察到明显的EGFP表达。结论成功构建表达EGFP的EGFP—pBABE逆转录病毒载体,为进一步利用该载体制备逆转录病毒并观测逆转录病毒感染细胞的效率奠定了良好的实验基础。     

经HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞后HIF-1α基因的表达

常规经基因修饰腺病毒转染细胞后,即进行细胞的移植,并不对病毒转染效果进行检验。本研究采用HIF-1α与EGFP双基因重组腺病毒转染大鼠心肌干细胞,察看基因重组腺病毒是否能够转染心肌干细胞内;研究经HIF-1α与EGFP(enhanced green fluorescence protein,EGFP)双基因重组腺病毒转染心肌干细胞后HIF-1α基因的表达情况。在荧光显微镜下,去察看病毒转染细胞的情况,确定病毒最适MOI值;采用实时定量PCR(real-time quantitative PCR,RT-qPCR)评价HIF-1α基因表达变化;应用Western blotting方法检测HIF-1α蛋白表达变化,进而获得良好的实验结果。     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

禽腺病毒QU株一个复制非必需区的鉴定

禽腺病毒QU弱毒株属于鸭腺病毒1型病毒, 可作为潜在的重组疫苗载体。为确定QU株的复制非必需区, 参照鸭腺病毒1型病毒基因组右侧E4区附近序列设计引物, 扩增QU株基因组的一段3.4 kb片段, 插入来自pEGFP-C1质粒的增强型绿色荧光蛋白(EGFP)基因表达盒片段, 构建了含EGFP基因的重组质粒pADGFP。采用脂质体介导法, 将重组质粒pADGFP与QU株共转染CEF细胞, 用96孔板稀释法筛选纯化表达绿色荧光蛋白的重组QU病毒株rQUGFP。该重组毒的生长曲线与亲本毒一致, 连续传代后病毒滴度稳定。结果表明, QU株基因组右侧E4区附近一段包括ORF1、ORF8和ORF9三个开放阅读框的区域为病毒的复制非必需区, 且插入的EGFP基因可以稳定表达。为进一步以禽腺病毒QU株为载体构建重组疫苗的研究打下基础。     

反义前S/S基因转移表达抗乙型肝炎病毒的研究

为了观察反义基因转录表达抗乙型肝炎病毒(HBV)的作用,将HBV ayw前S/S基因(preS/S)片段反向插入逆转录病毒载体质粒,构建preS/S反义基因重组体,经转染PA317细胞后,获得重组逆转录病毒颗粒.用重组病毒转导2.2.15细胞后,第3天即可见细胞培养上清HBV表面抗原(HBsAg)和e抗原(HBeAg)表达量减少,到转导后第5天,细胞培养上清中的HBsAg和HBeAg表达量降到最低水平,HBsAg抑制率为71%,HBeAg抑制率为27%,抑制作用至少可持续到转导后第11天.空载及正向插入基因的重组逆转录病毒转导对2.2.15细胞内HBV抗原表达无抑制作用.     

Lentiviral vector-mediated gene transfer to endotherial cells compared with adenoviral and retroviral vectors

Human immunodeficiency virus (HIV, lentivirus) vector has attractive features for gene therapy, including the ability to transduce non-dividing cells and long-term transgene expression. We have already reported that lentivirus vector can transduce well-differentiated rat cardiac myocytes. Endothelial cells (EC) are an attractive target for gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. There are several reports regarding application of adenovirus and retrovirus based vectors to EC. However, there have been few reports which show the effect to lentivirus-mediated gene transfer efficiency, compared with adenovirus and retrovirus. In this study, bovine aortic endothelial cells (BAECs) were infected, in vitro, with these virus vectors. Transduction efficiency (TE) of beta-Gal gene transfer in BAECs by adenovirus, lentivirus, or retrovirus at MOI10 (Multiplicity of infection) (determined on Hela cells) is 69+/-11, 33+/-8, or 22+/-6% respectively. In adenovirus and lentivirus, almost 100% of BAECs were transduced at MOI 50. However, in retrovirus, TE showed only 48+/-6% at MOI 50 and no increase at MOI 100. The percentage of beta-Gal positive cells was decreased rapidly at longer passage of cells after being transduced by adenovirus. However, lentivirus and retrovirus showed sustained higher percentage of positive cells. Furthermore, transduction by lentiviral vectors had no significant effect on viability of BAECs. Our results indicate that lentivirus showed high-level and long term gene expression in BAECs. Lentivirus can be an effective vector for the ex vivo, genetically modified EC implantation and in vivo gene therapy.     

应用绿色荧光蛋白研究外源基因在造血细胞的表达调控

以增强型绿色荧光蛋白为报道分子,研究双基因逆转录病毒载体介导小鼠骨髓细胞的基因转移中,SV(simianvirus)40启动子和内部核糖体进入位点(internalribosomeentrysite,IRES)对双基因共表达的影响。构建双基因中间序列分别为SV40启动子和IRES的载体pLESN和pLEIN。经包装获得较高滴度的病毒上清,以共培养的方式感染5-氟尿嘧啶预刺激的小鼠骨髓细胞。流式细胞仪检测表明转染效率约25%,PCR证明EGFP基因整合至骨髓细胞基因组。半固体培养转基因细胞7d,LEIN组获得具有G418抗性的集落中98%表达绿色荧光,而LESN组54%。结果表明:在双基因逆转录病毒载体介导的小鼠骨髓细胞的基因转导中,IRES与内部SV40启动子相比,更能保证双基因的共同表达。     

脂质体DC-Chol/DOPE对HEK293FT细胞的高效转染及其在腺病毒制备中的应用

重组病毒载体系统因为具有高效的基因转移能力得到了广泛应用,而病毒包装细胞的转染是重组病毒制备过程中的关键步骤。优化了脂质体DC-Chol/DOPE介导的转染常用的病毒包装细胞系HEK293FT的实验条件,比较了DC-Chol/DOPE、Lipofectamine2000和磷酸钙共沉淀法转染细胞的效率,并且比较了用DC-Chol/DOPE和磷酸钙共沉淀法转染293FT细胞制备重组腺病毒的结果,发现DC-Chol/DOPE对293FT细胞的转染效率以及最终收获的病毒滴度都远高于磷酸钙共沉淀法转染。所以,利用DC-Chol/DOPE转染293FT细胞制备重组病毒是一种简单、高效、成本低廉的方法。     

Chicken adenovirus (CELO virus) particles augment receptor-mediated DNA delivery to mammalian cells and yield exceptional levels of stable transformants.

Delivery of genes via receptor-mediated endocytosis is severely limited by the poor exit of endocytosed DNA from the endosome. A large enhancement in delivery efficiency has been obtained by including human adenovirus particles in the delivery system. This enhancement is probably a function of the natural adenovirus entry mechanism, which must include passage through or disruption of the endosomal membrane. In an effort to identify safer virus particles useful in this application, we have tested the chicken adenovirus CELO virus for its ability to augment receptor-mediated gene delivery. We report here that CELO virus possesses pH-dependent, liposome disruption activity similar to that of human adenovirus type 5. Furthermore, the chicken adenovirus can be used to augment receptor-mediated gene delivery to levels comparable to those found for the human adenovirus when it is physically linked to polylysine ligand-condensed DNA particles. The chicken adenovirus has the advantage of being produced inexpensively in embryonated eggs, and the virus is naturally replication defective in mammalian cells, even in the presence of wild-type human adenovirus.     

Expression of PiT1 and PiT2 retroviral receptors and transduction efficiency of tumor cells

Recombinant retroviral vectors are still the most common gene delivery vehicles for gene therapy purposes, especially for construction of genetically modified tumor vaccines (GMTV). However, these vehicles are characterized by relatively low titre and in the case of many tumor cell lines, low transduction efficiency. We constructed bicistronic retroviral vector pseudotypes of amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV), encoding enhanced green fluorescent protein (EGFP) as a rapid and easily detectable reporter gene. Transduction of five different human melanoma and four renal carcinoma cell lines by these two virus pseudotypes revealed differences in transduction efficiency, which wase markedly lower for the renal carcinoma cell lines. Stimulation of retroviral receptor expression (PiT1 and PiT2) by phosphate depletion induced a limited increase of receptor mRNA levels, but did not improve the gene transfer efficiency. In contrast, simultaneous transduction with both vector pseudotypes markedly increased the transduction efficiency, compared to GaLV or A-MuLV alone. The same effect could be achieved by several repeated exposures of target cells to fresh vector preparation. Overexpression of GaLV receptor (PiT1) in target cells significantly increased the transduction rate and enabled retrovirus mediated gene transfer into the cells which normally are not transducible by GaLV pseudotypes. We demonstrated that, using different transduction strategies, the relatively inefficient, widely used retroviral vector systems could be significantly improved.     

Production of retrovirus and adenovirus vectors for gene therapy: a comparative study using microcarrier and stationary cell culture

In gene therapy, retrovirus and adenovirus vectors are extensively used as gene-delivery vehicles and further large-scale processing of these viral vectors will be increasingly important. This study examined stationary and microcarrier cell culture systems with respect to the production of a retrovirus vector (encoding a monounit hammerhead ribozyme gene with an intron) and an adenovirus vector (encoding a reporter lacZ gene). Cytodex 1 and Cytodex 3 solid microcarriers were found to be able to provide good cell growth and high-titer vector production in suspension cultures. Porous microcarriers such as Cytopore 2 gave slightly lower but still efficient growth but produced significantly lower titers of retrovirus and adenovirus vector from the producer cells. The specific retrovirus production was not proportionally related to the specific growth rate of the producer cells. High MOI infection was essential for high-titer production of adenovirus vector in 293 cells. Hydrodynamic shear forces on microcarrier-grown cells increased the production yield for retrovirus vector but decreased for adenovirus vector. The cellular productivity was much more efficient for adenovirus vector produced in 293 cells as compared to the retrovirus vector produced in PA317-RCM1 cells. These findings can provide further insight into the feasibility of applying microcarrier cell culture technology to produce gene-therapy virus vectors.     

小鼠精原干细胞不同转染方法效率的比较

本研究采用腺病毒感染、慢病毒感染、脂质体转染和电穿孔转化方法将含有绿色荧光蛋白（GFP）的质粒转入经过差异贴壁法初步分离纯化的小鼠精原干细胞(SSCs)中,转染48 h后通过流式细胞仪检测GFP阳性细胞比例比较4种方法在体外转染精原干细胞的效率.结果显示,脂质体转染效率最高仅为8.64%,不能满足对精原干细胞进一步实验的要求;电穿孔法效率最高达到25.27%,但转化后细胞大量死亡;腺病毒转染细胞的效率达到了32.4%;慢病毒转染效率最高,达到74.25%. 因此,慢病毒转染法是体外转染小鼠精原干细胞的有效方法.     

Mo MLV gag-pol基因在NIH3T3细胞中的表达和鉴定

目的 构建含MoMLV gag-pol基因的重组表达载体,实现其在NIH3T3细胞中稳定表达。方法 应用RT-PCR方法反转录并扩增gag-pol基因,克隆入真核表达载体pcDNA4/HisMaxA上,构建重组表达载体pcDNA4/HisMaxA-gag-pol,用脂质体法转染NIH3T3细胞,Zeocin筛选稳定表达细胞株,通过SDS-PAGE分析检测表明, gag-pol基因在NIH3T3细胞实现了表达,产物相对分子质量(kD)为194.78×103。然后,将逆转录病毒载体导入此细胞系,包装逆转录病毒。用PCR与标记基因补救分析法检测野生型辅助病毒。结果 酶切鉴定的片段大小分别为5.2-kb,与预期大小一致,经Zeocin筛选后获得稳定表达细胞株,SDS-PAGE实验表明产物融合蛋白相对分子质量(kD)为194.78×103,与预期相符。脂质体转染包装细胞,嘌呤霉素加压筛选出高病毒滴度(4.0×106CFU/ml)的细胞克隆,且未检测到辅助病毒。结论 本工作构建的融合表达载体pcDNA4/HisMaxA-gag-pol及其在NIH3T3细胞中的表达,构建成功具有靶向性的逆转录病毒包装细胞系,该细胞系能够包装出高滴度的逆转录病毒,为肝细胞的基因治疗提供了一种新的基因转移系统。