Pseudotype-dependent lentiviral transduction of astrocytes or neurons in the rat substantia nigra

Gene transfer to the central nervous system provides powerful methodology for the study of gene function and gene–environment interactions in vivo, in addition to a vehicle for the delivery of therapeutic transgenes for gene therapy. The aim of the present study was to determine patterns of tropism exhibited by pseudotyped lentiviral vectors in the rat substantia nigra, in order to evaluate their utility for gene transfer in experimental models of Parkinson's disease. Isogenic lentiviral vector particles encoding a GFP reporter were pseudotyped with envelope glycoproteins derived from vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), or Moloney murine leukemia virus (MuLV). Adult male Lewis rats received unilateral stereotactic infusions of vector into the substantia nigra; three weeks later, patterns of viral transduction were determined by immunohistological detection of GFP. Different pseudotypes gave rise to transgene expression in restricted and distinct cellular populations. VSV and MV pseudotypes transduced midbrain neurons, including a subset of nigral dopaminergic neurons. In contrast, LCMV- and MuLV-pseudotyped lentivirus produced transgene expression exclusively in astrocytes; the restricted transduction of astroglial cells was not explained by the cellular distribution of receptors previously shown to mediate entry of LCMV or MuLV. These data suggest that pseudotyped lentiviral vectors will be useful for experimental gene transfer to the rat substantia nigra. In particular, the availability of neuronal and astrocytic-targeting vectors will allow dissociation of cell autonomous and cell non-autonomous functions of key gene products in vivo.     

慢病毒短发夹RNA介导的多形性腺瘤基因样蛋白2沉默对肝癌细胞MHCC97-L增殖、迁移及侵袭的影响

目的 探讨慢病毒短发夹RNA(shRNA)介导的多形性腺瘤基因样蛋白2(PLAGL2)沉默对肝癌细胞恶性行为的影响及其机制.方法 Real-time PCR与Western blotting分别检测肝癌组织及癌旁组织中PLAGL2的表达水平;体外培养肝癌细胞MHCC97-L,构建慢病毒载体质粒PLAGL2-shRNA与...     

超级白细胞介素-6重组慢病毒的构建及其对肝细胞的促增殖作用

目的 观察超级白细胞介素-6重组慢病毒对人正常肝细胞L-02的促增殖作用.方法 利用慢病毒表达质粒及其包装系统构建超级白细胞介素-6重组慢病毒(FIV-Hyper-IL-6)、IL-6重组慢病毒(FIV-IL-6)和空慢病毒载体(FIV),用嘌呤霉素筛选的方法测定其病毒滴度.将人肝细胞L-02分为4组:FIV-Hyper-IL-6组、FIV-IL-6组、FIV组及未感染组,各组分别感染其相应的重组慢病毒,采用四甲基偶氮唑盐法检测其对L-02细胞生长状态的影响;并用RT-PCR法检测重组慢病毒感染L-02细胞后产生的急性时相蛋白一结合珠蛋白mRNA表达量的变化.应用方差分析进行统计学分析.结果 构建出的各重组慢病毒能成功感染靶细胞,嘌呤霉素筛选法测得的各重组慢病毒的病毒滴度均为107pfu/ml.病毒颗粒感染L-02细胞后48h,细胞的促增殖作用(A值)FIV-Hyper-IL-6组为0.6267±0.0256,FIV-IL-6组、FIV组及未感染组分别为0.5563±0.0112、0.5040±0.0078、0.4790±0.0201,F=41.09,P值均<0.01,且随着时间的延长,对细胞的促增殖作用有所增加,并于感染后72h达到最高,为0.8000±0.0166.FIV-Hyper-IL-6组细胞结合珠蛋白mRNA吸光度值为0.7030±0.0106,FIV-IL-6组、FIV组及未感染组分别为0.3355±0.0093、0.1143±0.0153、0.1145±0.0076,q=57.5007,P值均<0.01.结论 构建的Hyper-IL-6重组慢病毒能够显著刺激L-02细胞表达结合珠蛋白,对L-02细胞有较强的促增殖作用.     

慢病毒介导I型胶原短发夹RNA转染对大鼠系膜细胞生长行为的影响

目的通过观察慢病毒介导的I型胶原（COLI）短发夹（sh）RNA感染大鼠系膜细胞后其增殖、凋亡及细胞周期的变化,探索未来应用于动物实验,甚至临床时RNA干涉（RNAi）药物对靶细胞生长行为的影响。方法利用感染增强剂（对照组）、空慢病毒载体及感染增强剂（pSC-GFP组）、COLIshRNA慢病毒表达载体及感染增强剂（pSC—GFP／COLI组）分别感染大鼠系膜细胞,用噻唑蓝法检测细胞增殖,利用AnnexinV／PE法检测细胞凋亡,利用流式细胞仪检测细胞周期,进而观察慢病毒表达载体对细胞上述能力的影响。结果pSC—GFP／COLI组和pSC—GFP组均能显著抑制细胞增殖,且慢病毒表达载体抑制能力又显著高于空病毒,分别于感染后24h、48h和72h计算抑制效率为22．52％、28．57、33．33％。凋亡实验结果表明,慢病毒感染细胞后一定程度诱导了细胞凋亡,但作为载体携带的COLIshRNA未引起进一步的细胞凋亡加剧。细胞周期实验发现慢病毒引起细胞周期阻滞于G2／M期。而在72hCOLIshRNA对s期的阻滞逐渐突出。结论慢病毒介导I型胶原短发夹RNA转染大鼠系膜细胞后,在一定程度上影响了细胞生长行为,但总体来说仍是安全的,为进一步动物实验及药物研制奠定基础。     

Genetic analysis of small ruminant lentiviruses following lactogenic transmission

Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission.     

小鼠Mmp-9 shRNA重组慢病毒包装、筛选及干扰效果检测

目的:设计、筛选、构建并鉴定Mmp-9小干扰RNA(small interfering RNA,siRNA),并构建其慢病毒表达载体,为血管化疾病的防治提供理论依据及实验室数据。方法:针对基质金属蛋白酶9靶基因设计siRNA序列,并合成小发卡RNA(short hairpin RNA,shRNA)结构的DNA,与经AgeΙ和EcoRΙ酶切的pSIL-RFP连接、转化大肠杆菌感受态细胞,构建成携带Mmp9-shRNA慢病毒质粒载体(pSIL-RFP/MMP9-shRNA),PCR及测序鉴定后,Western blot筛选出携带最佳siRNA的慢病毒质粒载体,并利用慢病毒包装质粒(pHelper1.0和pHelper2.0)将其制备成MMP9-shRNA慢病毒,并测定病毒滴度为1×1010pfu/l。结果:PCR和DNA测序证实合成的含基质金属蛋白酶9短发卡RNA慢病毒载体寡核苷酸链正确插入pSIL-RFP载体,表明实验成功构建基质金属蛋白酶9RNA干扰慢病毒表达载体。结论:成功的构建并筛选出针对小鼠Mmp9基因沉默的特异性shRNA重组慢病毒,为血管化性疾病的防治研究提供基础。     

plenti6.3-hHGF-IRES-hrGFP慢病毒表达载体的构建和鉴定

目的构建人肝细胞生长因子(hHGF)基因慢病毒表达载体并进行鉴定。方法采用聚合酶链反应(PCR)技术扩增IRES和hrGFP,拼接成IRES-hrGFP,并克隆到pLenti6.3载体;进一步采用PCR技术扩增hHGF,并克隆到plenti6.3-IRES-hrGFP载体,然后BamHI/AscI双酶切和测序鉴定构建的plenti6.3-hHGF-IRES-hrGFP重组载体。脂质体法转染293T细胞,24h、48h、72h后显微镜下观察细胞的绿色荧光蛋白表达情况和ElISA测定细胞上清液中HGF蛋白表达情况。最后利用ViraPowerTM慢病毒表达系统,将表达质粒与包装混合物(PackagingMix)共转染293T细胞产生包装病毒颗粒,以293T细胞hrGFP表达水平(荧光显微镜下对hrGFP表达阳性细胞进行计数)测定病毒滴度。结果 IRES,hrGFP和hHGF基因片段重组到plenti6.3载体,电泳结果和双酶切鉴定均能得到与理论大小相符的片段,测序结果与Genebank序列完全一致。质粒转染293T细胞后绿色荧光蛋白表达量和上清中HGF蛋白含量随时间增长而增多,72h最高。24h、48h、72h后HGF蛋白含量分别为(477±19),(1424±78),(4274±128)μg/L。测得病毒的滴度为1.9×108TU/mL。结论携带hHGF基因的慢病毒载体构建成功,在293T细胞中正确表达,并获得较高的病毒滴度,为其体内外研究提供基础。     

Vector platforms for gene therapy of inherited retinopathies

Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina's compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs' limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations.     

小鼠NMNAT1基因的过表达和RNAi重组慢病毒的构建包装和检测

目的:针对小鼠烟酰胺单核苷酸腺苷酰转移酶1( NMNAT1)基因构建质粒并进行慢病毒包装,同时检测其表达水平和干扰效率,为进一步探讨该基因的功能提供研究工具和实验基础.方法:根据NMNAT1基因信息,用慢病毒载体pLenti6构建三种重组质粒,分别包含NMNAT1 cDNA全长、一个针对NMNAT1的小干扰序列和一个用于干扰对照的阴性序列.把这些质粒包装进慢病毒载体,并检测病毒滴度,再用慢病毒感染Hela细胞检测NMNAT1表达量和RNA干扰效率.结果:测序结果证明目的序列正确地插入到载体内.通过qPCR方法鉴定慢病毒包装成功,病毒滴度均为2×108 TU/ml以上.表达NMNAT1的重组慢病毒感染Hela细胞,该细胞能够高水平表达NMNAT1蛋白,而携带RNAi序列的慢病毒能够显著抑制其表达,干扰效率在70％以上.结论:针对NMNAT1的过表达和RNAi重组慢病毒制备成功,为进一步研究NMNAT1基因的功能和用慢病毒进行基因治疗提供了良好的研究工具.