人乳头瘤病毒治疗性活载体疫苗的研究进展

高危型人乳头瘤病毒(HPV)-16和HPV-18在宫颈癌的致病过程中起着重要作用.近年来,HPV预防性疫苗已成功上市,但其费用较高,且不能治疗已感染的患者及相关的损伤.多种靶向E6/E7抗原的HPV治疗性疫苗已进入临床前模型和临床试验,包括活载体疫苗,多肽、蛋白疫苗,核酸疫苗及细胞疫苗.此文就HPV治疗性活载体疫苗进行...     

表达CTGF shRNA的pBSHH1质粒载体的构建和鉴定

目的构建表达CTGFshRNA的pBSHH1重组真核表达载体,为获得最佳CTGF基因的siRNA序列和RNAi治疗肾小管间质纤维化提供实验基础。方法从CTGF基因中筛选出4个符合RNAi条件的片段,分别设计4对寡核苷酸,退火后形成双链,两端带有BglII和HindⅢ酶切位点,将上述4对寡核苷酸依次连入BglⅡ和HindⅢ双酶切后线形化的pBSHH1载体,构建成能在细胞内产生CTGFshRNA的质粒,依次命名为pBSHH1-T-1、2、3、4。酶切鉴定,测序证实。结果pBSHH1-T-1、2、3、4经限制性内切酶HindⅢ和EcoRI酶切,电泳后显示连接了双链DNA的280bp条带,进一步测序并证实重组质粒连接正确。结论成功构建了四个能在细胞内表达CTGFshRNA的pBSHH1质粒载体,为获得最佳的CTGF基因的siR-NA序列和进一步RNAi治疗肾小管间质纤维化奠定了基础。     

腺病毒载体介导NGF基因转染大鼠肌源性干细胞的实验研究

目的:探讨腺病毒载体介导神经生长因子(NGF)体外转染大鼠肌源干细胞(MDSCs)应用于基因治疗的可行性。方法:培养并纯化MDSCs细胞株,采用具有高效转染能力的腺病毒载系统将NGF基因导入MDSCs中,采用绿色荧光蛋白(EGFP)荧光技术检测转染率、实时荧光定量PCR (qPCR)检测目的基因NGF的表达情况。结果:...     

人白细胞介素24真核表达载体的构建及其在HepG2细胞中的表达

目的 构建人白细胞介素24(human interleukin-24,hIL-24)真核表达载体,并在HepG2细胞中稳定表达,为进一步研究其抗肿瘤作用奠定基础.方法 采用逆转录聚合酶链反应(RT-PCR),从植物血凝素活化的人外周血单个核细胞中克隆得到hIL-24基因目的 片段.应用DNA重组技术将IL-24PCR产物双酶切后定向克隆至真核表达载体pcDNA3.1(+),转化大肠杆菌DHSα获得重组载体,进行PCR、酶切及测序鉴定.应用脂质体将鉴定正确的重组质粒转染至HepG2细胞,用G418筛选稳定转染细胞株.采用RT-PCR检测稳定转染细胞HepG2中IL-24 mRNA的表达.结果 通过RT-PCR获得与预期大小一致约600 bp的IL-24基因片段;重组载体pcDNA3.1(+)-IL-24经PCR、双酶切及测序证实,IL-24 eDNA片段已正确插入真核表达载体中;在稳定转染的HepG2细胞株中可见到IL-24 mRNA表达.结论 成功构建了hIL-24真核表达载体pcDNA3.1(+)-IL-24,并获得了稳定转染该重组质粒的HepG2细胞株.     

Hepatocyte-based gene therapy

Hepatocyte-based gene therapy may be used to replace a missing gene product, confer proliferating ability to cultured hepatocytes, prevent allograft rejection, massively repopulate the host liver, or grow xenogeneic hepatocytes in mammalian liver. Gene transfer into isolated hepatocytes can be accomplished via nonviral or viral vectors, the viral vectors being more useful at this time. Common recombinant viruses that integrate into the host genome include murine leukemia retroviruses and lentiviruses, adenoassociated virus, and the T-antigen-deleted SV40 virus. Episomal viruses, such as adenoviruses, permit efficient gene transfer, but the transgene is lost upon proliferation of the transplanted hepatocyte in the host. Hybrid viruses that combine the high transduction efficiency of adenoviral vectors and the integrative capacity of other vectors, such as adenoassociated viruses, have been designed. Massive repopulation of the liver by transplanted hepatocytes can be achieved if a mitotic stimulus to the transplanted cells is combined with prevention of proliferation of the host hepatocytes. Treatment with a plant alkaloid or retrorsine, or preparative irradiation of the liver can be used to inhibit host hepatocellular proliferation, while partial hepatectomy, expression of Fas ligand, or thyroid hormone administration can be used as a mitotic stimulus to the transplanted cells. Received: July 4, 2000 / Accepted: October 13, 2000     

Analysis of T cell responses to chimpanzee adenovirus vectors encoding HIV gag–pol–nef antigen

Adenoviruses have been shown to be both immunogenic and efficient at presenting HIV proteins but recent trials have suggested that they may play a role in increasing the risk of HIV acquisition. This risk may be associated with the presence of pre-existing immunity to the viral vectors. Chimpanzee adenoviruses (chAd) have low seroprevalence in human populations and so reduce this risk. ChAd3 and chAd63 were used to deliver an HIV gag, pol and nef transgene. ELISpot analysis of T cell responses in mice showed that both chAd vectors were able to induce an immune response to Gag and Pol peptides but that only the chAd3 vector induced responses to Nef peptides. Although the route of injection did not influence the magnitude of immune responses to either chAd vector, the dose of vector did. Taken together these results demonstrate that chimpanzee adenoviruses are suitable vector candidates for the delivery of HIV proteins and could be used for an HIV vaccine and furthermore the chAd3 vector produces a broader response to the HIV transgene.     

靶向载脂蛋白M基因小干扰RNA表达载体构建及其功能

目的设计和构建载脂蛋白M基因特异性的小干扰RNA体内表达载体,筛选抑制载脂蛋白M表达的有效小干扰RNA,并初步探讨载脂蛋白M的功能。方法以载脂蛋白M为目的基因,以产生小干扰RNA质粒载体pSilencerTM2.1-U6为表达模板,细胞内转录合成4条小干扰RNA,并构建携带荧光素酶报告基因的重组质粒载体plucF-载脂蛋白。将重组质粒载体plucF-载脂蛋白与产生小干扰RNA的质粒pSilencerTM2.1-U6共转染293T细胞,定量测量荧光素酶活性,初步筛选出抑制荧光素酶表达的有效小干扰RNA,然后将小干扰RNA转染L-02,逆转录聚合酶链反应检测载脂蛋白M mRNA的表达,进一步证实小干扰RNA对载脂蛋白M表达的抑制效果,酶联免疫吸附法检测有效小干扰RNA对载脂蛋白AⅠ、载脂蛋白B和载脂蛋白E合成和分泌的影响。结果合成的4条小干扰RNA中有2条抑制荧光素酶表达,抑制效率分别为86%和91%,并特异性抑制肝细胞载脂蛋白M mRNA的表达;有效小干扰RNA能够有效抑制载脂蛋白AⅠ合成和分泌(P<0.05),而载脂蛋白B和载脂蛋白E的含量无明显改变(P>0.05)。结论成功构建并筛选到针对载脂蛋白M基因表达的有效小干扰RNA质粒,为研究冠心病的发病机制奠定基础。     

Host preferences of phlebotomine sand flies at a hypoendemic focus of canine leishmaniasis in central Italy

A survey was carried out on phlebotomine sand flies and their feeding habits at a hypoendemic focus of Leishmania infantum in Macerata province, central Italy. During two consecutive years (2000-2001), 1465 sand fly specimens (42.5% of which were males) were collected from a variety of diurnal resting sites in the municipality of Camerino. The most prevalent species was Phlebotomus perniciosus (76.6%), followed by P. papatasi (10.4%), Sergentomyia minuta (9.1%), Phlebotomus perfiliewi (3.3%) and P. mascittii (0.5%). Among the 842 females collected, 578 (68.6%) were blood-fed. Based on the results of blood meal analyses, P. perniciosus fed on man, dogs, equines, sheep and birds; P. perfiliewi on dogs, equines, sheep and birds; P. papatasi on dogs, equines and birds. Two specimens of P. mascittii fed on equines. Forage ratios (FRs) and host selectivity indices gave different results for the large domestic animals. More than 95% of the specimens collected inside a stable, dog kennel, sheep pen and chicken house were found to have fed on the animals housed in the respective shelters. In addition, at one collecting site where almost all the hosts mentioned above were present simultaneously, both P. perniciosus and P. perfiliewi were found to have fed on all five species, indicating that host choice was probably related to its availability (i.e. number and size) rather than specific attractiveness. The feeding habits of the two Leishmania vectors may have implications for the epidemiology of leishmaniasis in urban and peri-urban areas, where sand fly females deprived of other vertebrate hosts (particularly the larger species) may begin to bite humans and dogs more frequently.     

Lentiviral-mediated let-7d microRNA overexpression induced anxiolytic- and anti-depressant-like behaviors and impaired dopamine D3 receptor expression

Generalized anxiety and major depression disorders (MDD) are severe debilitating mood disorders whose etiology are not fully understood, but growing evidence indicates that microRNAs (miRNAs) might play a key role in their neuropathophysiological mechanisms. In the current study, we investigate the role of Lethal-7 (let-7d) miRNA, and its direct target dopamine D3 receptor (D3R) gain-of-function, in the hippocampus, in preclinical models of anxiety and depression in mice. For this purpose, we have constructed a lentiviral vector carrying let-7d miRNA and its anxiolytic effect was investigated by employing the open-field (OF) and the elevated plus maze (EPM) tests. The anti-depressant activity was evaluated using the tail suspension and the forced-swim tests (TST & FST). Our results show that let-7d overexpression significantly improved the measures of anxiety in the OF and EPM tests. In addition, let-7d increased the mobility time in the TST and FST. Interestingly, gene expression interaction analysis shows that the D3R mRNA negatively correlates with let-7d expression. In a different set of experiments, we used a tetracycline-inducible (tet-off) lentiviral vector to overexpress D3R to assess its gain-of-function in the hippocampus on anxiety- and depression-like behaviors. In line, we found that in the absence of doxycycline, D3R produced a significant anxiogenic and depressant-like response. Most importantly, these effects were abrogated when mice were fed doxycycline in drinking water. Our results provide the first evidence for an anxiolytic and anti-depressant-like action of let-7d through a potential D3R target-mediated mechanism which might open new avenues for anxiolytic and anti-depressant therapies.