Investigation of hrDNA targeting vector-mediated tumor-specific suicide gene therapy for hepatocellular carcinoma

Vectors pose most pivotal problem of gene therapy[1]. Because of the high transfection efficiency both in vitro and in vivo, the viral vector has been employed in 70% clinical trials of gene therapy (http://www.wiley.co.uk/ genmed/clinical). However, thei…     

运用HSV－tk／ACV系统进行肿瘤基因治疗研究

构建了含ｐｇｋ启动子驱动的ＨＳＶ－ｔｋ基因的反转录病毒载体ｐＬＮＴＫ，将ＨＳＶ－ｔｋ基因转移至人脑胶质瘤ＳＨＧ４４细胞（命名为ＳＨＧＬＮＴＫ）及小鼠黑色素瘤细胞Ｂ１６（命名为Ｂ１６ＬＮＴＫ）．体外实验证实核着类似物ＡＣＶ对ＳＨＧＬＮＴＫ细胞和Ｂ１６ＬＮＴＫ细胞的杀伤敏感性分别高于亲本细胞１０００和４００倍．转ＨＳＶ－ｔｋ基因细胞与亲本细胞按不同比例共培养时，亲本细胞对ＡＣＶ的敏感性明显增高，存在旁观者效应．首次应用人脑胶质瘤细胞株进行裸鼠体内实验，结果表明：ＡＣＶ能完全抑制ＳＨＧＬＮＴＫ细胞在裸小鼠体内肿瘤的形成，对裸小鼠体内已形成的ＳＨＧＬＮＴＫ肿瘤的治疗效果与对照ＳＨＧ４４肿瘤相比，肿瘤体积缩小８０％；用Ｂ１６ＬＮＴＫ细胞接种同系Ｃ５７／ＢＬ小鼠，经ＡＣＶ治疗后，Ｂ１６ＬＮＴＫ组小鼠的肿瘤较对照组Ｂ１６肿瘤小９５％．ＨＳＶ－ＴＫ／ＡＣＶ系统原位基因转移治疗ＳＨＧ荷瘤裸小鼠、Ｂ１６荷瘤小鼠，原位注射病毒悬液／ＡＣＶ治疗组的ＳＨＧ肿瘤、Ｂ１６肿瘤分别较对照组肿瘤小５０％，４３％，原位注射ＰＡ３１７／ＬＮＴＫ细胞，ＡＣＶ治疗的ＳＨＧ肿瘤较对照组肿瘤小９０％，以上实验结果，统计学上差异极显著，Ｐ＜０．０１．实验     

锌指蛋白在多位点基因打靶中的应用

综述了锌指蛋白的功能特性.可以与切割DNA的无特异性切割结构域组成人工锌指核酸酶,可以造成细胞染色质指定序列特异性双链断裂,促使细胞启动重组修复机制,在诱导产生的重组酶的作用下,可以将外源基因片段重组插入染色质的效率提高上万倍。锌指蛋白技术与多位点基因打靶技术相结合,可以在不需药物筛选的情况下,进行人体细胞基因治疗。     

Non-viral gene carrier mediated short hairpin RNA interference for inhibition of tumor cells growth

A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine （PEI） with polyethyleneglycol （PEG） and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low （30%） in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA （shRNA） specific for nucleostemin gene （NS） was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.     

Progress of gene targeting in mouse

Gene targeting is a powerful approach of studying the gene function in vivo. Specific genetic modifications, including simple gene disruption, point mutations, large chromosomal deletions and rearrangements, targeted incorporation of foreign genes, could be introduced into the mouse genome by gene targeting. Recent studies make it possible to do the gene targeting with temporal and spatial control.     

TNF基因治疗肿瘤的研究进展

肿瘤坏死因子 (TNF)是一种既能直接杀死肿瘤细胞 ,又能通过激活免疫系统发挥抗肿瘤作用的细胞因子 .其基因工程药品临床应用后 ,具有严重的毒副作用 ,因此 ,人们开始了TNF基因冶疗的研究 .TNF基因治疗为TNF在肿瘤治疗中的应用开拓了新的途径     

使用病毒载体的罕见病基因疗法临床进展

 很大一部分的罕见病由遗传因素决定,难以用普通的小分子或大分子药物治愈,而基因治疗有望从根本上修正人体功能的缺失或异常,给罕见病患者带来改善生活质量的希望。目前许多基因疗法的临床试验正在开展,病毒载体是常用的基因递送方法,本文讨论了用于临床基因递送的多种病毒载体,包括腺相关病毒、逆转录病毒和慢病毒,重点列举了这些病毒在罕见病临床试验中的研究、应用和进展,评价了这些病毒的优缺点,并简述了基因疗法的研究方向及应用前景。     

HSVTK gene therapy for carcinoembryonic antigen-producing human lung cancer cells

The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells. Foundation item: Supported by the National Natural Science Foundation of China Natural Science Foundation of Hubei Province Biography: XIAO Geng-fu(1966-), male, phD graduate candidate, Lecturer.     

线粒体病的基因治疗

线粒体基因突变导致的线粒体病仍是不治之症,目前只有针对症状的支持性疗法.根治线粒体病只能寄希望于基因治疗.基因治疗的主要目标是修补特定基因突变导致的功能缺陷,或促使异质性的转变,以降低突变型与野生型基因组的比例.所探讨的途径主要包括:①在肽核酸和锌指肽的引导下选择性抑制突变基因组的复制;②利用特定的限制性内切酶去除突变...     

Gene therapy for cancer by mutant HSV deleted apoptosis-inhibited gene

A mutant HSV (mtHSV) deleted icp34.5, an apoptosis-inhibiting gene was constructed. It is supposed that the mtHSV can replicate in p53-deficient cells selectively and lead to oncolysis targetedly. Mice tumor model harboring sarcoma cell line s-180 was developed and the mtHSV was injected into the tumors. We found that the mean volume and weight of tumors of early therapeutic group(ETG) were reduced 49.29% and 38.31% of that of control tumors. In the mid-term group (MTG), the redutcion rate were 26.9% and 24.52% respectively. Foundation item: Supported by Foundation of 863 New Hi-technique Research (39880031); Research Fund for the Doctoral Programm of Higher Education (RFDP98048615); Chen-guang Project of Wuhan City (20005004024). Biography: Lan Ping (1971-), female, Ph.D candidate, research direction: molecular virology.     

Prophylaxis of tumor through oral administration of IL-12 GM-CSF gene carried by live attenuated salmonella

A live attenuated AraA^- autotrophic mutant ofSalmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVhIL-12, pCMVmGM-CSF and pCMVhGM-CSF and was administered orally to BALB/c and C57BL/6 mice. After 6 weeks, these mice were challenged with 4T1 and Lewis tumor cells respectively. GFP expression and gene integration could be detected in mice’s livers, spleens, intestines, kidneys and tumors. The serum level of cytokines increased significantly in treated mice, so did the ratio of CD ₈ ⁺ /CD ₄ ⁺ , which resulted in the tumor regression and prolongation of the survival time of those mice. These researches laid an experimental foundation for the tumor gene therapy using live attenuated salmonella.     

慢病毒载体及其介导的RNA干扰在基因治疗中的应用研究

主要就慢病毒载体及其介导的RNA干扰技术在基因治疗中的应用研究进行了综述,并对其在该领域具有的广阔前景进行了展望.慢病毒载体作为一种新型的载体,可有效地将携带的目的基因导入宿主细胞,并将其整合到宿主细胞基因组中,从而使目的基因得以持久稳定地表达.该载体因具有高感染性、高表达效率及不易诱发宿主免疫反应等优点,已成为基因治疗研究中的一个重要工具.RNA干扰技术可以特异性抑制或关闭特定基因的表达,因此该技术可广泛应用在基因功能探究和恶性肿瘤治疗等领域.由慢病毒载体介导的RNA干扰技术能持久、稳定、特异性地抑制各类细胞中特定基因的表达,在病毒感染、肿瘤等疾病的基因治疗中被广泛应用,成为生物医学领域的新热点.     

新型Bola两亲分子作为基因治疗药物输递载体的研究

为检测新型Bola两亲分子(Orn-C16-G)用作基因载体的性能,制备了Bola与siRNA的复合物(Bolaplexes),用原子力显微镜观察其在不同复合比下的形貌;并转染Hela细胞,用CCK-8检测Bolaplexes的细胞毒性,用荧光显微镜、流式细胞仪表征Bolaplexes的细胞内吞效果。实验结果显示,复合比1∶1条件下,Bola分子可以有效地压缩siRNA分子形成较小尺寸的复合物(100～200 nm)。Bolaplexes可高效地进入Hela细胞,并且毒性低于商售转染试剂,有较好的应用前景。本研究为下一步Bola两亲分子用于基因治疗的临床研究奠定了基础。     

四例血友病B患者基因治疗的临床试验

报道了对四例血友病Ｂ患者基因治疗的临床试验，取自四例血友病Ｂ患者的自体皮肤成纤维细胞，通过反转录病毒介导的基因转移，能高效地分泌人凝血因子Ⅸ。用胶原包埋经遗传修饰的自体皮肤成纤维细胞，再移植到血友病Ｂ患者皮下。四例患者经治疗后，自发出血症状得到了减轻，血浆中凝血因子Ⅸ蛋白浓度和凝血活性明显地增加，其中两例患者增加较大，达到正常值的４％￣５％。另外两例患者体内的凝血因子Ⅸ和凝血活性也有不同程度的提高     

Research advances in gene therapy for Parkinson's disease

During the long period of time when people have been seeking for an effective therapeutic method for PD, the gene therapy has shown greater and greater advantages over other methods. It can be performed mainly in two ways: ex vivo and in vivo. With the former, TH gene as well as some neurotrophic factor genes (such as GDNF, BNDF genes) can be invited to some cell lines or primary cells thus forming engineered cells and then implanting them into brain. While with the latter, viral vectors including HSV-1, Ad, AAV that can be utilized to construct recombinant viruses, or non-virus vectors can be used to delived DNA into brain directly. The present review summarizes the recent research advances in the gene therapy for PD, and it is reasonable for us to predict a notable progress in prevention and treatment for PD in the next decade.     

Silica nanoparticle is a possible safe carrier for gene therapy

In order to develop a safe and effective gene therapy carrier, some toxicological and biodynamical experiments were carried out on silica nanoparticles （SiNPs）. First we prepared SiNPs with appropriate portions of cyclohexane, deionized water and ethyl silicate, and then transfected the modified SiNPs and GFP plasmid DNA complex into the HT1080 cells to test the effectiveness of transfection for gene therapy. At the same time, we injected the SiNPs into a number of mice through tail vein. Then we made the mice crossed to evaluate the acute, long-term and reproductive toxicity. In vivo distribution analysis and pathological examination were made on both adult mice and their offspring. SiNPs were uniform and had an average diameter of 40 nm, and the modified SiNPs carried exogenous DNA molecules into target cells and the transferred GFP fusion gene was effectively expressed in the cells. The SiNPs injected via tail vein were widely distributed in almost all of tissues, and the injected mice had the ability to reproduce normally. The in vivo and in vitro results of this study clearly show that SiNPs can be used as a safe and effective carrier for gene transfection and gene therapy.     

Interaction of hepatitis B virus with tumor suppressor gene p53: its significance and biological function

The mechanism of the interaction of hepatitis B virus (HBV) with tumor suppressor p53 and its role in the hepatocar-cinogenesis have been studied by PCR-directed sequencing, gel shift assays and in situ ultraviolet cross-linking assay. The biological function of the interaction of HBV with p53 gene was investigated by co-transfection of chloramphenicol acetyltransferase ( CAT) reporter gene. p53 and HBV DNA. and quantitative PCR. Among the 16 primary hepatocellular carcinoma (PHC) samples. 13 were HBV-DNA positive. 10 HBxAg positive and 9 p53 protein positive. The p53 gene point mutation was found in 5 samples, one of which had a G to T substitution located at codon 249. After analyzing the HBV genome by a computer program, a p53 response element binding sequence was found in HBV genome at upstream of enhancer I. from 1047 to 1059 nucleotides. This sequence could specifically bind to p53 protein, increase p53 protein accumulation in the PHC cells and stimulate the transactivating activity of p53 and HBV replication . The results also revealed that HBxAg could combine with p53 protein to form a complex in the cells and enhance CAT expression. Immunocytochemical staining showed that p53 protein complex was located in the cytoplasm and the process of p53 entry to nuclei was. in part, blocked. From our results, we conclude that the mutation of p53 gene at codon 249 is infrequent in HBV-associated PHC. the DNA-protein binding between HBV and p53. and the protein-protein binding between HBxAg and p53 might lead to the reduction or inactivation of p53 protein, which in turn resulting in HBV-associated hepatocarcinogenesis.     

基于化学动力学疗法的联合治疗在肿瘤中的研究进展

由于肿瘤微环境(TME)的复杂性,单一的治疗策略难以有效地消除肿瘤,因此需要多种方法以克服单一治疗策略的缺陷.化学动力学疗法(CDT)是一种新兴的癌症治疗策略,通过芬顿或类芬顿反应,将内源性过氧化氢(H₂O₂)转化为羟基自由基(·OH)杀死癌细胞.该治疗方法因其侵袭性小、肿瘤特异性高而受到广泛研究.但是,温和的酸性pH值、低H₂O₂含量,以及TME中过表达的还原物质严重抑制了CDT效率.因此,基于CDT的联合治疗策略得到了广泛的发展.文章对近年来CDT联合治疗策略进行了概述,并对其进行了展望.