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Liposomes Complexed to Plasmids Encoding M protein of Vesicular Stomatitis Viruses Inhibits Formation of Malignant Ascites in Mice

目的:水泡口炎病毒(Vesicular Stomatitis Virus,VSV)基质蛋白(Matrix protein, M 蛋白)具有诱导肿瘤细胞凋亡的作用,本研究探讨水泡口炎病毒基质蛋白对癌性腹水形成的抑制和治疗作用。方法:采用旋转蒸发仪法制备纳米脂质体,检测其体外转染效率;采用脂质体转染技术将已构建的水泡口炎病毒基质蛋白(VSVM)重组真核表达质粒pcDNA31M 转入MethA肿瘤细胞,转染后6小时将细胞接种于小鼠腹腔,观察小鼠腹水的形成情况;腹水治疗组,则先将MethA肿瘤细胞接种于小鼠腹腔,将小鼠随机分成4组,于接种后第二天分别用脂质体包裹的pcDNA31M、pcDNA31空载体、单纯脂质体及生理盐水治疗,观察腹水的形成情况。结果:自制的DOTAP:DOPE脂质体与Sigma公司购买的Lipofectamine 2000的转染效率相似,pcDNA31M明显抑制MethA腹水的形成,对已经形成的腹水也有明显的治疗作用,与对照组比较有统计学意义(Plt;005),同时明显延长了小鼠的存活期。结论: VSVM蛋白真核表达质粒pcDNA31M对小鼠腹水的形成有抑制作用,能延长小鼠的存活期,对于恶性腹水的治疗具有一定的意义,值得进一步研究。

An Improved Technique of Construction of Recombinant Adeno-Associated Virus 2 Mediated Gene Transfer to TIMP1 and Identification

Objective To use an improved technique to construct the recombinant adeno-associated virus 2 (rAAV2) mediated gene which can transfer human tissue inhibitor of metalloproteinase-1 (TIMP1). Methods Human TIMP1 gene was amplified from pDNR-LIB plasmid by PCR and cloned into the rAAV2 vector pSNAV to recombinant pSNAV-TIMP1, then was transferred into BHK-21 cells by means of lipofectamine. Using G418 selection, a mixed cell named BHK-21/rAAV2-TIMP1 was isolated, which was capable to express TIMP1. The cell was subsequently infected with recombinant herpes simplex virus 1 (rHSV1-rc/△UL2) that was able to package the rAAV2-TIMP1. After purification, rAAV2-TIMP1 was obtained. Results The rAAV2 carrying human TIMP1 gene was constructed successfully. The viral titer of the rAAV2-TIMP1 was 1×1012 v.g./ml. Conclusion rAAV2-TIMP1 was constructed successfully, which would provide experimental basis for carrying the TIMP1 into hepatocellular carcinoma effectively and inhibiting the invasiveness and migratory capacity of hepatocellular carcinoma in vitro and in vivo models.

Application and Prospection of RNAi in Colorectal Cancer Gene Therapy

【Abstract】Objective To explore the application of RNA interference (RNAi) in colorectal cancer gene therapy. Methods The related literatures in recent years were reviewed. Results RNAi causes a high effective and distinctive degradation of mRNA homologous in sequence to the dsRNA. This new technology has been successfully applied to research the genesis and the growth of colorectal cancer.Conclusion RNAi has been a new focus in gene therapy for colorectal cancer.

Effects of DNA Electrotransfer in Muscle on the Implanted Tumor Growth in Nude Mice

【Abstract】ObjectiveTo study the antitumor effects of DNA electrotransfer in muscle (ETM) by using established animal model for human anaplastic thyroid cancer cell line TA-K.MethodsNude mice with implanted TA-K were divided into five groups including: control group, pcDNA-3 plasmid electrotransfered into muscle (pcDNA ETM group), TIMP-3 plasmid injected into muscle (TIMP-3 IM group), TIMP-3 plasmid electrotransfered into muscle (TIMP-3 ETM group), TIMP-3 plasmid electrotransfered into implanted tumor (TIMP-3 ETT group). Electrical parameters used in electrotransfer were: 200 V/cm, 20 ms/pulse; 8 pulses, 1 Hz in muscle and 600 V/cm, 20 ms/pulse; 1 pulses, 1 Hz in implanted tumor respectively. ResultsThe growth of TA-K was inhibited more significantly in the groups of TIMP-3 plasmid electrotransfered into muscle and TIMP-3 plasmid electrotransfered into implanted tumor than the other groups (P＜0.05), and the content of TIMP-3 protein in tumor tissues was higher in these two groups . ConclusionAnti-oncogene has the antitumor effects by DNA electrotransfer in muscle.

Advances in Vascular Prosthesis in Which Lined with Genetically Modified Endothelial Cells

Objective To discuss the endothelial cell which was modified by exogenous anticoagulant genes contribute to the increase of antithrombosis activity of lined vascular prosthesis and the influence to other physiological functions of endothelial cells. Methods This summarized paper was made on literature review of recent years. Results The transfection of genes, including plasminogen activator (tPA, uPA, Urokinase), thrombomoduline (TM) and hirudin, etc, to endothelial cells resulted in not only the increase of antithrombosis activity of local vascular, but also the decrease of endothelial cell function in adherence and proliferation. Conclusion The increase of antithrombosis activity of lined vascular prosthesis has been done by exogenous genes. However, this technique ought to be studied, intensively.

Construction of Recombinant Adenovirus Vectors Carrying Antisense Matrix Metalloproteinase-2

【Abstract】Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack-CMV,with the resultant plasmid and the backbone plasmid pAdEasy-1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad-MMP2AS) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP. Results The recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the b green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×108/ml. Conclusion The recombinant adenovirus Ad-MMP2AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.

Construction and Study of Specific HSVTK/GCV Reconstructed AdenoAssociated Virus Plasmid of Human Hepatocellular Carcinoma

ObjectiveTo investigate target gene therapy for hepatocellular carcinoma (HCC). MethodsHerpes simplex virus thymidine kinase (HSVTK) gene was inserted into the gene of AFP enhancer/ALB promoter with adenoassociated virus (AAV) plasmid (WAV2) as a carrier, and a hybrid plasmid pWAV2/AFPALB/HYTK was constructed. Besides, plasmid pEGFP1/AFPALB was also constructed. Two kinds of plasmids were transferred into AFP positive cells HepG2 and AFP negative cells 7721, SPC and 7901.ResultsIt was found that enhance green fluorescence protein could only be seen in AFP positive cells HepG2. 710 bp DNA was amplified only in AFP positive HepG2 cells.ConclusionPlasmid pWAV2/AFPALB/HYTK for HCC demonstrates specificity in vitro.

The Background and History of Human Gene Therapy and Advances in the Study of Gene Therapy of Human Hepatocellular Carcinoma

ObjectiveTo know the background of human gene therapy and the advances in gene therapy study of human hepatocellular carcinoma (HCC). MethodsThe related literatures were reviewed. ResultsAs a substitute measure gene therapy was of great superiority and had got a good result in the clinical study of treatment of human genetic diseases. But it was mainly limited to the study of preclinical phase when it was used to the treatment of tumor. As a carrier of gene transfer， the newly modified retrovirus might have superiority in the study of gene therapy of HCC when compared with other viruses such as adenovirus and adenoassociated virus (AAV). The genes TK,p53 and p16 etc. had been used to the study of therapy of HCC and had some good results.ConclusionThe future of human gene therapy is optimistic, but there is a long way to go when gene therapy is widely used in the clinical study of treatment of HCC.

The Advances of Hepatocyte-Directed Gene Transfer Vectors

Objective To investigate the stable, high effective and low toxicity gene transfer vectors’ basics in research and clinical application.Methods Current status of hepatocytedirected gene transfer vectors were reviewed by history document and contemporary experimental advances analysis.Results Viral and nonviral vector systems could both transduce target genes to liver effectively with various transduction rates based on their corresponding biological characteristics.Conclusion Hepatocyte is the effective target for gene therapy of liverrelated genetic, neoplastic and infectious diseases, although the security needs to be further evaluated.

THE PROBLEM AND ASPECT OF GASTRIC CANCER GENE THERAPY