Experimental Study On Brain Glioma Therapy With The Combination Gene Of HSV-tk And Endostatin Mediated By PIRES

Glioma is the most frequently occurring malignant tumor in central nervous system. It is characterized by its powerful invasion and high malignancy. It is difficult to make early diagnosis, and the therapeutic efficacy is bad. The curative effect of the conventional treatment methods, including surgery, radiotherapy and chemotherapy, still show the poor efficacy. In recent years, glioma gene therapy has become a research hotspot. However, a number of studies have shown that single gene therapy is not always a effective way to inhibit tumor proliferation and recurrence. The result of single gent therapy is not satisfied. The occurrence and the development of malignant glioma is the result of the combined effect of genes. Combined different methods of gene therapy, especially those have the synergistic effect genes can not only overcome the single-gene's defects, but also can improve the therapeutic effect greatly. It will open up a new way for improving the patient prognosis and curing the tumor ultimately.Glioma is a typical angiogenesis dependent tumor. Inhibiting tumor angiogenesis has become a new hotspot in recent years. Endostatin is the most intensively angiogenesis inhibitor now. It can inhibit vascular endothelium cell proliferation, migration specificly and inhibit tumor angiogenesis. So based on these functions, Endostatin can reduce the tumor blood supply and nutrition to inhibit tumor growth and metastasis effectively. It has shown a good clinical application prospect.Suicide gene therapy, also known as drug sensitivity gene therapy, is another hot spot in tumor gene therapy field. HSV-tk/GCV system is the most spectacular one. Herpes simplex virus thymidine kinase gene (HSV-tk) can encode a particular enzyme, which can convert nontoxic prodrug GCV into highly toxic metablism and interfere the DNA synthesis of tumor cell. It can not only leads to the apoptosis of tumor cells which has been transfected, but also can kill the close tumor cells which is not been transfected with suicide gene by bystander effect.pIRES vector allow two open reading frame for translation in the same mRNA. So it is possible for a dual-gene to co-expressed in one vector.In the view of the above research background, this study take full advantage of the superiorities of the suicide gene therapy and anti-angiogenesis therapy and construct a dual-gene co-expression vector with HSV-tk and Endostatin gene mediated by pIRES. Our goal is to explore the biological behavior effects of the glioma cell and vascular endothelial cell, such as proliferation, migration and apoptosis, made by the combined gene therapy in vitro. At the same time, establish rat brain glioma animal model to study the inhibition ability to glioma by the double-gene recombinant vector in vivo. Through all the research above, we analyze the feasibility and characteristics of HSV-tk gene and Endostatin's treatment of glioma and make a foundation for glioma's gene therapy. In this study, the experiments were carried out as follows:1. Expression of ES in human gliomaObjective: Investigate the expression of ES in the serum and glioma tissues, as well as its relationship with clinical pathological features in order to lay the foundation for ES gene therapy. Methods: Fourty-six patients with glioma treated at the Institute of Neurosurgy in the present hospital, from September 2006 to October 2007 were studied respectively. There were 31 male and 15 female patients in the present study, aging between 15~64 years, and the average age is 41.6. The glioma tissue specimens were chosen according to the classification standard of central nervous system tumors of WHO in1999. There were 7 cases I grade specimen and 18, 13, 8cases II, III, IV grade respectively. ELISA method was used to detect the expressions of ES in the glioma patients'serum preoperative and postoperative respectively. Immunohistochemistry method was used to detect the ES protein expression and microvessel density in the human glioma tissues. At the same time, using semi-quantitative RT-PCR method to detect ES mRNA expression in glioma tissue samples. Results:â‘ The expression of ES in glioma patients'serum is higher significantly than that in normal person's serum and it shows close correlation with glioma pathological grade(P<0.01);â‘¡The expression of ES in glioma patients'serum postoperative is slower significantly than preoperative.â‘¢The expression of Endostatin in glioma tissues are significantly higher than in normal brain tissues in both mRNA and protein levels(P<0.01). The expression intensity enhanced with the increase of malignancy degree;â‘£Non-parameter Kruskal-Wallis analysis was used to analyze the correlation of glioma pathogenic grade with MVD and the correlation of ES protein expression intentsity with MVD, and the results showed that MVD increased significantly with the glioma pathogenic grade and ES protein expression intensity increased. Conclusions: There is high level ES expression in glioma patients'serum preoperative and it has close correlation with glioma pathogenic grade. suggesting that ES may be a useful marker for diagnosis of glioma tumor. The decreased ES expression level in serum postoperative suggests that ES can be a effective indicator for judgement of surgical treatment and prognosis. The high level ES expression in glioma tissues and its cloth correlation with glioma pathogenic grade and angiogenesis provide a theoretical basis.for the application of surgery combined with endostatin gene therapy.2. Construction of recombinant vector co-expressing HSV-tk and EndostatinObjective: To construct a recombinant vector co-expressing the suicide gene HSV-tk(TK) and Endostatin (ES) genes mediated by pIRES vector, and to lay the basis for the following experiments in vitro and in vivo. Methods:â‘ Using Ad-tk vector carrying HSV-tk gene as template, the full length sequence of HSV-tk gene was amplified and cloned into pMD18-T simple vector to construct pMD18-T-TKNheI/EcoRI, and which was further confirmed by PCR, restriction enzyme digestion and sequence analysis methods.â‘¡Using RT-PCR method, the full length sequence of endostatin gene was amplified from human normal brain tissue and cloned into pMD18-T simple vector to construct pMD18-T-ESSalI/NotI, and which was further confirmed by PCR, restriction enzyme digestion and sequence analysis methods.â‘¢TK gene and ES gene were inserted into the upstream and downstream of pIRES vector, respectively, to construct the single-gene recombinant plasmids pTK-IRES, pIRES-ES and double-gene co-expression recombinant vector pTK-IRES-ES, and which were confirmed by PCR, restriction enzyme digestion and sequence analysis. Results: The cloning vector pMD18-T-TKNheI/EcoRI, pMD18-T-ESSalI/NotI and the expression vector pTK-IRES, pIRES-ES, pTK-IRES-ES were all confirmed by restriction enzyme digestion and sequence analysis. Conclusions: The recombinant eukaryotic vector co-expressing HSV-tk and endostatin genes was constructed successfully.3.pTK-IRES-ES double-target treatment on brain glioma in vitroObjective: To explore the inhibition effect of pTK-IRES-ES on glioma cells and vascular endothelial cells. in vitro. Methods: pTK-IRES-ES plasmids were transfected into C6 and ECV304 with lipo2000. RT-PCR and Western bolt were used to detect the gene expression of recombinant plasmid; Cell proliferation ability was measured by MTT method in C6 and ECV304 cells after they were transfected with pTK-IRES-ES; Scratching method and Transwell chamber were used to observe the transfected ECV304 cells migration ability; Ultrastructure of transfected C6 cells was observed by electron microscopy; The change of C6 and ECV304 apoptosis percentage were detected with Hoechst33258 and Annexin V-FITC kits. Results:â‘ ES gene can be transcripted and expressed at ECV304 cell transfected with pTK-IRES-ES. Scratch test showed that the migration speed of ECV304 cell was significantly slow down and the migration distance was lower than control group. Transwell migration chamber experiments showed that the number of cell migrated to the next room is significantly lower in transfected ECV304 group than in control group. Apoptosis test results showed that the apoptosis rate of ECV304 cell was 35.93% after transfected with pTK-ES, which is significantly higher than control group.â‘¡TK gene could be expressed in C6 glioma cell after it was transfected with pTK-ES, and the survival rate of C6 cell was reduced gradually with the increase of the concentration of GCV. C6 cell was detected of apoptosis when it was transfected with pTK-ES combined with GCV by electron microscopy,Hoechst33258 and Annexin V-FITC apoptosis detection, and the apoptosis rate up was 40.1%, which is significantly higher than the control group. Conclusions: The recombinant plamid pTK-IRES-ES, carrying suicide gene and endostatin gene, can inhibit transfected ECV304 and C6 proliferation and migration ability and facilitate cell apoptosis.4.pTK-IRES-ES double-target treatment on brain glioma in vivoObjective: Verify the application effect of pTK-IRES-ES dual-target gene therapy on glioma in vivo, for providing a theoretical basis of gene therapy on gliomain the future. Methods: Rat brain glioma model is established by stereotaxic technique and then divided into five groups randomly (pTK-IRES-ES/GCV group,pTK-IRES/GCV group,pIRES-ES group,pIRES group and control group). Recombinant plasmid was injected intratumor. The difference of treatment effect in each group are contrasted by the rat general state,survival time,tumor inhibition rate,tumor general pathological change and MVD. Results:â‘ The tumor volume in pTK-IRES-ES/GCV group is significantly smaller than the other group. The tumor inhibition rate of pTK-IRES-ES/GCVgroup,pTK-IRES/GCV group,pIRES-ES group,pIRES group is 63%,46%,43% respectively;â‘¡The rat nervous system symptoms is reduced significantly and the survival time is significantly prolonged compared with other groups;â‘¢Necrosis and apoptosis increases in pTK-IRES-ES/GCV group by HE staining;â‘£The microvessel density in pTK-IRES-ES/GCV group is reduced significantly compared with other groups. Conclusions: The application therapeutic effects of Endostatin gene combined with suicide gene therapy dual-target treatment on gliomas is superior significantly than a single target one. It provides a far-reaching and broad prospects for clinical application .