摘要
脑胶质瘤是中枢神经系统最常见的恶性肿瘤,现有治疗方法疗效不佳,迫切需要开发新的治疗手段。自杀基因疗法和抗肿瘤血管生成治疗是近年来胶质瘤基因治疗领域的研究热点,其中以单纯疱疹病毒胸苷激酶(Herpes simplex virus thymidine kinase, HSV-tk,简称TK)自杀基因疗法和内皮抑素(Endostatin,简称ES)基因治疗方法颇具应用前景。本研究在探讨Endostatin在人脑胶质瘤中表达情况的基础上,以TK和ES作为双靶点,构建双基因共表达载体,探讨其对脑胶质瘤的体内外治疗作用。研究结果显示:1、脑胶质瘤患者血清中ES含量及胶质瘤组织中ES mRNA和蛋白表达量均显著高于正常脑组织,且表达强度随肿瘤恶性程度增高而增强。Pearson相关性分析表明,随着ES表达强度的增高,肿瘤微血管密度呈显著上升趋势。2、成功构建了pTK-IRES-ES双基因共表达载体。3、体外实验:转染pTK-IRES-ES重组质粒的人脐静脉内皮细胞ECV304和大鼠C6胶质瘤细胞可同时表达TK和ES基因;双基因重组质粒可明显抑制肿瘤细胞及血管内皮细胞增殖,促进细胞凋亡,同时可抑制血管内皮细胞的迁移能力。4、体内实验:成功建立了大鼠胶质瘤脑内动物模型,pTK-IRES-ES重组质粒治疗组与单基因治疗组及模型对照组相比,大鼠神经系统症状明显减轻,生存期延长,肿瘤生长受到明显抑制,肿瘤细胞发生凋亡坏死,肿瘤组织微血管密度降低。
以自杀基因和血管内皮抑素基因作为双靶点对脑胶质瘤进行联合基因治疗,其效果显著优于单基因治疗。高效、靶向、低毒副作用,协同作用强的双靶点基因治疗策略为未来胶质瘤治疗提供了新的思路。目前国内外尚未见联合应用HSV-tk和Endostatin双基因治疗脑胶质瘤的相关研究报道。
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 glioma
Objective: 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:①The 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);②The expression of ES in glioma patients’serum postoperative is slower significantly than preoperative.③The 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;④Non-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 Endostatin
Objective: 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:①Using 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.②Using 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.③TK 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 vitro
Objective: 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:①ES 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.②TK 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 vivo
Objective: 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:①The 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;②The rat nervous system symptoms is reduced significantly and the survival time is significantly prolonged compared with other groups;③Necrosis and apoptosis increases in pTK-IRES-ES/GCV group by HE staining;④The 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 .
引文
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