Dissertation > Excellent graduate degree dissertation topics show

Effects of Adenovirus Ad-Canstatin on the Treatment of Liver Cancer

Author: ZuoMin
Tutor: LiJianSheng
School: Zhengzhou University
Course: Clinical
Keywords: Primary liver cancer canstatin gene caspase-3 Flk-1
CLC: R735.7
Type: PhD thesis
Year: 2010
Downloads: 152
Quote: 0
Read: Download Dissertation

Abstract

Primary liver cancer is one of the global high incidence of malignancy, According to World Health Organization statistics, the world each year newly discovered cases of over 26 million people, the morbidity and mortality of liver cancer account for cancer in China . Occurrence of liver cancer through the regulation of a number of links formed multiple steps from the start, tumor promotion and development, involving multiple genes are involved, and mutations, eventually leading to liver cells by the atypical hyperplasia until hepatocellular carcinoma, the cause is not entirely clear. Currently, most studies suggest that the incidence of hepatocellular carcinoma in cirrhotic nodules, hepatitis B and C virus infection, aflatoxin gene variation is closely related to the geographical environment factors. Early metastasis of primary liver menstrual blood and lymph, locally invasive, has been developed to most when diagnosed in late, the prognosis is poor. In the vast majority of patients with liver cancer has entered a definite diagnosis is often late, the cancer had occurred both inside and outside the liver extensive transfer traditional surgery, intervention, chemotherapy and radiotherapy treatment made some significant progress in the treatment of liver cancer, but liver cancer The near-term survival and long-term prognosis of patients no significant improvement, therefore, become the greatest concern of clinicians and scientists find new treatments. In addition to the the hospital commonly used traditional surgery, chemotherapy, and radiation therapy, as the technology of genetic engineering has become more sophisticated, many hospitals are gradually carried out a variety of gene-mediated therapy, gene therapy techniques include suicide gene therapy, immune gene treatment import tumor suppressor gene therapy, anti-tumor vascular gene therapy, which has become one of the current research focus adenovirus-mediated anti-tumor vascular gene therapy approach. As early as in the 1970s, Folkman first proposed malignant tumor growth depends on the formation of new blood vessels, neovascular supply malignant nutrients needed for growth, this view was subsequently confirmed by numerous studies. When tumor diameter 1mm, if no neovascularization to support its growth, the tumor will enter a period of dormancy, or degradation, If the length of the new blood vessels into the tumor tissue, the tumor will be rapid growth reaches the volume control is lost. The new blood vessels to the tumor oxygen and nutrition and take away the metabolic waste. Thus, anti-angiogenesis therapy can directly cut off the tumor growth and metastasis depends on the nutrient supply, this method has become a new bright spot of liver cancer research field. Recent studies have shown that neovascularization exist only in a small number of organizations, transient phenomenon of neovascularization in addition to the female reproductive endometrial, placental tissue and wound repair parts, most of the time the body's vascular system remains stationary state, the body vascular part of the organization depends on the local pro-angiogenesis factor and vascular rejection equilibrium between the factor. On the one hand to promote the up-regulation of vascular endothelial growth factor to promote angiogenesis, on the other hand vascular growth inhibitory factor levels down to inhibit angiogenesis. The present study demonstrate that all endogenous vascular growth inhibitory factor can inhibit tumor growth in animal models, but the direct application of angiogenesis inhibitor treatment of malignant tumors is still facing many problems: 1) require long-term application of angiogenesis inhibitor; 2) large dose of harmful immune response might be interested in the human body. 3) the high cost of production led to the huge cost of treatment in which patients bear the difficulty. Gene therapy is via biological carrier or biotechnology objective DNA or RNA transferred to a host cell so that it can effectively be expressed, the recombinant protein synthesized in the host cell, the role of the local tissue play an anti-tumor therapeutic effect. The advantage of this kind of gene therapy is: 1) with the introduction of exogenous genes, the host cell itself may be synthesized recombinant protein, thus reducing the cost of production; 2) gene therapy is the gene into tumor cells, so that it can be effectively expressed recombinant protein content in the local tumor area increased significantly improve the therapeutic effect. Today, over two hundred clinical gene therapy trials ongoing worldwide project, about half of them are for cancer treatment, which have half of the gene therapy, and both are against the tumor cells themselves. Application to inhibit angiogenesis gene therapy directed against vascular endothelial cells, because very stable hereditary vascular endothelial cells, the target gene can be stably expressed in the human body for months or years, and this gene therapy vector than the manufacture of a large number of high-purity protein easier and reduce the number of spending. Currently, adenovirus as gene therapy vectors tool having the advantages of high efficiency and transfection of exogenous gene expression, recombinant adenovirus to a large number of expression of recombinant proteins in the local tumor tissue, in the local inhibition of tumor angiogenesis, while adenovirus infected tumor cells can induce host anti-tumor immune response can play from a variety of aspects of the anti-tumor effect. In recent years, the researchers found a new endogenous angiogenesis inhibitor - canstatin protein of canstatin non-collagen protein derived from collagen type IV a2 chain C-terminal region (NC1) domain, proliferation and migration of endothelial cells has The inhibitory effect can be induced endothelial cell apoptosis, which play to inhibit the formation and growth of new blood vessels. Of canstatin protein can significantly inhibit the growth of human renal cell carcinoma, prostate cancer, esophageal cancer, and ovarian cancer growth, but how canstatin inhibition of protein on liver cancer therapeutic effect of genes on human hepatocellular carcinoma by adenovirus-mediated canstatin ? these problems at home and abroad has not been reported. In the present study, we first cloned human canstatin gene and its original expression vectors and recombinant canstatin protein was constructed, and then confirmed the canstatin protein inhibition on Liver Cancer, then we also build the carrying canstatin gene adenovirus vector, finally recombinant adenovirus injected into mouse liver tumor carry canstatin gene, observed changes in mouse liver tumor volume, pathological changes, caspase-3 and Flk-1 Tat microvascular density and liver cancer tissue, and its role in the treatment of liver cancer . The purpose of the first part of the experiment canstatin cDNA Cloning: The Clone canstatin cDNA gene and analysis of its gene sequence. Methods: RT-PCR technique to extract purpose genes of canstatin cDNA from fresh human placental tissue, then cloning vector pGEM-T, and then obtain the recombinant plasmid pGEM-T/canstatin final transformation E.coliDH 5a, positive clones were screened out genes Determination of its sequence. Results: 1% agarose gel electrophoresis showed three distinct bands (5S, 18S, 28S) mRNA in human placental tissue; spectrophotometer, the mRNA the A260 = 0.878, A280 = 0.411, ratio of A260/A280 = 2.136, calculate the total RNA concentration was 1.76g / L. RT-PCR amplification of the object and purpose of the gene canstatin length is basically the same. RT-PCR amplification product with the vector PGEM-T connection processing, transforming E.coli DH5a, seen white and blue colonies on LB plates with ampicillin, and the selection of relatively good five white colonies Bam HI and Hind Ⅲ restriction enzyme digestion, confirmed the positive clones. Shanghai Biological Engineering Company determination of the gene sequence, the same the target gene recombinant plasmid positive clones in GenBank canstatin gene sequences. Conclusion: We successfully cloned the canstatin genes, anti-liver cancer treatment research laid the foundation for the next step using canstatin protein. Prokaryotic expression of the second part of the experiment and recombinant human canstatin protein purpose: to build canstatin gene prokaryotic expression vector, expression and purification of of recombinant human canstatin protein. Methods: double digestion from the recombinant plasmid pGEM-T/canstatin cut canstatin gene, inserted into the target plasmid pET-22b () corresponding sites, transforming E. coli BL21, recombinant human canstatin protein after IPTG induction, using SDS -PAGE electrophoresis to analyze the human canstatin protein. Ni-NTA affinity chromatography and purification of of recombinant human canstatin protein renaturation with PBS its dialysis. Results: The recombinant plasmid PGEMT-T/canstatin cut the target genes of canstatin cDNA inserted into plasmid pET-22b () the appropriate sites, transforming E. coliBL21 pick five good colony Bam HI and Hind Ⅲ digested do electrophoresis identification of all colonies contained two specific bands, respectively, corresponding to the target gene and the original plasmid. SDS-PAGE electrophoresis to analyze the results show that the new protein bands appear 24KD induced after 1h, 2h, 3h and 4h expressed protein ratio of total bacterial protein was 17.2%, 17.8%, 21.0% and 22.4%, respectively. Ni-NTA affinity chromatography purified 125Mm and 250mM eluent the human canstatin proteins. Conclusion the: canstatin prokaryotic expression vector can be successfully constructed and purified a of recombinant human canstatin protein. The third part of the experiment is to explore the recombinant human canstatin proteins in vivo treatment of liver cancer Objective: To investigate the treatment of liver cancer the recombinant human canstatin protein in the body. Methods: SMMC-7721 hepatoma cell line to establish nude mouse hepatoma subcutaneous xenograft model of 30, when the tumor grew to 2-3mm in diameter, were randomly divided into three groups, that PBS control group ,5-Fu the treatment group canstatin protein treatment group groups were confirmed by intratumoral the multipoint injection administration. During treatment regularly measuring the transplanted tumor size and weight; remove the tumor after treatment do routine pathological examination of immunohistochemical staining, the total course of 21 days. Results: 3 days of treatment, the tumor volumes of canstatin treatment group and 5-Fu treatment group was significantly less than the control group (P lt; 0.05); six days of treatment, there was a significant difference P lt; 0.01. The end of treatment, although the efficacy of 5-Fu treatment group the most significant, but the systemic side effects obvious pathologically confirmed serious liver damage. Conclusion: recombinant human canstatin protein can significantly inhibit the growth of liver cancer tissue, no significant side effects, is the ideal treatment for liver cancer patients. Construction of the fourth part of the experiment the adenovirus vector carry canstatin genes purpose: using genetic engineering techniques constructed adenovirus vector carry canstatin gene the Ad-canstatin. Methods; using the polymerase chain reaction (PCR) technology, amplified PET / canstatin carrier on canstatin gene, inserted into the double digested sites pCA13 plasmid shuttle vector was successfully constructed pCA13-Cans; pCA13-Cans with adenovirus the skeleton pBGHE3 total of 293 cells were transfected with the recombinant adenovirus by viral plaque purification using the polymerase chain reaction (PCR) identification of recombinant adenovirus. Cesium chloride density gradient centrifugation and purification of recombinant adenovirus 293 cells amplified recombinant adenovirus, and use the TCH150 detection of recombinant adenovirus titer. Results: from PET / canstatin carrier successfully amplified a 709bp DNA fragment containing canstatin gene insert to pCAl3 plasmid successfully constructed pCA13-Cans plasmid by PCR and restriction analysis is correct, the adenovirus backbone pBGHE3 homologous recombination in 293 cells to obtain recombinant adenovirus was constructed correctly, and then identified by PCR recombinant adenovirus Ad-named of canstatin TCH150 detect recombinant virus titer of 3.1 × 109pfu/ml. Conclusion: The high-titer recombinant adenovirus the Ad-canstatin for further study of its anti-tumor effect laid the foundation. The fifth part experiment the Ad-Canstatin treatment liver cancer experiment research purposes: observation the Ad-Canstatin tumor in vivo in mouse hepatoma inhibition of hepatocellular carcinoma. Method: the growth of human hepatoma SMMC-7721 human hepatocellular carcinoma tumor cells to establish subcutaneous xenograft model. First of cultured human liver tumor cell suspension, 24 mice were randomly divided into three groups, each nude mouse the left axillary subcutaneous injection of tumor cell suspension 0.1ml (concentration of 2 × 107/ml) until the tumor volume of nude mice growth to about 50mm3 bearing nude mice were randomly divided equally into the Ad-canstatin treatment group and PBS control group and GFP no-load groups of eight tumor-bearing nude mice. Each tumor underwent two intratumoral injection interval of 72 hours, the the Ad-of canstatin group, and the amount of GFP no-load group nude intratumoral injection of virus were 8 × 109PFU nude mice injected PBS control group, 0.1 ml of PBS. MAIN OUTCOME MEASURES are: 1, the rate of tumor growth inhibition and tumor volume growth curve. Pathological examination: the injection of the drug 30 days after the nude mice were killed, compared histopathological changes. 4, immunohistochemical methods check: detection of each group in the tumor tissue of caspase-3 and Flk-1 expression. Results: 1. 3 days after injection the Ad-canstatin gene therapy, tumor volume of the Ad-of canstatin group of nude mice was significantly less than the PBS and GFP group (P lt; 0.05); subsequently observed that, with time in each group nude mice tumor volume increases, but the Ad-canstatin groups of nude mice tumor growth and tumor volume were significantly smaller than the PBS and GFP group (P lt; 0.05). Pathology detected groups of nude mice tumors in vivo were seen to have different degrees of necrotic tissue, Ad-of canstatin treatment group nude intratumoral necrotic tissue was significantly more than PBS and GFP groups. Treatment group 3.Ad-Canstatin Flk-1 expression was significantly lower than the PBS group and GFP group, the difference was statistically significant (x2 = 7.778, p lt; 0.01); 4.Ad-of canstatin treatment group, caspase-3 expression higher than GFP and PBS group, the difference was statistically significant (x2 = 6.563, x2 = 10.50, p lt; 0.01). Conclusion: adenovirus vector the Ad-Canstatin able to successfully inhibit the growth of liver cancer tissue in nude mice, and be able to reduce the Flk-1 expression and caspase-3 expression in hepatoma tissues. We think that the mechanism of action of the Ad-canstatin the treatment of liver cancer: on the one hand, the expression in tumor tissue the canstatin protein to inhibit liver cancer angiogenesis, the other hand, it may reduce the Flk-1 expression and increase caspase-3 expression in HCC tissues, promote apoptosis of liver cancer cells, which also play a role in inhibiting the growth of liver cancer cells. In short, canstatin protein as endogenous angiogenesis inhibitors is expected to become a new drug for the treatment of liver cancer, the use of adenovirus-mediated the canstatin gene therapy for liver cancer, not only can play a powerful anti-tumor effect and can reduce the cost of treatment, opened up for the treatment of liver cancer The new method, the future will have a good prospect.

Effects of Adenovirus Ad-Canstatin on the Treatment of Liver Cancer

Abstract

Related Dissertations