A Conditional Replication-competent Adenovirus Mediated By Cox-2 Promoter To Target The Brain Glioblastoma

Among the Neurologic Tumors, Glioblastoma is the most common cancer, because it has stronger invasiveness, higher severity, and easy relapse, treatment effects are usually very poor. At present the most commonly used treatment methods are operation, radiotherapy, chemotherapy, and traditional Chinese medicine auxiliary treatment, etc, however, the side effects are strong, and treatment effects are little. With the unceasingly recognition of tumor by people, gene therapy comes. Gene therapy has high selective in treating humor, and the harms are small and light. it has effect on advanced cancer and metastases cancer, and therefore valued by people.Conditionally replicative adenovirus, i.e. tumor specific and proliferative adenovirus, is used in gene therapy. Conditionally replicative adenovirus has the characters of killing the infection tumor cell directly and carrying massive foreign aid gene expressions and has little damage to the periphery normal cell. therefore, it is becoming a hot spot of gene therapy.This research is the study of the inhibiting effect of COX-2 promoter with conditionally replicative adenovirus to the growth of brain Glioblastoma cell. this thesis is divided into the following five parts:1. Construction and Identification of A Conditional Replication-competent Adenovirus Mediated by Cox-2 PromoterObjective To construct conditional replication-competent adenovirus mediated by Cox-2 promoter, establish the foundation which use it to treat glioma. Methods①To construct pRsetB-E1A pronucleus expression plasmid;②To express and purify recombination E1A protein;③To produce and detect multiclone antibody;④To detect expression level of COX-2;⑤To construct and identity recombination adeno virus plasmid. Results①E1 protein can be expressed effectually, the tite of anti-E1A polyclonal antibody is very high;②The higher level Cox-2 is detected by flow cytometer and RT-PCR in glioma cell A-172 and LN-18;③Recombination adenovirus plasmid Ad-Cox-2-El A is constructed successfully which is identifyed in gene and protein level. Conclusion Ad-Cox-2-E1A is constructed successfully and it has favourable expression activity, it can be good carrier in glioma gene therapy in future2. Obtaining of Conditionally Replicative Adenovirus under Restructuring ConditionsObjective Obtaining COX-2 promoter with the conditionally replicative adenovirus (El A) through cloning, packing, amplifying and purifying. Method:①Structuring the adenovirus plasmid to be reconstructed.②Packing and amplifying the adenovirus reconstructed.③Purifying the adenovirus reconstructed.④Identifying the adenovirus reconstructed. Result:①Getting massive reconstructed adenovirus plasmids through PCR method.②successfully packing, amplifying and purifying the conditionally replicative adenovirus under the restructuring conditions.③identification of conditionally replicative adenovirus shows that, no loss of the adenovirus segments restructured, and exogenous gene expression normal. Conclusion: Ad-Cox-2-E1A is obtained massively, expression of exogenous gene is normal, and could be used for the subsequent experiments.3. Cox-2 Promoter's Specific Activity In Human Glioblastoma CellObjective Use the adenovirus restructured to infect human brain Glioblastoma cell and normal human brain fibroblast to test the promoting specificity of Cox-2 promoter on the adenovirus vector in brain Glioblastoma. Method:Use Fluorescein enzyme to report gene principle and to determine the promoting activity of Cox-2 promoter in the test. Result:The promoting activity of Cox-2 promoter cannot be found in the normal cells almost. In the brain Glioblastoma cells the Cox-2 promoter has obvious promoting activity. Conclusion:in adenovirus vector the Cox-2 promoter do not have promoting activity in normal brain cell, and this only represent specific expression to gene which it carries in tumor cells, therefore comparatively therapy effect are gain, at the same time setting a foundation for reducing the side effect to the surrounding cells.4. Specific Activity of Ade-Cox-2-E1 to Human Brain Glioblastoma Cells In VitroObjective Use reconstructed adenovirus Ad-COX-2-E1A to infect human brain Glioblastoma cell and normal human brain fibroblast, through testing livability of cells to test the specificity killing of conditionally replicative adenovirus against brain Glioblastoma cell. Method:①Test the specificity expression of Ade-Cox-2-E1 infecting tumor-cell (E1A) Protein by using Western.②Test the specificity inhibiting of Ade-Cox-2-E1 to tumor-cell by using MTT method. Result:①The reconstructed adenovirus (Ad-Cox-2-E1) infects the experimental cells, in the normal cells the expression of E1 A protein is hard to be found while in brain Glioblastoma cells, expression of E1 A protein can be found.②The reconstructed adenovirus do not have obvious inhibiting effect to growth of the brain normal cells, while have comparatively good specific inhibiting activity to brain Glioblastoma cells. Conclusion:The reconstructed adenovirus at the same time maintains the character of high infectious of wild type adenovirus, while realizes the specificity copying in brain Glioblastoma cells, therefore decreases the side effect to the surrounding normal cells obviously.5. Specific Activity of Ade-Cox-2-E1 In Human Brain Glioblastomal Tumor In VivoObjective Conduct local injection to tumor by using the reconstructed adenovirus Ad-COX-2-E1A, and test the inhibiting effect to human Glioblastoma cell A-172 in BalB/c nude mouse by using tumor growth curve. Method:①Adopting BalB/C nude mouse to design tumor model.②Showing the inhibiting effect of Ade-Cox-2-E1 to the growth of nude mouse through the contrast with the control group according to change in volume.③Observing the effect of Ad-Cox-2-E1 to the major viscera of nude mouse by pathological section. Result:①The Glioblastoma model of BalB/C nude mouse has been successfully set up.②The experimental group has obvious effect to the inhibiting nude mouse in growth, and since the 22nd day, the tumor has no significant difference in volume, effect of inhibiting tumor is obvious.③the pathological sections show that, it has no obvious side effect to liver and kidney tissues. Conclusion:Ade-Cox-2-E1A reconstructed adenovirus can effectively inhibit the growth of human brain Glioblastoma cell, however it has no obvious side effect to the surrounding and main visceral organs.