In Vivo And In Vitro Inhibition Of Glioma Growth Resulting From RAAV Vector Delivery Of INOS MRNA Antisense

Glioma is the most common of primary brain tumor and exhibit infaust prognosis.Glioma grows rapidly, invasively and displays non-significantly border, which make it difficultly complete resection. Chemotherapy and radiotherapy could not highly specially kill the glioma cells, moreover, they could induce toxic and side effects of central nervous system beyond systemic tolerance. To date, there are some aspects of advancement in the conventional therapy of brain glioa, but remain less efficacy and poor prognosis. The survival average of malignant glioma is 0.6-0.7 year. In the present, the occurrence and development mechanism of glioma remain unclear.Therefore, the study on molecular biology mechanism of glioma may provide a more effective therapeutic target. Improving the therapeutic effect of glioma is considered to be the focus of current neurosurgery. With the development of molecular biology , gene therapy, as a novel therapeutic technology, already provoke extensive attention. Gene therapy is a new concept way against disease,it can let target cells die on the basis of the distinction of molecule mechanism between tumor cell and normal cell. Previous study show that inos involved the course of angiogenesis, proliferation and apoptosis in glioma. Therefore,the target treatment to aim directly at iNOS is possibly a new way of Comprehensive treatment of gliomas.In present research, we would explore the roles of inos, VEGF and PCNA gene in the pathopoiesis mechanism of glioma, construct the rAAV-AsiNOS, investigate the effect of glioma proliferation when rAAV-AsiNOS was in vivo and in vitro.一.TO investigate the expression of iNOS, VEGF and PCNA in gliomaObjective To investigate the expression of iNOS, VEGF and PCNA in glioma and the correlation withen them.Methods To collect 93 cases of human brain neurogliocytoma(49 cases of low potential malignancy group,44 cases of high potential malignancy group) and 14 cases of fresh brain tissue. INOS,VEGFandPCNA expression were detected by immunohistochemistry , RT-PCR and Western Blot. to analyze the correlation withen iNOS, VEGF and PCNA.Results The immunohistochemistry results showed that iNOS was not expressed in the fresh brain tissue group , positive expressed in the cytoplasm of low potential malignancy group and strong expressed in the cytoplasm of high potential malignancy group.It had a statistical significancen between low potential malignancy group and high potential malignancy group(P <0.05);VEGF,PCNA andⅧ-Rag were all expressed in the cytoplasm of normal brain tissue group and neurogliocytoma group.They were all positive weakly positive expressed in the normal brain tissue group, positive expressed in the low potential malignancy group and strong expressed in the high potential malignancy group. It had a statistical significancen withen two groups(P <0.05);Analysis of immunohistochemistry showed iNOS,VEGF,PCNA andⅧ-Rag had a statistical significancen not only in low potential malignancy group but also in high potential malignancy group(P <0.05);Analysis of RT-PCR showed iNOS mRNA was not expressed in the fresh brain tissue group , positive expressed in the low potential malignancy group and strong expressed in the high potential malignancy group. .It had a statistical significancen between low potential malignancy group and high potential malignancy group(P <0.05); The RT-PCR results also showed that VEGFand PCNA mRNA was low expressed in the fresh brain tissue group , positive expressed in the low potential malignancy group and strong expressed in the high potential malignancy group. .It had a statistical significancen withen three groups(P <0.05); Analysis of Western Blot showed that iNOS proteinum was not expressed in the fresh brain tissue group , positive expressed in the low potential malignancy group and strong expressed in the high potential malignancy group. .It had a statistical significancen between low potential malignancy group and high potential malignancy group(P <0.05). The Western Blot results also showed that VEGF and PCNA proteinum was low expressed in the fresh brain tissue group , positive expressed in the low potential malignancy group and strong expressed in the high potential malignancy group. It had a statistical significancen withen three groups(P <0.05).Conclusions INOS,VEGF,PCNA andⅧ-Rag were all participate in the tumorigenesis and development.The genetic expressions of iNOS,VEGF,PCNA andⅧ-Rag were correlated with the malignant degree of glioma. Meanwhile, iNOS,VEGF,PCNA andⅧ-Rag mediated the glioma cell proliferation and growth.二,TO construct the rAAV vector delivery of iNOS mRNA antisense Objective TO construct and titrate the the rAAV vector delivery of iNOS mRNA antisense.Methods gene sequence of iNOS were replicated by RT-PCR. After its construction, the rAAV-AsiNOS was packaged in 293cells in culture and then purified, concentrated, and titrated.Results rAAV-AsiNOS was successfully constructed. The titer of virus preparations was 6~12×107/ml and the infection rate was up to 70%. The peak fraction of rAAV preparations contained a concentration of 2×1010 viral particles per milliliter excluding influential factors.Conclusions rAAV-AsiNOS was constructed successfully. PC12 cells could be transfected with rAAV vectors and the transfection rate was up to 70%.三,To explore the mechanisms of resistance to the proliferation of C6 rat glioma cellsObjective To explore the effect of glioma proliferation and the change of iNOS,VEGFand PCNA gene expression when rAAV-AsiNOS was in C6 rat glioma cells .Methods The study were divided into 3 groups: nontreated,rAAV-LacZ and rAAV-AsiNOS groups.To observe C6 cell growth curve. After cultivate for 1h,3h,6h,12h and 24h,FCM was used to determine the percentage of NT positive cells and the apoptosis rate,the expression of nNOS, iNOS, p38 MAPK and Caspase-3 mRNA was semi-quantified by RT-PCR. Results rAAV-LacZ was proved to have no distinct effect on C6 glioma cell growth, but rAAV-AsiNOS group showed that there was a tendency of cell growth torpidity, there was a statistic significance compared to control group. FCM measurement showed that NT positive cell percentage increased in the C6 cells compared with control group and AAV-LacZ group after C6 cells were cultivated in the medium having AAV-AsiNOS. Cultivating 2 hours later, apoptosis rate dropped in the C6 cells of rAAV-AsiNO group than control group and AAV-LacZ group. There were different percentages of Inos,VEGF and PCNA positive cell in different groups. In rAAV-iNOS group, cultivating 3h later,the percentage of iNOS,VEGF and PCNA positive cell significantly dropped,RT-PCR showed that rAAV-iNOS transfected C6 cells had low mRNA expression of iNOS,VEGF and PCNA.Conclusions rAAV-iNOS can restraint C6 cell proliferation by inhibiting VEGFand PCNA genes expression on account of repressing the expression of iNOS.四,To explore the mechanisms of resistance to C6 cell proliferation after rAAV-AsiNOS transfection in vivoObjective To establish the rat cell line C6 brain model. To explore the influence of glioma proliferation and iNOS,VEGF and PCNA gene expression when rAAV-AsiNOS was in vivo.Methods C6 cells were transfected to the right caudate nucleus areas of SD rats by stereotactic technology.After 7 days, rats were divided into 4 groups: control, rAAV-LacZ, rAAV-AsiNOS and blank groups,every group had 8 rats. Before the establishment of rat cell line C6 brain model , after 7 days of the establishment of rat cell line C6 brain model and after 7 days of transfection,every rat was to give a Neurological Deficit Score. After 7 days of transfection, determine the weight and volume of the C6 gliomas, observe the ultramicrostructure of C6 cells.The expression of iNOS,VEGFand PCNA antigens were detected by means of immunohistochemistry. The expression of iNOS,VEGFand PCNA mRNA was semi-quantified by RT-PCR., Results The tumor-bearing rats of rAAV-iNOS group improve the Neurological Deficit Score but that rats in control and rAAV-LacZ groups had opposite results. MR scan showed focus of infection of 15d were exceed than that of 8d in both control group and rAAV-LacZ group, at the same time, focus of infection in rAAV-iNOS group rats were obviously diminuted. Under the scanning electronmicroscopy, there were apoptotic bodys in the C6 cells in rAAV-AsiNOS group.Immunohistochemistry and RT-PCR showed the expression of iNOS,VEGFand PCNA in control and rAAV-LacZ groups was degraded than that in rAAV-AsiNOS group with statistical significance.Conclusions rAAV-iNOS can inhibit iNOS expression , regulate VEGF synthesis, decrease angiopoiesis, degrade PCNA expression and relief the effect of inhibiting tumour cell apoptosis by iNOS in rat c6 brain glioma.