Clinic Experimental Research On Endostatin Gene Therapy For Glial Brain Tumor

Objective: To study localization of endostatin (ES) in human gliomas and relationship between endostatin and different WHO grade gliomas; explore the inhibitory role of endostatin gene(HSV-1-EScDNA) on the endothelial cell proliferation and the growth of rat C₆ gliomas and alteration of microvessel densities (MVD) in remaining tumors after therapy. Methods: â‘  Endostatin was immunostained by SP immnohistochemical method in the sections formalin fixed paraffin embedded tumor tissue from 30 patients with gliomas. â‘¡Different grades gliomas cells immunohitochemical staining are analyzed with cell-accounting and gray level with computer-aided image analysis system. â‘¢After the sequence of EScDNA in the pSecTagA-EScDNA wasceritifed correctly, human umbilical vein endothelial cells(EVC-304) would be transfected with pSecTagA-EScDNA and pSecTagA by the lipofection method . â‘£The postive clones were confirmed to have expression of exogenous EScDNA by RT-PCR and Western Blot. ⑤In order to assess the suppressive effect on the growth of EVC-304, FCM test for cell cycle and MTT test for cell activity effecy were performed. â‘¥ Rat C₆ glioma models were built by subcutaneously implanting C₆ cells in axillary fossa. ⑦HSV-1-EScDNA was injected intratumor directedly with virus titer 1×10⁵ PFU. ⑧ The tumor volume and tumor growth curve were observed by graphs and the tumors microvessel densities (MVD) were enumerated before and after therapy. Results â‘  Endostatin immunoreactivity was detected in glioma cells, endothelial cells, macrophages, and lymphocytes; the percentage of cells labeled with the endostatin antibody was significantly lower in the tumor parenchyma of human glioblastomas than in WHO Grade II astrocytomas(p =0.009). â‘¡Average gray level: WHOII26.83, WHOIII 69.63, WHOIV 86.19, Present for significant deviation (p<0.01). â‘¢EScDNA were dectected in those transfected cells by RT-PCR and Western Blot. â‘£FCM and MTT test suggest that cell cycle was extended longer and cell activity effecy lower in test group than in control group. ⑤The tumor growth curveshow that the tumor growth was markedly supressed in test group, which accordingly demonstrates that EScDNA application in vivo produce obvious inhibitory effect on the rat C₆ glioma. â‘¥HSV-1-EScDNA had less toxic side effects. ⑦Microvessel density (MVD) in rats gliomas of endostatin therapy group was significantly lower than MVD in control group(p<0.01). Conclusion Endostatin were present in various cell types in human gliomas in vivo; Lower levels in glioblastomas than WHO II astrocytomas might have reflected the shift of a probable regulatory balance between promoters and inhibitors of angiogenesis towards facilitation of neovascularization; Endostatin can remarkably inhibit the growth of the C₆ gliomas by inhibiting the glioma angiogenesis. These findings can support the clinical antiangiogenic gene therapy approach for gliomas with experiment testimonies.