ABSTRACT
Aim To investigate the influence of endostatin on pathological morphology of C_6 glioma and its molecular pharmacologic mechanism.Methods The C_6 cells,the C_6 clone stably transfected with endostatin cDNA(endo-C_6),which the endostatin with biological activities can be secreted from,and the C_6 clone stably transfected with empty vector pBudCE4.1 cDNA(pBud-C_6) were injected subcutaneous in nude mice to establish different tumoral model respectively.The morphologies of these tumoral tissues were observed and compared to each other under light and electron microscope.The expression of VEGF in tumor tissue was determined by ELISA.Results The expression of VEGF in endo-C_6 glioma(endo-C_6G) tissues was lower than that in C_6 glioma(C_6G) and pBud-C_6 glioma(pBud-C_6G).Moreover,endo-C_6G tissue was characteristic of a mimetic envelope,no intratumor bleeding and cystis degeneration,apoptosis of tumor cells,and edema in and around tumor.Rarefied vessels were found in tumor,and no vessel like structure formed by tumor cells was observed.The large irregular necrosis focus was showed in tumor,but mild vascular reaction around necrosis focus and peritumor,rare surrounding invasion.The basal lamina was discontinued.The basemembrane(BM) was loose.Few vesicular vacuolar organelle(VVO) structures were observed in plasma of endothelial cells.In C_6G and pBud-C_6G,tumor lesions demonstrated significant vascular reaction,intratumor bleeding,necrosis,edema in and around tumor,and surrounding infiltration.Vessel like structure formed by tumor cells was also observed.When examined with electron microscope,plenty of VVO structures were observed in plasma of endothelial cells,the worse the edema,the more the VVO were,which coincided with the expression of VEGF.Mostly,loose basal lamina surrounded by small amounts of collagen fibers was multilayer and integrated and continuous.No correlation between gene transfection and fenestra formation or cleft of capillary endothelial cell was observed,no apoptosis of endothelial cells were found.Conclusion In glioma,the apoptosis of endothelial cells tissue was not induced directly by endostatin,but the angiogenesis and vascular reaction can be inhibit by endostatin by down-regulation of the expression of VEGF in C_6 glioma cells.
ABSTRACT
Aim To construct eukarytic vector for rat endostatin(endos) cDNA and observe its expression in C6 cell.Methods cDNA encoding rat endostatin was amplified from newborn brain tissue with RT-PCR and inserted into the eukarytic vector pBudCE 4.1.Recombinant was identified with KpnI,XhoI double digestion,PCR and nucleotide sequencing of the target gene.After successful reconstruction of the genes of endostatin,the recombinants was transfected into C6 cells with lipofectintechniques.The positive clones were screened out through zeocin resistance test.The endostatin in supernate of the positive clones was identified with Western-blot and MTT method.With immunocytochemistry,the endostatin in the positive clones was located.The quantities of VEGF in supernate of the positive clones were quantified with ELISA assay.Results The size of the amplified endostatin gene fragment was in accord with that we expected.And the gene sequence inserted into the eukarytic vector pBudCE 4.1 was consistent with the known sequence.Endostatin was secreted from the positive clone.Down-requlation of vascular endothelial growth factor(VEGF) was found in the positive clones.Conclusion The recombinant of rat endostatin gene clone had been established and inserted into the eukarytic vector pBudCE 4.1 successfully and endostatin was expressed in C6 cells.This provides a basis for further studies of endostatin effects in vivo,and creates the conditions for final clinical trial