The Study For The Role Of TrkA Gene In The Proliferation-differentiation And The Angiogenesis Of Human Neuroblastoma

(1) To investigate the feasibility of inhibiting proliferation and inducing differentiation in human neuroblastoma ( NB ) with TrkA gene. ( 2 ) To investigate the role of TrkA gene inhibiting the synthesis and secretion of vascular endotheli-al growth factor ( VEGF) in human neuroblastoma cell. ( 3 ) To investigate the feasibility of gene therapy for human neuroblastoma with TrkA gene inhibiting angionenesis.Methods(1) Recombined TrkA cDNA plasmid was transfected into human neuroblastoma cells(SH -SY5Y cell line) by lipofect method. (2) The expression of TrkA gene of cells before and after transfection was detected by RT - PCR. (3) Comparing the proliferation rate and differentiation extent of parent SH - SY5Y cells with TrkA - transfectant SH - SY5 Y cells by Trypan blue exclusion experiment and phase -control microscope. (4) The expression of VEGFmRNA and VEGF protein in parental SY5Y NB cell line and SY5Y -TrkA NB cell line was detected by RT - PCR and immuno - cell - chemistry. (5 ) The quantity of VEGF in cell culture supernatants was determined by ELISA assay. (6) The tu-morigenesis of cells before and after transfection and the tumor volume was compared. The angiogenesis of tumors in nude mices was determined and analysedby RT-PCR and immunohistochemistry and microvessel density ( MVD) counting.Results(1) TrkA gene was successfully transfected into SH - SY5 Y cells and can stably expressed. (2) The capability of proliferation of the transfected cells was greatly reduced (p <0. 001) , the differentiation extent of the transfected cells was increased( p <0. 001). (3) The expression of VEGF mRNA( p <0. 01) and VEGF protein ( p < 0.001) was greatly decreased because of the high - level expression of TrkA gene in SY5Y - TrkA ceUs. (4) The quantity of VEGF in cell culture supernatants of SYS Y - TrkA cells was much lower than that of parent SY5Y( p<0.001). (5) The capability of tumorigenesis and the tumor volume of the SY5Y - TrkAcells in nude mices was greatly reduced ( p < 0. 01). (6) The distinction of VEGF expression and MVD between experiment group and control group is significant (p <0.01).Conclusion(1) The proliferation of neuroblastoma are effectively inhibited by transfect-ing TrkA gene and the SY5Y - TrkA cells can produce fine differentiation. (2) The synthesis and secretion of VEGF in human neuroblastoma cells can be effectively inhibited by high - level expression of TrkA gene. The angiogenesis and tumor growth of human neuroblastoma can also be effectively inhibited by TrkA gene. This experiment provides a theoretical basis for neuroblastoma angiostatic gene therapy.