Experimental Research Of Gene Therapy Treats Glioma Using Neural Stem Cells For Vectors

One of the impediments to the treatment of brain tumors (e.g., gliomas) has been the degree to which they expand, infiltrate surrounding tissue, and migrate widely into normal brain, usually rendering them "elusive" to effective resection, irradiation, chemotherapy, or gene therapy. Whether or no can effectively treat expanding, infiltrating tumor cells is key point of all therapy methods successfully. Neural stem cells (NSCs) are one focus of neuroscience studies currently. NSCs used gene therapy vector have many advantages, e.g., abundant sources, can be engineered easy, can carry multi-exogenous gene and stably express a exogenous gene in vitro and in vivo,and so on. When NSCs were implanted into experimental adult rats intracranial, NSCs can distribute themselves quickly and integration themselves to host cells, while continuing to stably express a exogenous gene. So NSCs is an ideal vector of gene therapy. NSCs can be applicationed in the gene therapy of intracranial tumors. There are many investigations about the gene therapy of intracranial tumors. And some researchs have gained progress greatly. But there also are some problems that needs to be solved. For instance, ① Pathology mechanism of gliomas has not be understanded. Specific target genes that gene therapy treats gliomas are deficiency.② Gliomas cellsexistence heterology. Transfection single gene can no treat all kind tumor cells. (3) Heredity behavior of tumor cells is not stable.Tumor cells easy develop resistance to gene therapy. (4) Transfection gene vector systems emploied existence deficiency in transfection efficiency, orientation , quantity of carning target therapeutic genes , manipulation of target therapeutic genes, immunogenicity and reliability at present.⑤ Existing concrete technic difficulties in transfection gene therapy. To aim directly at these problems and flaws, we have doed some investigation and observation with NSCs origin from Wistar foetus rat cerebral cortex and subcortex tissues.Firstly To establish the methods of harvest and culture of NSCs and to observe the characteristics of proliferation and induce differentiation of NSCs. Wistar foetus rat cerebral cortex and subcortex tissues were dissociated mechanically and enzymatically. The suspension culture technique was performed to acquire cell clones. Immunocytochemical staining was used to identify the NSCs. We have found that cell clones could be acquired from Wistar foetus rat cerebral cortex and subcortex tissues after primary and passage culture. There clones were nestin-positive and possessed the ability of proliferation and self renewal.The differentiatied cells express neurons , astrocytes and oligodendrocytes specific characteristics.Secondly To establish a cryopreservation method for NSCs from Wistar foetus rats and to study of viability and biological properties of these cells after cryopreservation.NSCs in 10% BSA+8% DMSO were stored in liquid nitrogen. Cells culture and immunocytochemical staining were used toidentify the viability, morphology, differentiation and specific antigen expression of NSCs after cryopreservation. We have found that different time of cryopreservation , different passages do not affect NSCs survival(P>0.05) after cryopreservation.And NSCs could survive and expansion in vitro for many passages.Thirdly To transfection NSCs using enhanced green fluorescent protein (EGFP) expression vector pIRES2-EGFP, to detect transfection efficiency and expression of this eukaryon cells expression vector and offer experiment datas about NSCs as a delivery vehicle for target therapeutic genes, that gene therapy treats the diseases of central nervous system.In vitro to amplification, enzyme cutting and evaluation pIRES2-EGFP plasmid. Suspension method cultures NSCs.That Lipofectamine 2000 liposomes conduct pIRES2-EGFP plasmids transfection NSCs. G418 screens transfectioned NSCs. Fluorescence microscope observes transfection efficiency and expression. Immunocytochemical evaluation NSCs transfectioned EGFP genes. We found that after transfectioning 6 hour, fluorescence proteins expressioned by chance; after transfectioning 24 hour, fluorescence proteins phanero-expressioned; 48 hour, fluorescence proteins expressioned at summit; after one month, resistance cells have developed great neurosphere.Fourthly To observe the NSCs migration behavior and expression of exogenous gene in vitro, while gliomas cells is present. To investigation the NSCs migration behavior and expression of exogenous gene, while NSCs implanted into experimental intracranial gliomas in adult rat. To harvest, culture NSCs.That Lipofectamine 2000 liposomes conduct pIRES2-EGFPplasmids transfection NSCs. BrdU labels NSCs. Co- cultures glioma cells and NSCs to observe NSCs migration behavior in vitro. Inoculation of established intracranial gliomas with NSCs transfectioned by EGFP gene and labeled by BrdU to observe NSCs migration behavior and expression of exogenous gene in vivo. We demonstrate that neural stem cells, when implanted into experimental intracranial gliomas in adult rat, distribute themselves quickly and extensively throughout the tumor bed and migrate uniquely in juxtaposition to widely expanding and aggressively advancing tumor cells, while continuing to stably express a foreign gene.Conclusion ① The NSCs isolated and cultured by using the present methods have the ability of self — renewal and differentiation .② Cryopreservation and resuscitation have not changed biological properties of NSCs, including their morphology and capability for expansion and differentiation. ③ To transfection NSCs using pIRES2-EGFP plasmids conducted by lipofectamine 2000 liposomes is a simple, efficient, easy success transfection method. And is a fairly ideal transfection gene method. (4) NSCs display extensive tropism for gliomas cells, while continuing to stably express a foreign gene in vivo and in vitro.