| Objective Glioma is the most common malignant tumors of the central nervous system,the tumor showed invasive growth,whether surgery or other treatment,are unable to completely cure glioma,even combined radiotherapy and chemotherapy after surgery,the tumor will relapse,lead to poor prognosis.Therefore,it become a new and important research topic to search for new therapies that can effectively kill glioma cells.In recent years,glioma stem cells has important significance for treatment of glioma,because glioma stem cells has the characteristic of self-renewal and differentiation potential,which is the root of brain glioma recurrent after surgery and chemotherapy resistance.Therefore,glioma stem cells have become potential targets for the treatment of gliomas.Although temozolomide has been demonstrated to have anti-tumor effect,but some patients have drug resistance,so the treatment effect is not so good,it cannot prevent tumor recurrence and further growth,so there will be a new way to treat glioma treatment.In this experiment we obtained specimen from patients with fresh glioma tissue,isolated and using culture methods of stem cells to culture glioma stem cells from fresh specimens,and studied the feasibility of establishing the animal model of glioma invasion.Oncolytic virus provided a new treatment method for the treatment of glioma.Our experiments in vitro had confirmed the recombinant vesicular stomatitis virus(VSV△M51)and double-deletion vaccinia virus(vvDD)can infect and kill glioma cell lines,animal experiments also confirmed that the animal can prolong the survival time of the model.In this experiment,we further confirmed that recombinant vesicular stomatitis virus(VSV△M51)and vaccinia virus(vvDD)is effective in the treatment of glioma stem cells in vitro.Methods Glioma stem cells were cultured from 12 surgically resected fresh brain glioma tissues.The proliferation and differentiation of the glioma stem cells that formed the cell spheres were examined and the surface markers of the glioma stem cells forming the cell spheres were detected,and their stem cell attributes were determined.Enhanced green fluorescent protein(EGFR)was transfected into glioma stem cells by retroviral transfection.An animal model of tumor growth was made by using the stem cells transfected with enhanced green fluorescent protein(GFP),and an animal model of glioma invasion was established.Then we examined the sensitivity of cultured glioma stem cells to temozolomide in vitro.After that we used different doses of recombinant vesicular stomatitis virus(VSV△M51)and vaccinia virus(vvDD)to infect these glioma stem cells,especially on temozolomide resistant stem cells,observing the killing effect on stem cells in vitro.Results Glioma stem cell spheres could be successfully cultured in 9 of 12 surgically resected glioma specimens.These cultured stem cells of glioma have the ability of self proliferation and differentiation.All glioma stem cell spheres expressed specific markers of neural stem cell nestin(Nestin),and 7 of 9 cases expressed neural stem cell marker CD 133.The glioma stem cell spheres can form adherent glioma cells after differentiation,which could express markers of mature nerve cells:the human glial fibrillary acidic protein(GFAP)and the anti human microtubule associated protein-2(MAP2).Glioma stem cells could be transfected with enhanced green fluorescent protein(EGFR).After transfection of EGFR,glioma stem cells formed gliomas in intracranial areas of immunodeficient mice,similar to those of human gliomas,with invasive growth characteristics.VSVAM51 and vvDD oncolytic virus could infect on glioma temozolomide resistant stem cells,and cause cytopathic effect and destruction of glioma stem cell self-renewal capacity,and effectively kill the glioma stem cells and differentiated cells(glioma cells).Conclusion Glioma stem cells can be successfully cultured with neural stem cell cultures.These stem cells had self-renewal,multilineage differentiation and tumor formation ability.The transfected glioma stem cells could form tumors in animal models and the formed tumors have the characteristics of invasive growth similar to those of human gliomas,therefore,it can be a better representation of human gliomas and will have a very wide range of applications in the future study of glioma treatment.The experiment confirmed that oncolytic virus VSV△M51 and vvDD can successfully infect and effectively kill the temozolomide resistant glioma stem cells,which may provide a new method for the treatment of glioma patients with temozolomide resistant. |
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