Establishment Of The Rat Glioma Model By C6 Glioma Cells With EGFP Gene And Effects Of Hyaluronic Acid On Proliferation And Invasion Of Rat Glioma

Glioma is the most frequent malignant tumor in central nervous system. Although many kinds of therapeutic methods, including operation, radiotherapy, chemotherapy and genetherapy, are exploited to treat this tumor, the prognosis of glioma patients are still poor. The reason for the poor prognosis may be that glioma usually invades the surrounding brain and is easy to recur. Nowadays, the study on the mechanism of invasion of glioma in vivo has been paid more emphasis. Excellent research works on invasion of glioma are based on the successful experimental model. Green fluorescent protein, one stable reporter molecular with low molecular weight, has been used to localize functional protein and trace interesting cells in several kinds of cells.The invasion of glioma is a multi-step process involving interaction between tumor cells and brain ECM. HA, one kind of main components in central nervous system, may be involved in the invasion and metastasis of glioma cells. The specific receptors of HA, such as CD44 and BEHAB, are highly expressed in invasive glioma cells, which may suggest that HA is able to induce in vivo invasion of glioma cells. The direct proof that HA induces glioma invasion is still absent. The study on the effects of HA on the proliferation and invasion of glioma may help detect the molecular mechanism of HA on theglioma.Firstly, EGFP gene was transfected into C6 glioma cells in vitro and stable transfecants were selected. The cells transfected with interesting genes and empty vector were respectively named as C6-E-N and C6-N cells. By flowcytometry, electron microscope and MTT method, the effects of EGFP gene on the cell cycle, ultraultrastructure and proliferative rates of C6 cells were detected. The expression of EGFP gene was detected under fluorescent microscoope. The changes of tumorgenesis ability of transfected cells were determined by tumorgenesis test in nude mice.Secondly, stable transfectants were stereotactically implanted into caput nuclei caudati of SD rats to establish transplanted brain glioma. The survival duration of rats and the growth of transplanted rumor were monitored. The transplanted tumor sections were made and stained respectively with HE and immunohistochemistry staining. The invasion of glioma cells were also detedted under fluorescent microscope. The transplanted tumor was primarily cultured to determine the storage of exotic gene in vivo.Thirdly, the effects of HA on the proliferation and invasion of glioma were determined by the transplanted rumor established as the above.The results showed that stable C6 cells which expressed EGFP gene were obtained by selection and named as C6-E-N cells. There were not obvious differences in cell cycle, ulrtastructure and proliferative rates between C6-E-N and C6 cells. The transplanted glioma model were successfully established by using C6-E-N cells, and no evident differences in tumorgenesis rate, growth rate and living duration were found compared with control group. In vivo EGFP gene loss was not detected. The fluorescence of EGFP could visualize the invasive glioma cells, which is superior to the HE and immunhistochemical staining. The HA with higher concentration than lOmg/ml could promote the proliferation and invasion of glioma. Our study will helps to discover the molecular mechanism of glioma invasion and probe into new anti-invasion treatment method.