| Background: Glioblastoma(GB),a common intracranial malignant tumor,is a central nervous system disease.It is classified into four grades according to the World Health Organization standard,in which high-grade glioma is difficult to completely cure due to its high malignancy,metastasis ability and recurrence rate.Generally it takes less than 18 months from the initiation of treatment to the death of patients.Therapeutic methods currently available include surgical extraction,radiotherapy,chemotherapy,and the combination of these approaches.Although effective drugs are applied in clinical practice,the toxicity and side effects of some drugs increase the burden to patients.Therefore,it is an urgent medical problem to find safe and effective therapeutic drugs without toxic side effects.Resveratrol(Res),a kind of natural polyphenolic drug found in plant stems,leaves,and fruits,has cardiovascular protection and broad-spectrum antibacterial effects and functions as an antioxidant and antitumor agent that can pass the blood-brain barrier without toxic side effects.Astrocytes(AS)are structural cells which massively reside in the brain and play a role in supporting and protecting neurons.In the microenvironment of the tumor,growth factor and cytokines are released by AS around glioma to influence the growth of the rumor.In the present study,it was designed to investigate the interaction between the glioma cell line RG2 and AS and the effect of Res on the two types of cells,and to explore the effect of tumor microenvironment on RG2 cells.Methods:(1)Establishment of the co-culture system: cortical cells were removed from neonatal SD rats(within 24 hours)and cultured.After purified by passage,the cortical cells were co-cultured with glioma RG2 cells and then treatedwith 80?M Res for 48 hours.(2)Numbers of AS and RG2 cells were measured with a cell counter to detect the proliferation of the two cells.(3)By using the scratch test,mechanical damage was applied to the adherent cells to detect the migration ability of RG2 cells.(4)Transcriptional changes of GFAP and STAT3 in AS as well as that of the MMP2,MMP7,and CyclinD1 in RG2 cells were determined by qRT-PCR.(5)Cell morphology and the expression of GFAP were observed by immunohistochemical staining and immunofluorescence staining.(6)Expressions of GFAP and STAT3 in AS were detected by Western blot.Results:(1)Cell numbers of the AS and RG2 cells were increased after the co-culture.(2)qRT-PCR results showed that the expressions of GFAP and STAT3 in co-cultured AS were increased compared with the AS that was cultured alone,and the relative expressions of MMP2,MMP7 and CyclinD1 in the co-cultured RG2 cells were increased compared with the ones without co-culture.(3)Cell numbers of the AS and RG2 cells in the co-culture system were significantly decreased after treatment with Res.(4)Results of immunohistochemical staining demonstrated that GFAP was expressed in the AS around the tumor.Immunofluorescence showed that the number of AS and the expression of GFAP in AS were increased after co-culture,and both of them were decreased after the addition of Res.(5)Results of qRT-PCR analysis showed that the relative expressions of GFAP and STAT3 in the AS were decreased 48 hours after Res treatment,and the relative expressions of MMP2,MMP7 and CyclinD1 were decreased in the RG2 cells.(6)Western blot revealed that the expressions of GFAP and STAT3 proteins were decreased in co-cultured AS after Res treatment.Conclusions:(1)The proliferation and invasion of glioma RG2 cells are enhanced by AS in vitro,which is reflected in the increased cell number and the upregulated expressions of MMP2,MMP7 and CyclinD1.(2)RG2 cells activate the surrounding AS in the co-culture system,which is shown in the increased cell number and GFAP expression.(3)Res inhibits both the proliferation of glioma RG2 cells and the activation of surrounding AS.(4)The modified co-culture system we used can better simulate the internal environment compared with other co-culture studies. |
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