Research On Expression And Effect Of Toll Receptor 4 On Brain Glial Cells

Objective: To observe the expression profile of TLR4 and explore its influence on biological behavior of glial cells.Methods:1 The expression profile of TLR4 m RNA was assessed in U251-MG cell line by Reverse Transcription-Polymerase Chain Reaction.2 The expression of TLR4 protein level was detected by Western Blot in U251-MG cell line.3 TLR4 si RNA recombinant plasmid was established and transfected into U251-MG cell line. The expression profile of TLR4 m RNA was assessed in TLR4-downregulated U251-MG cell line by Reverse TranscriptionPolymerase Chain Reaction.4 The expression profile of TLR4 protein was detected in TLR4-downregulated U251-MG cell line by Western Blot.5 The impact on glial cell cycle before and after TLR4 knockdown was analyzed by flow cytometry method.6 MTT method was used to analyze proliferation activity of glial cells after TLR4 down-regulation.7 The contents changes of IL-6, IL-10 and VEGF in culture supernatant of glial cells were analyzed when TLR4 was down-regulated.Results:1 RT-PCR analysis showed that TLR4 m RNA was expressed in U251-MG cell line.2 TLR4 protein was observed in U251-MG cell line according to Western Blot assessment.3 According to RT-PCR analysis, the level of TLR4 m RNA in TLR4 si RNA-transfected cells was significantly down-regulated(0.138±0.0004) relative to non-transfected(1.034±0.0006) and null vector-transfected group(1.003±0.0005)(P<0.01). There was no significant difference between non-transfected and null vector-transfected group(P>0.05).4 According to Western Blot assay, the level of TLR4 protein in TLR4 si RNA-transfected cells was significantly down-regulated(0.200±0.0042) relative to non-transfected(1.007±0.0029) and null vector-transfected group(0.900±0.0062)(P<0.05). There was no significant difference between non-transfected and null vector-transfected group(P>0.05).5 Flow cytometry method showed that the proportion of cells decreased in phase G0/G1 and increased in phase S in TLR4 si RNA-transfected, non-transfected and vector-transfected group in the 48 hours’ stimulation of LPS with the concentration of 10μg/ml; in the context of LPS, the proportion of cells was relatively decreased in phase S and increased in phase G0/G1 relativelly in TLR4-downreglated group compared with untransfected and null vector- transfected group(P<0.05).6 After stimulation of LPS with the concentration of 10μg/ml, the proliferation rate of TLR4-downreglated group was relatively lower compared to non-transfected and null vector-transfected group(P<0.05).7 The contents of IL-6, IL-10 and VEGF of TLR4 si RNA-transfected cells was relatively lower than non-transfected and null vector-transfected group in the 12 hours’ stimulation of LPS with the concentration of 10μg/ml(P<0.05).Conclusion:TLR4 was expressed in glial cells U251-MG, TLR4-downregulation can inhibit cell proliferation and cytokine secretion stimulated by LPS.