To First Engineer HEK293 Cell-line To Stably Overexpress HGRβ, And Then Explore The Effect Of HGRβ Overexpression In Their Sensitivity To LPS And Dexamethasone

General objective:To provide new insights into the biologic functions of the human glucocorticoid receptor beta(hGRβ) isoform,during inflammation.Specific objectives:To construct hGRβrecombinant HEK293 cells(hGRβ-HEK293 cells) exogenously overexpressing hGRβ;to progressively assay cellular biologic properties of these recombinant cells in response to both inflammatory and anti-inflammatory reactions,following treatment with lipopolysaccharide(LPS) and Dexamethasone respectively.Methodology:The hGRβ-HEK293 cells were constructed by infecting HEK293 cell-line with hGRβrecombinant retroviruses.PCR and semi-quantitative RT-PCR techniques were then employed to respectively identify the integration and expression of hGRβgene in the hGRβ-HEK293 cells.Finally,MTT and flow cytometry techniques were applied to study cellular properties of these recombinant cells in response to an inflammatory reaction triggered by LPS,and anti-inflammatory action of Dexamethasone.Results:PCR amplifications of neo and hGRβgene fragments indicated successful integration of the hGRβgene in the hGRβ-HEK293 cells;semi-quantitative RT-PCR showed apparently higher quantities of hGRβmRNA in the hGRβ-HEK293 cells than in HEK293 cells,the normal control(P＜0.05).MTT assay detected statistical difference in proliferation in response to LPS stimulation,between the hGRβ-HEK293 cells and the normal control in which the former showed comparatively higher proliferation rates.Finally,the MTT and flow cytometry analyses demonstrated that the hGRβ-HEK293 cells were less sensitive to Dexamethesone induced inflammatory proliferation repression(antiinflammation) as compared to the normal control(P＜0.05).Conclusions:The hGRβ-HEK293 cells over-expressing hGRβisoform,were successfully constructed as identified by PCR and semi quantitative PCR techniques; over-expression of the hGRβisoform increased sensitivity of the hGRβ-HEK293 cells to LPS triggered inflammatory proliferation and decreased their sensitivity to antiinflammatory action of Dexamethasone.