Septin2 Rat Mesangial Cell Proliferation

Objective:To research the in vitro effects of Septin2 on Mesangial Proliferation in Rats.Methods:Cultured primary rat mesangial cells, processing the following experiment:1)The plasmids pEGFP-C 1 which could express the green fluorescent protein (GFP)were transfected into the cells. The transfection efficiency were determined by observing under fluorescent microscope 24 hours subsequently.2)Transfection of Septin2 plasmids by electroporation: Groups dividing:①pRK5 transfection group;②pRK5-Septin2 group. The plasmids pRK5 and pRK5-Setin2 were transfected into the mesangial cell under the optimal transfection conditions. The expression of Septin2 protein were examined by Western blot, cell cycle profiles were determined by flow cytometric analysis, the expressions of cell cycle regulatory proteins (cyclin D1,cyclin E,p21) were examined by Western blot.3) Design and synthesis the siRNA of GAPDH, according to different concentrations cells were divided into 4 groups:①siCon group,②200 nmol/L group,③400 nmol/L,④800 nmol/L group.48h later, Western blot to detect the expression of GAPDH protein.4) Electransfection of siSeptin2:Groups dividing:①siCon group,②siSeptin2 group. Western-blot detects the expression of Septin2 protein, flow cytometric analysis checks the cell cycle profiles.Results:1)When plasmids is 80μg, voltage is 340V, capacitor is 550μF, the electroporation transfection efficiency in primary rat mesangial cells can reach more than 50%.2)Compared to pRK5 transfection group, plasmids pRK5-Septin2 increased protein expression level of Septin2(P<0.05). Cell cycle analysis showed that the percentage of the cells in S stage were markedly reduced in pRK5-Septin2 group (P<0.05), the percentage of the cells in G0/G1 stage were markedly increased (P<0.05). The expression of cell cycle regulatory protein cyclin D1, cyclinE decreased and P21 reduced(P<0.05).3)When the concentration of siGAPDH was 800nmol/L, the inhibitory effect of protein level was the most significant (P<0.05).4) The synthesized siRNA of Septin2 gene inhibited the Septin2 protein expression levels (P<0.05), flow cytometry analysis didn't show any difference of significance.Conclusion:Overexpression of Septin2 is able to arrest cell cycle progression in the G0/G1 phase,the expression of positive regulators driving the G0/G1 phase, for example cyclin D1,cyclin E, were downregulated in cells transfected with Septin2, whereas expression of negative regulators p21 were upregulated. This is suggested that Septin2 may inhibite proliferation of mesangial cell by regulating the expression of cell cycle proteins. This may have provided a new target of mesangial proliferative glomerulonephritis therapy.