Based On RNAi To Study The Effect Of TGF-β Signaling Pathways Caused By Sodium Fluoride

[Objective] This paper cultivated mice testis sertoli cells in vitro, building siRNA inhibition of sertoli cells TGF-β lmodel. Dying fluorine to detect the expression of related genes and proteins, studied the effects of TGF-β signaling pathways in fluorine impact the regulating mechanism in the cellular immunity function.[Method] Builded RNAi inhibiting the sertoli cells TGF-β1 model, using the qRT-PCR and Western blot detected respectively after transfection of TGF-p 1 mRNA and proteins. Then dyed fluorine, applying qRT-PCR detected the expression of TGF-β1, Smad2 and Smad4, Smad6 and Smad7mRNA, and ELISA detected sertoli cells secrete immune related proteins interleukin-6, and tumor necrosis factor alpha respectively.[Results] (1) The identification of sertoli cells after culture:Bying HE dyeing, and Giemsa staining observating the morphology and growth of sertoli cells. For cell count found 90% purity, and cell activity was good.They can be used for model establishment.(2) The best transfection concentration screening of siRNA:Utilized different concentrations (20,30, 50,100 nM) of fluorescent FAM-siRNA, according to green fluorescent strength screened the best transfection of siRNA concentrations, determined the optimal transfection of 50 nM of siRNA concentrations.(3) The screening of effective si-TGF-β1jamming sequence:three kinds of si-TGF-β1 by the concentration of 50 nM transfection, detected the quantity of TGF-β1 mRNA and protein expression. Compared with the control group, the results showed that the expression of mice testis SCs TGF-β1 in siRNA1 (si-TGF-β1-1) group and the siRNA2 (si-TGF-β1-2) group are significantly decreased, which siRNA1 group difference was significant.(4) qRT-PCR results:Compared with the control group, the expression of TGF-β1, Smad2 and Smad4, Smad6 and Smad7 mRNA in experimental group were no statistical significance, but showed a reduced tendency.(5) ELISA:Compared with the control group, the result showed that expression level of IL-6 protein in 10-4 M (mol/L) NaF and 10-3M(mol/L) NaF two groups was significant difference (**P<0.01); Compared with the control group, the expression level of TNF-α protein shows the tendency of decrease in every group, but the difference was not significant.[Conclusion] TGF-β signaling pathways play a regulatory role in fluorine impacting the immunity function of SCs, which may be through the TGF-β super family members (except TGF-β1), Smad proteins family and immunity related proteins IL-6 and TNF-α.