Fluoride Impact TGF-β Signaling Pathways Based On Overexpression Technology

[Objective] In order to study the mechanism of testicular sertoli cell immune privilege fuction, we transfect TGF-β1 gene overexpression vector into mouse testis sertoli cells which exposed to fluoride. This study want to provide theoretical foundation for the toxicology of fluoride on reproduction system.[Method] In this study, we use varies of methods to prove that fluorine influnce testis sertoli cell immune privilege faction. Firstly, we set different groups with vary dose of fluoride to simulate fluoride influnce testis sertoli cell in vivo. Secondly, we use many methods to prove the fluoride affects smad2、 smad3、smad4、smad6、smad7 which is the very important proteins in TGF-β system. The methods like double enzyme digestion; agarose gel recycling; Real-time-PCR; western blotting; enzyme-linked immunosorbent assay（ELISA）.[Result]（1） Electropheresis and sequencing results show that:pcDNA3.1 （+）-TGF-β1 overexpression vector had successfully constructed.（2） Real time-PCR and Western blotting results show that:pcDNA3.1 （+）-TGF-β1 overexpression vector had successfully transfected into mouse testis sertoli cells.（3） Real time-PCR method proves that:Compared with the control group, the level of smad2、smad4 mRNA expression showed no significant difference. However, when compared with the control group and the 10^-3mM、10^-5mM NaF group, smad3 in 10^-4mM NaFgroup had remarkably reduced; when compared with the control group and the 10^-5mM NaF group, smad6 in 10^-3mN、10^-4mM NaF group had remarkably reduced; when compared with the control group, smad7 in 10^-3mM、10^-4mM、10^-5mM NaF group had remarkably reduced.（4） Enzyme-linked immunosorbent assay（ELISA） shows that:under the two-factor TGF-β1 overexpression and vary dose fluoride, when compared with the control group, IL-1β、 IL-6 and TNF-a factors all had significant raise in the highest fluorine concentration. Among them, IL-1β and TNF-a in 10^-3mM NaF group had remarkably change; IL-6 in 10^-3mN、10^-4mM NaF group had remarkably change.[Conclusion] Fluoride damaged testis sertoli cell immune privilege fuction through influnce TGF-β1 expression. It can cause reproductive toxicity.