Primariry Research On SMAD Protein In Transforming Growth Factor Beta Signaling

PART I SMAD EXPRESSION IN MURINE LUNG CELLS AFTER HDM AIRWAY EXPOSUREObjective: To explore whether there's difference in Smad expression of lung cells in mice of different genetic background after HDM airway exposure.Methods: A/J and C3H mice were treated i.t with HDM.Sixteen hours after the last airway challenge,mice were sacrificed and lung tissue were taken for homogenization.Nuclear protein after homogenization were extracted and then western blot were carried out to detect Smad2/3 and Smad6 expression.Results: The difference was significant in Smad protein expression after 16 hours of HDM exprosure in A/J and C3H murine lung.A/J mice expressed higher level of Smad,while in C3H Smad6 level was less.While Smad2/3 expression was opposite.In A/J,Smad2/3 level was less than in C3H. Conclusions: After HDM exposure,Smad protein expression in different genetic background mice is different which needs more rerearch works to testify its role in TGF-βsignaling transduction.PART II RNA INTERFERENCE ON MURINE BMDC SMAD6 GENEObjective: To design siRNA sequence according to CDS area of target gene of Smad6, to construct expression vector, and to explore Smad6 gene and protein expression in murine BMDC after infection with this lentiviral vector.Methods: Three siRNA(siRNA1,siRNA2,siRNA3)sequences were designed according to CDS area of target gene of Smad6.Expression vector,pSIH1-H1-copGFP shRNA vector,were construct and extraction of plasmid DNA de-endotoxin were carried out.And then the vector constructed before were used to transfect L929 cell lines.Thereafter,Smad6 gene expression in BMDC were dected by real time PCR.After that,lentivirus pakage system were used to pack and produce lentiviral vector.At last,primary BMDC of C3H/HeJ were got to define best MOI level and infection condition.After the lentiviral vector infection of BMDC,RT-PCR and western blot were carried out to detect gene and protein expression of Smad6.Results: Results from RT-PCR showed that,siRNA1 corresponding to Smad6 had the best effect on gene silence with a effect of silence around 67%.But considering the effect of transfection was only 75%,the real efficiency of gene silence may reach 90%(mRNA) and the best MOI was 20.Results from RT-PCR showed that,infection of BMDC by lentiviral vector,siRNA1 showed an efficiency of gene silence around 85%.And Smad6 protein was inhibited significantly in murine BMDC in comparing to control group.Conclusions: The siRNA designed corresponding to Smad6 CDS area and the lentiviral vector following constructed showed a significant and effective inhibition on gene and protein expression of Smad6 which laid the first stone for our future research work.