Experimental Research Of The Function Of CYP1A1 Gene In Pathogenesis Of Mycoplasmal Pneumonia Of Swine

Mycoplasma pneumonia of swine is a chronic-touching respiratory disease caused by Mycoplasma pneumoniae.Cytochrome P450 are a class of high-heme-containing proteases,which are mainly involved in the catalytic oxidation of endogenous substances and exogenous substances,the oxidative synthesis of steroid substances and the oxidative metabolism of drugs in vivo.CYP1A1 is one of CYP1 subfamily,which is the main enzyme that have functions in the metabolism and activation of procarcinogens,including alkyl aromatics and aromatic amines.The latest research shows that cytochrome P450s also play a role in the inflammatory response,the subject of our preliminary studies indicate that CYP1A1 gene expression and mycoplasma pneumonia of swine correlated.Objective:To explore the CYP1A1 gene's function in the process of inflammatory reaction caused by Mycoplasma pneumoniae of swine by establishing the porcine alveolar model with different expression level of CYP1A1 gene and constructing mycoplasma pneumonia infection model in vitro.Methods:Amplificated the CYP1A1 gene with RT-PCR,connected the fragment with pMD18-T vector after purified to cloned recombinant plasmid,The PMD18-T-CYP1A1 was verified by DNA sequencing.The PMD18-T-CYP1A1 recombinant plasmid and pcDNA3.1(+)/zeo vevtor plasmid was respectively digested with Notl and Xhol,ligated the fragment and pcDNA3.1(+)/zeo vevtor with T4 DNA ligase enzyme to obtain the pcDNA3.1(+)/zeo-CYP1A1 plasmid after verified.Amplificate pcDNA3.1(+)/zeo-CYPIAI by transforming into the competent cells.The plasmid was transfected with Lipofectamine 2000 into porcine alveolar macrophage cell line 3D4/21.After screened with Zeocin for 28 days The CYP1A1-overexpressing cells were left.Detected the expressive level of CYP1A1 in cells by means of RT-PCR and Western-blot;in adition,transfected the siRNA into cells to obtain the macrophage cell lines model of CYP1A1 inhibited.Study the expression of IL-1??TL-6?IL-8?TNF-??PPAR-? among each group after mycoplasma hyopneuminiae stimulation at different time.Results:1.Successfully cloned the sequence of CYP1A1 in swine,NCBI sequence blast result shows that the sequence is the target gene sequence we need.Successfully constructed the eukaryotic expression vector and the digestion result identified it.2.To determine the optimal concentration of Zeocin.PAMs were screened with zeocin of five gradient of concentrations.The group of 300 300?g/ml were dying in one week;the group of 250?g/ml were dying on the 14th days;other groups survived to the third week.3.The cell transfection experiments were performed in 6-well plates,Stable resistant clones were observed on the 28th days since the cells were selected by Zeocin,We got the best transfection effects in the condition of the dose of Lipofectamine 2000 is 3.75ul/hole and the amount of recombinant plasmid is 2.5ug/hole.4.To obtain the cells model of CYP1A1-innhibited PAMs,a group of PAMs were interfered by siRNA.The result of Western-blot indicate that the expression level of CYP1A1 in CYP1A1-inhibited PAMs is lower than normal PAM.5.Successfully detected the p3 6 gene of Mycoplasma pneumonia by means of PCR diagnostic.6.With the time going on after stimulation,The expression levels of TNF-??IL-1??IL-6?IL-8 of all treatment groups were increasing in different degree.The expression of TNF-??IL-1??IL-6?IL-8 in overexpression group were significant higher occurred at 4h?24h?12h?4h;the control group and negative control group were occurred at 4h?12h?4h?4h.while the Si group were occurred at 4h?4h?12h?4h.Compared with other group at the same time points after infection,the expression level of inflammatory fators of overexpression group were lower at majority of time(P<0.05),the expression level of TNF-a was significant lower than control group and negative control group at 4h(P<0.01),the expression level of IL-1? was significant lower than control group and negative control group at 12h,the expression level of IL-6 was significant lower than control group and negative control group at 4h and 12h and the the expression level of IL-8 was significant lower than control group and negative control group at 12h.The expression level of inflammatory fators of Si group were higher at majority of time,the expression level of TNF-a was significant higher than control group and negative control group at 12h,the expression level of IL-1? was significant higher than control group and negative control group at 4h and 24h,the expression level of IL-8 was significant higher than control group and negative control group at 24h.7.The expression level of PPAR-y were significant rise occurred at 12h after stimulation.The expression levels of PPAR-y of overexpression group were significant higher than other groups at majority of time.The expression levels of PPAR-y of Si group were significant lower than other group.Conclusions:1.We successfully constructed the CYP1A1-overexpressed cells which can effectively express CYP1A1.2.The siRNA has significant Interference effect on cells.3.The expression level of CYP1A1 was negatively correlated with the degree of inflammatory and may inhibit inflammation by promoting the expression of PPAR-?.