哮喘气道重塑大鼠尾加压素Ⅱ促转化生长因子-β1表达的研究

UrotensinⅡ Promotes the Expressions of TGF-β1 on Airway Smooth Muscle Cell of Asthmatic Ariway Remodeling Rats

目的研究哮喘气道重塑大鼠尾加压素Ⅱ(UⅡ)和转化生长因子(TGF)-β1的表达变化,探讨UⅡ对哮喘大鼠气道平滑肌细胞(ASMC)中TGF-β1表达的调控作用。方法以卵清白蛋白(OVA)致敏与激发建立哮喘大鼠气道重塑模型,16只雄性SD大鼠随机分为对照组和哮喘组,每组8只。分别采用免疫组织化学法(IHC)和反转录-聚合酶链反应(RT-PCR)法测定UⅡ和TGF-β1的蛋白和mRNA的表达。体外培养哮喘大鼠ASMC,分为对照组和UⅡ刺激组,其中UⅡ刺激组根据UⅡ干预时间的不同分为:4、8、12、24和48h组;按加入UⅡ浓度的不同分为:0.4、4、10、40和400nmol/L组。酶联吸附试验(ELISA)检测细胞培养液中TGF-β1蛋白含量,实时定量PCR(real-time PCR)检测ASMC中TGF-β1mRNA表达变化。结果哮喘组肺组织UⅡ蛋白、TGF-β1蛋白表达较对照组均明显升高,具有显著性统计学意义(P<0.01);哮喘组肺组织中UⅡmRNA和TGF-β1mRNA含量与对照组比较均明显增加,具有显著性统计学意义(P<0.01)。体外实验显示:UⅡ不同时间点促哮喘大鼠ASMC中,TGF-β1 mRNA的表达4h开始上升,24h达到高峰;TGF-β1蛋白表达于12h开始增加,24h达到高峰。UⅡ不同浓度促哮喘大鼠ASMC中,TGF-β1mRNA和蛋白表达均于4nmol/L开始上升,40nmol/L达到高峰。结论哮喘大鼠肺组织中UⅡ和TGF-β1呈高表达;UⅡ对哮喘大鼠ASMC中TGF-β1的表达可能存在调控作用,且呈一定时间-浓度依赖性。

Objective To investigate the relationship of urotensin 11 （UⅡ ） and TGF -β1 expressions in asthmatic remodeling rats. Methods Rats were sensitized and challenged by OVA to establish asthmatic model. 16 males Sprague -Dawley （SD） rats were ran- domly divided into two groups：control group and asthmatic group. The UⅡ and TGF -β1 epressions were detected by immunohistochemistry. The UⅡ mRNA and TGF-β1mRNA contents were determined by RT- PCR. In vitro experiments were conducted to determine the direct effect of UⅡ on TGF -β1 expression by ASMC. ASMC were then incubated with UⅡ （40nmol/L） for 4h to 48h （time course） and at variable concentrations （0.4nmol/L to 400nmol/L, dose -dependent study） to modulate the expression of TGF-β1. Real -time PCR was employed to detect the expression of TGF -β1 at mRNA levels. TGF -β1 concentrations were determined by sandwich enzyme - linked immunosorbent assay（ELISA）. Results The UⅡ and TGF -β1 expressions in the asthmatic group increased significantly compared with the control group （P 〈0.01 ）. The U lI mRNA and TGF-β1mRNA contents in the asthmatic group was also higher compared with the control group （ P 〈 0.01 ）. In vitro experiments, purified ASMC were incubated with UⅡ for 4, 8, 12, 24 and 48h. RNA levels of TGF-β1 started increased as early as 4h after UⅡ treatment, peaked at 24h. Interestingly, protein levels of TGF -β1 started rose at 12h, peaked at 24h. UⅡ caused significant increase of TGF -β1 in a dose - dependent manner from 4nmol/L to 40nmol/L. They reached plateau after 40nmol/L. Conclusion The UⅡ and TGF -β1 expressions increase in the process of airway remodling in asthmatic rats. UⅡ may rendered its effect on ASMC through the upregulation of TGF -β1 at the time and concentration dependence.