UGBP在Antiflammin-1抗成纤维细胞增殖中的作用及机制的初步探讨

发布时间：2018-03-23 16:05

  本文选题：UGBP　切入点：AF-1　出处：《中南大学》2010年硕士论文

【摘要】： 子宫珠蛋白(uteroglobin, UG)是一种具有抗炎、抗氧化等多种生物活性的小分子分泌性蛋白,它的生物学作用可能有赖于子宫珠蛋白结合蛋白(UG结合蛋白,Uteroglobin-binding protein, UGBP)的介导。但目前对UGBP的基因结构学功能还了解甚少,对其研究远远滞后于对其天然配体UG的研究,这必将限制对UG生物学功能全面深入的认识,也必将限制其可能的临床应用,因此,针对UG结合蛋白进行探索性研究具有重要意义。我室前期研究结果显示：UG活性片段Antiflammin-1(简称AF-1,对应于UG分子中第39-47位的氨基酸序列)能够与主要在质膜上的UGBP特异性结合,并且AF-1通过UGBP的介导可影响小鼠肺成纤维细胞(NIH3T3)内ERK磷酸化水平,提示UGBP可能做为AF-1的受体参与其功能的发挥。但AF-1对肺成纤维细胞增殖有何影响未见报道。为此,本课题观察了AF-1对肺成纤维细胞增殖的影响,并进一步探讨了UGBP在介导AF-1有抗肺成纤维细胞增殖效应中的作用,为揭示AF-1抗肺纤维化的受体机制研究提供新的启示。 方法：①BrdU-ELISA法测定小鼠肺成纤维细胞(NIH3T3)的细胞增殖；②流式细胞术检测细胞周期变化；③采用RT-PCR和流式细胞术分别检测各组细胞cyclinDl及p27 mRNA和蛋白的表达水平；④采用抗UGBP抗体作为工具药物探讨UGBP的生物作用。 结果： 1. TGF-β1(0.5～15ng/ml)作用12h,呈剂量依赖性促进NIH3T3细胞增殖,其中以10ng/ml促增殖效应最为显著(P0.01)。 2.单独加入AF-1(4×10-7～2.5×10-4mol/L)对NIH3T3细胞增殖均无显著影响(P(?)0.05)。AF-1 (4×10-7～2.5×10-4 mol/L)预处理可呈剂量依赖性抑制TGF-β1 (10ng/ml)促NIH3T3细胞增殖作用(P0.01),且该抑制作用可被抗UGBP抗体所阻断(P0.01)。 3.流式细胞术结果显示,TGF-β1 (10ng/ml)处理NIH3T3细胞12h可使增殖指数(PrI)达到峰值(P0.01)；若向TGF-β1诱导的NIH3T3细胞中提前孵育AF-1 (10-5mol/L)预处理可抑制TGF-β1所致PrI的增高(P0.01),该抑制效应可被抗UGBP抗体阻断。 4.单独比较各组细胞G1、S、G2期之间的差异,可以发现TGF-β1+AF-1组比TGF-β1组S期细胞百分比显著下降(P0.01)；而TGF-β1+AF-1组与TGF-β1组之间G2期细胞百分比没有显著性差异(P0.05)。 5. PT-PCR结果显示,cyclinD1和p27 mRNA在各组NIH3T3细胞上均有表达。TGF-β1可促进cyclinD1 mRNA表达(P0.01)：AF-1可抑制TGF-β1促cyclinD1 mRNA表达(P0.01),且该抑制效应可被抗UGBP抗体预处理所取消(P0.01l)。p27 mRNA的表达结果与上述情况相反,TGF-β1可降低p27 mRNA水平(P0.01), AF-1对TGF-β1减弱p27 mRNA表达有抑制效应(P0.01),且该抑制效应可被抗UGBP抗体预处理所取消(P0.01)。 6.流式细胞术检测NIH3T3细胞核中cyclinDl和p27蛋白水平结果显示,TGF-β1组cyclinDl表达水平显著高于空白组(P0.01)；AF-1抑制TGF-β1促cyclinDl表达作用(P0.01),且该作用可被抗UGBP抗体预处理所取消(P0.01)。p27的表达结果显示,TGF-β1组p27表达水平显著低于空白组(P0.01); AF-1对TGF-β1减弱p27表达起抑制作用(P0.01),且该作用可被抗UGBP抗体预处理所取消(P0.01)。 结论： 1.AF-1对TGF-β1诱导NIH3T3细胞的增殖和细胞周期的促进均有抑制效应,该效应有赖于UGBP的介导。 2.AF-1对细胞周期的调控点主要阻滞G1向S期的转化,而对细胞周期另一个限制点S期向G2期的转化无明显影响。 3.AF-1抑制TGF-β1促NIH3T3细胞周期的机制与cyclinD1和p27的表达变化有关,并有赖于UGBP介导。
[Abstract]:Uteroglobin (Uteroglobin, UG) is a small molecule with anti-inflammatory, antioxidant and other biological activity of the secreted protein, its biological role may depend on Uteroglobin binding protein (UG binding protein, Uteroglobin-binding protein, UGBP) mediated. But the genetic structure of the UGBP function is poorly understood and the research is far behind the research on its natural ligand of UG, which will limit the comprehensive and deep understanding of the biological function of UG, will also limit its clinical application, may therefore, according to the UG binding protein is important for exploratory research. Our previous study showed that: the activity of UG fragment Antiflammin-1 (AF-1 that corresponds to the amino acid sequence of the 39-47 UG molecules) can be combined with mainly in the plasma membrane of UGBP specificity, and AF-1 mediated by UGBP can affect the small rat lung fibroblasts (NI H3T3) ERK phosphorylation, suggesting that UGBP may act as a AF-1 receptor involved in its function. But AF-1 on the proliferation of lung fibroblasts impact has not been reported. Therefore, the effect of AF-1 on the proliferation of fibroblasts. The lung, and to further explore the UGBP AF-1 mediated anti lung fibroblast proliferation effect, provide new Enlightenment for the research of AF-1 anti receptor mechanism of pulmonary fibrosis.
Methods: mouse lung fibroblasts of BrdU-ELISA (NIH3T3) cell proliferation; cell cycle was detected by flow cytometry changes; the expression level of cyclinDl and p27 cells were mRNA and protein were detected by flow cytometry and RT-PCR; using anti UGBP antibody as a tool to investigate the biological effects of UGBP drugs.
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