本文選題：UGBP　切入點：AF-1　出處：《中南大學(xué)》2010年碩士論文

【摘要】： 子宮珠蛋白(uteroglobin, UG)是一種具有抗炎、抗氧化等多種生物活性的小分子分泌性蛋白,它的生物學(xué)作用可能有賴于子宮珠蛋白結(jié)合蛋白(UG結(jié)合蛋白,Uteroglobin-binding protein, UGBP)的介導(dǎo)。但目前對UGBP的基因結(jié)構(gòu)學(xué)功能還了解甚少,對其研究遠遠滯后于對其天然配體UG的研究,這必將限制對UG生物學(xué)功能全面深入的認識,也必將限制其可能的臨床應(yīng)用,因此,針對UG結(jié)合蛋白進行探索性研究具有重要意義。我室前期研究結(jié)果顯示：UG活性片段Antiflammin-1(簡稱AF-1,對應(yīng)于UG分子中第39-47位的氨基酸序列)能夠與主要在質(zhì)膜上的UGBP特異性結(jié)合,并且AF-1通過UGBP的介導(dǎo)可影響小鼠肺成纖維細胞(NIH3T3)內(nèi)ERK磷酸化水平,提示UGBP可能做為AF-1的受體參與其功能的發(fā)揮。但AF-1對肺成纖維細胞增殖有何影響未見報道。為此,本課題觀察了AF-1對肺成纖維細胞增殖的影響,并進一步探討了UGBP在介導(dǎo)AF-1有抗肺成纖維細胞增殖效應(yīng)中的作用,為揭示AF-1抗肺纖維化的受體機制研究提供新的啟示。 方法：①BrdU-ELISA法測定小鼠肺成纖維細胞(NIH3T3)的細胞增殖；②流式細胞術(shù)檢測細胞周期變化；③采用RT-PCR和流式細胞術(shù)分別檢測各組細胞cyclinDl及p27 mRNA和蛋白的表達水平；④采用抗UGBP抗體作為工具藥物探討UGBP的生物作用。 結(jié)果： 1. TGF-β1(0.5～15ng/ml)作用12h,呈劑量依賴性促進NIH3T3細胞增殖,其中以10ng/ml促增殖效應(yīng)最為顯著(P0.01)。 2.單獨加入AF-1(4×10-7～2.5×10-4mol/L)對NIH3T3細胞增殖均無顯著影響(P(?)0.05)。AF-1 (4×10-7～2.5×10-4 mol/L)預(yù)處理可呈劑量依賴性抑制TGF-β1 (10ng/ml)促NIH3T3細胞增殖作用(P0.01),且該抑制作用可被抗UGBP抗體所阻斷(P0.01)。 3.流式細胞術(shù)結(jié)果顯示,TGF-β1 (10ng/ml)處理NIH3T3細胞12h可使增殖指數(shù)(PrI)達到峰值(P0.01)；若向TGF-β1誘導(dǎo)的NIH3T3細胞中提前孵育AF-1 (10-5mol/L)預(yù)處理可抑制TGF-β1所致PrI的增高(P0.01),該抑制效應(yīng)可被抗UGBP抗體阻斷。 4.單獨比較各組細胞G1、S、G2期之間的差異,可以發(fā)現(xiàn)TGF-β1+AF-1組比TGF-β1組S期細胞百分比顯著下降(P0.01)；而TGF-β1+AF-1組與TGF-β1組之間G2期細胞百分比沒有顯著性差異(P0.05)。 5. PT-PCR結(jié)果顯示,cyclinD1和p27 mRNA在各組NIH3T3細胞上均有表達。TGF-β1可促進cyclinD1 mRNA表達(P0.01)：AF-1可抑制TGF-β1促cyclinD1 mRNA表達(P0.01),且該抑制效應(yīng)可被抗UGBP抗體預(yù)處理所取消(P0.01l)。p27 mRNA的表達結(jié)果與上述情況相反,TGF-β1可降低p27 mRNA水平(P0.01), AF-1對TGF-β1減弱p27 mRNA表達有抑制效應(yīng)(P0.01),且該抑制效應(yīng)可被抗UGBP抗體預(yù)處理所取消(P0.01)。 6.流式細胞術(shù)檢測NIH3T3細胞核中cyclinDl和p27蛋白水平結(jié)果顯示,TGF-β1組cyclinDl表達水平顯著高于空白組(P0.01)；AF-1抑制TGF-β1促cyclinDl表達作用(P0.01),且該作用可被抗UGBP抗體預(yù)處理所取消(P0.01)。p27的表達結(jié)果顯示,TGF-β1組p27表達水平顯著低于空白組(P0.01); AF-1對TGF-β1減弱p27表達起抑制作用(P0.01),且該作用可被抗UGBP抗體預(yù)處理所取消(P0.01)。 結(jié)論： 1.AF-1對TGF-β1誘導(dǎo)NIH3T3細胞的增殖和細胞周期的促進均有抑制效應(yīng),該效應(yīng)有賴于UGBP的介導(dǎo)。 2.AF-1對細胞周期的調(diào)控點主要阻滯G1向S期的轉(zhuǎn)化,而對細胞周期另一個限制點S期向G2期的轉(zhuǎn)化無明顯影響。 3.AF-1抑制TGF-β1促NIH3T3細胞周期的機制與cyclinD1和p27的表達變化有關(guān),并有賴于UGBP介導(dǎo)。
[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.
Result錛，

本文編號：1654176