本文選題：苯并芘 + 骨髓間充質干細胞�。� 參考：《重慶醫(yī)科大學》2017年博士論文

【摘要】：背景在骨折的愈合過程中骨髓間充質干細胞(Mesenchymal stem cells,MSCs)對骨的再生起了重要作用。香煙中的主要成份苯并芘(Benzo[a]pyrene,BaP)是芳香烴受體(arylhydrocarbonreceptor,Ahr)的激動劑,可以負性調控骨量和成骨分化。目的通過臨床標本、動物模型、體外實驗的研究,分析BaP對MSCs分化能力的影響及作用機制,探討B(tài)aP是否會抑制大鼠骨折愈合的進程。方法本實驗總共分為三個部分1.收集來自吸煙人群的人骨髓間充質干細胞(human bone marrow mesenchymal stem,hBMSCs)檢測早期成骨標志物堿性磷酸酶(alkaline phosphatase,ALP)染色和活性,茜素紅S染色檢測晚期的鈣鹽沉積。檢測芳香烴受體和其靶基因細胞色素P4501B1(cytochrome p4501B1,CYP1B1)的表達情況,驗證長期吸煙人群的hBMSCs的Ahr信號通路是否被激活。用BaP處理C3H10T1/2和原代大鼠骨髓間充質干細胞(Bone marrow derived mesenchymal stem cells,BM-MSCs),利用MTT和克隆形成實驗檢測MSCs的增殖及自我更新能力,利用油紅O染色,檢測MSCs成脂分化能力,化學發(fā)光和細胞化學染色檢測ALP變化,茜素紅S染色檢測鈣鹽沉積,觀察MSCs的成骨分化能力。Western Blot檢測Ahr信號相關分子的表達情況。為進一步明確其具體機制,采用si Ahr干擾Ahr信號通路,檢測si Ahr對BaP作用下MSCs成骨分化能力:化學發(fā)光和細胞化學染色檢測ALP變化;茜素紅S染色檢測鈣鹽沉積;Western Blot檢測成骨標志物成骨特異性轉錄因子(Runt-related transcription factor 2,RUNX2),骨橋蛋白(Osteopontin,OPN)以及SMAD依賴信號通路TGF-β1/SMAD4和非SMAD信號依賴通路TGF-β1/ERK/AKT的表達情況。BaP激活Ahr信號通路的同時采用TGF-β1抑制劑(LY2157299)驗證TGF-β1通路的作用。2.白藜蘆醇作為Ahr的天然抑制劑,與BaP共同作用后分別檢測成骨分化能力和相關信號通路的表達。體內實驗,首先構建大鼠染毒和骨折模型,Western Blot檢測BM-MSCs的CYP1B1的表達情況,確保染毒模型構建成功,X線和HE檢測骨折愈合情況。用白藜蘆醇與BaP共同作用于大鼠,觀察骨折愈合情況。3.骨形態(tài)發(fā)生蛋白(Bone Morphogenetic Proteins,BMPs)作為成骨誘導因子,與BaP共同作用后檢測成骨分化能力的變化。結果吸煙人群的hBMSCs自我更新及成骨分化能力減弱,并且激活Ahr信號通路以及上調Ahr的靶基因CYP1B1的表達。體外實驗證實,BaP抑制MSCs的自我更新及多向分化能力,主要是通過激活Ahr信號通路抑制SMAD依賴信號通路TGF-β1/SMAD4和非SMAD信號依賴通路TGF-β1/ERK/AKT。白藜蘆醇作為Ahr的天然抑制劑,可以抑制BaP對MSCs的損害。BMP2和BMP9可以抵消BaP抑制成骨分化的作用。體內實驗結果顯示,BaP可以抑制大鼠骨折愈合的進程,并且這個作用會被白藜蘆醇挽救。結論1.BaP通過激活Ahr抑制TGF-β1/SMAD4和TGF-β1/ERK/AKT損傷MSCs的干性。白藜蘆醇可以拮抗Ahr抵消BaP的影響。2.BaP可以抑制骨折的愈合進程,并且這個作用會被白藜蘆醇挽救。
[Abstract]:Background Mesenchymal stem cells play an important role in bone regeneration during fracture healing. Benzo [a] pyrene-BaPin, the main component of cigarette, is an agonist of aromatic hydrocarbon receptor aryl hydrocarbon receptor Ahr. it can negatively regulate bone mass and osteogenic differentiation. Objective to analyze the effect of BaP on the differentiation ability of MSCs and its mechanism through clinical specimens, animal models and in vitro experiments, and to explore whether BaP can inhibit the process of fracture healing in rats. Methods the experiment was divided into three parts. Human bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells) from smoking population were collected to detect the early osteogenic marker alkaline phosphatase (ALP) staining and activity. Alizarin red S staining was used to detect the late calcium deposition. The expression of aromatics receptor and its target gene cytochrome P4501B1(cytochrome p4501B1CY CYP1B1) was detected to verify whether the Ahr signaling pathway of hBMSCs was activated in long-term smokers. C3H10T1/2 and primary rat bone marrow mesenchymal stem cells (BM-MSCs) were treated with BaP. The proliferation and self-renewal ability of MSCs were detected by MTT and clone formation assay. The ability of MSCs adipogenic differentiation was detected by oil red O staining. Chemiluminescence and cytochemical staining were used to detect the changes of ALP and alizarin red S staining to detect calcium deposition. The osteogenic differentiation ability of MSCs was observed. Western Blot was used to detect the expression of Ahr signal-related molecules. In order to further clarify its specific mechanism, si Ahr interference Ahr signaling pathway was used to detect the osteogenic differentiation ability of MSCs induced by BaP by si Ahr. The changes of ALP were detected by chemiluminescence and cytochemical staining. Detection of calcium Deposit by alizarin Red S staining Western Blot Detection of Osteopontinopontin Osteopontin Osteopontin Osteopontin (Osteopontin OPN) Osteopontin Osteopontin (Osteopontin OPN) Osteopontin Osteopontin (Osteopontin) and SMAD dependent signaling pathway TGF- 尾 1/ERK/AKT. TGF- 尾 1 inhibitor LY2157299) was used to verify the role of TGF- 尾 1 pathway. Resveratrol, as a natural inhibitor of Ahr, was combined with BaP to detect the osteogenic differentiation ability and the expression of signal pathway. In vivo experiments, the rat model of exposure and fracture was first constructed to detect the expression of BM-MSCs CYP1B1 by Western Blot, and to ensure the successful construction of the model by X-ray and HE detection of fracture healing. Resveratrol combined with BaP was used to observe fracture healing in rats. Bone morphogenetic protein bone Morphogenetic proteins (BMPs) were used as osteogenic inducers, and the changes of osteogenic differentiation ability were detected after combined action of BaP and bone morphogenetic protein (BMP). Results the ability of hBMSCs self-renewal and osteogenic differentiation was weakened in smoking population, and Ahr signaling pathway was activated and CYP1B1 expression of target gene of Ahr was up-regulated. In vitro experiments showed that bap inhibited the self-renewal and multidirectional differentiation of MSCs, mainly by activating the Ahr signaling pathway to inhibit the SMAD dependent signaling pathway TGF- 尾 1/SMAD4 and non-SMAD signaling dependent pathway TGF- 尾 1 / ERK-AKT. Resveratrol as a natural inhibitor of Ahr can inhibit the damage of BaP to MSCs. BMP2 and BMP9 can counteract the inhibitory effect of BaP on osteogenic differentiation. In vivo results showed that Bap inhibited the healing process of fracture in rats, and this effect was resuscitated by resveratrol. Conclusion 1.BaP inhibits the dryness of MSCs induced by TGF- 尾 1/SMAD4 and TGF- 尾 1/ERK/AKT by activating Ahr. Resveratrol antagonized the effect of Ahr on counteracting BaP. 2. Bap inhibited the healing process of fracture, and this effect was saved by resveratrol.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R683

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