苯并芘通过抑制骨髓间充质干细胞自我更新和分化能力影响骨折愈合的研究

发布时间：2018-05-21 07:01

  本文选题：苯并芘 + 骨髓间充质干细胞　； 参考：《重庆医科大学》2017年博士论文

【摘要】：背景在骨折的愈合过程中骨髓间充质干细胞(Mesenchymal stem cells,MSCs)对骨的再生起了重要作用。香烟中的主要成份苯并芘(Benzo[a]pyrene,BaP)是芳香烃受体(arylhydrocarbonreceptor,Ahr)的激动剂,可以负性调控骨量和成骨分化。目的通过临床标本、动物模型、体外实验的研究,分析BaP对MSCs分化能力的影响及作用机制,探讨BaP是否会抑制大鼠骨折愈合的进程。方法本实验总共分为三个部分1.收集来自吸烟人群的人骨髓间充质干细胞(human bone marrow mesenchymal stem,hBMSCs)检测早期成骨标志物碱性磷酸酶(alkaline phosphatase,ALP)染色和活性,茜素红S染色检测晚期的钙盐沉积。检测芳香烃受体和其靶基因细胞色素P4501B1(cytochrome p4501B1,CYP1B1)的表达情况,验证长期吸烟人群的hBMSCs的Ahr信号通路是否被激活。用BaP处理C3H10T1/2和原代大鼠骨髓间充质干细胞(Bone marrow derived mesenchymal stem cells,BM-MSCs),利用MTT和克隆形成实验检测MSCs的增殖及自我更新能力,利用油红O染色,检测MSCs成脂分化能力,化学发光和细胞化学染色检测ALP变化,茜素红S染色检测钙盐沉积,观察MSCs的成骨分化能力。Western Blot检测Ahr信号相关分子的表达情况。为进一步明确其具体机制,采用si Ahr干扰Ahr信号通路,检测si Ahr对BaP作用下MSCs成骨分化能力:化学发光和细胞化学染色检测ALP变化;茜素红S染色检测钙盐沉积;Western Blot检测成骨标志物成骨特异性转录因子(Runt-related transcription factor 2,RUNX2),骨桥蛋白(Osteopontin,OPN)以及SMAD依赖信号通路TGF-β1/SMAD4和非SMAD信号依赖通路TGF-β1/ERK/AKT的表达情况。BaP激活Ahr信号通路的同时采用TGF-β1抑制剂(LY2157299)验证TGF-β1通路的作用。2.白藜芦醇作为Ahr的天然抑制剂,与BaP共同作用后分别检测成骨分化能力和相关信号通路的表达。体内实验,首先构建大鼠染毒和骨折模型,Western Blot检测BM-MSCs的CYP1B1的表达情况,确保染毒模型构建成功,X线和HE检测骨折愈合情况。用白藜芦醇与BaP共同作用于大鼠,观察骨折愈合情况。3.骨形态发生蛋白(Bone Morphogenetic Proteins,BMPs)作为成骨诱导因子,与BaP共同作用后检测成骨分化能力的变化。结果吸烟人群的hBMSCs自我更新及成骨分化能力减弱,并且激活Ahr信号通路以及上调Ahr的靶基因CYP1B1的表达。体外实验证实,BaP抑制MSCs的自我更新及多向分化能力,主要是通过激活Ahr信号通路抑制SMAD依赖信号通路TGF-β1/SMAD4和非SMAD信号依赖通路TGF-β1/ERK/AKT。白藜芦醇作为Ahr的天然抑制剂,可以抑制BaP对MSCs的损害。BMP2和BMP9可以抵消BaP抑制成骨分化的作用。体内实验结果显示,BaP可以抑制大鼠骨折愈合的进程,并且这个作用会被白藜芦醇挽救。结论1.BaP通过激活Ahr抑制TGF-β1/SMAD4和TGF-β1/ERK/AKT损伤MSCs的干性。白藜芦醇可以拮抗Ahr抵消BaP的影响。2.BaP可以抑制骨折的愈合进程,并且这个作用会被白藜芦醇挽救。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R683

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