2型糖尿病股骨骨折后骨硬化蛋白及WNT信号通路相关性研究

发布时间：2018-03-20 10:22

本文选题：成骨细胞　切入点：2型糖尿病　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景高血糖导致的骨折不愈合主要原因是在骨折修复的过程中,高血糖促进软骨细胞凋亡和破骨细胞分化,减少成骨和软骨内骨化。以往对骨钙素、1型胶原氨基端肽(PINP)和Ⅰ型胶原羧基端肽β特殊序列(β-CTX)的研究主要是针对于糖尿病骨质疏松的研究,尚未有骨硬化蛋白及上述指标在2型糖尿病(Type2DiabetesMellitus,T2DM)股骨骨折愈合中相关变化规律的报道。有发现,Wnt信号通路在骨髓间充质干细胞向成骨细胞分化,及抑制成骨细胞凋亡方面起到积极的作用,但Wnt信号对于T2DM患者骨折修复是否发挥作用及其作用机制尚未见相关报道。研究目的1、股骨骨折合并T2DM患者相对于未合并T2DM患者在骨折愈合在急诊入院时血液骨代谢指标(骨硬化蛋白、骨钙素、PINP和β-CTX)的变化情况检测,通过对结果的分析比较,以确定候选主要参与骨折修复重建的关键因子。2、从细胞水平验证骨硬化蛋白对成骨细胞hFOB 1.19干扰后引起的成骨功能变化的规律,以确定骨硬化蛋白是否具有调节其余三个骨代谢标志物的作用。3、通过Wnt激动剂和骨硬化蛋白处理hFOB 1.19,来检测骨硬化蛋白是否通过调节Wnt信号通路上的β-连环蛋白(β-catenin)来调节骨重建的。4、沉默hFOB 1.19细胞β-catenin的表达,检测骨代谢指标骨钙素、PINP和β-CTX的变化规律,进一步揭示骨重建可能的调节机制,为研制治疗糖尿病导致的骨不愈合的药物提供新的药物治疗靶点。方法随机选取2014年6月到2015年6月在呼和浩特市第一医院骨科就诊的股骨骨折伴有T2DM患者32例作为实验组,同期在该时间段内股骨骨折未合并有糖尿病的患者27例作为对照组,全部患者在急诊入院时分别抽取静脉血液样本。采用酶联免疫吸附法(Elisa)测量骨折修复重建过程中骨钙素、PINP、β-CTX和骨硬化蛋白的含量变化。对hFOB 1.19细胞进行培养,骨硬化蛋白干扰hFOB 1.19细胞后,采用qRT-PCR检测骨钙素、PINP、β-CTX的mRNA表达量。Wnt激动剂和骨硬化蛋白处理hFOB 1.19细胞,采用免疫印迹(Western blot)法检测β-catenin和磷酸化β-catenin(p-β-catenin)含量变化。利用HiPerFect转染试剂盒转染 siRNA-β-catenin 到 hFOB 1.19 细胞中,Western blot 检测 β-catenin蛋白含量变化和qRT-PCR检测骨钙素、PINP、β-CTX的mRNA表达的变化。结果在股骨骨折急诊入院患者血清中,T2DM骨硬化蛋白的含量相对于对照组显著增高,骨钙素、PINP和β-CTX的含量显著降低,且骨硬化蛋白与骨钙素、PINP、β-CTX含量之间为线性负相关。骨硬化蛋白干扰hFOB 1.19细胞后,骨钙素、PINP和β-CTX的mRNA水平均随着骨硬化蛋白的浓度增高而逐渐降低,呈负相关。Wnt激活剂和骨硬化蛋白干扰hFOB 1.19细胞后,胞质中的β-catenin蛋白表达量无显著性差异,而Wnt激活剂可以下调细胞质中p-β-catenin的水平,骨硬化蛋白可以上调p-β-catenin的水平,而且骨硬化蛋白浓度越大p-β-catenin上调的水平越高。siRNA-β-catenin转染后成功干扰hFOB 1.19细胞β-catenin的表达,同时使骨钙素、PINP和β-CTX的mRNA水平显著降低。结论1、在股骨骨折早期愈合过程中,T2DM骨硬化蛋白的含量相对于对照组显著增高,骨钙素、PINP和β-CTX的含量显著降低,推测骨硬化蛋白可能参与T2DM骨折愈合过程。2、骨硬化蛋白可以下调骨钙素、PINP和β-CTX的mRNA表达水平,影响骨修复重建的进程。3、骨硬化蛋白可以上调p-β-catenin的水平,而且其浓度越大p-β-catenin上调的水平越高。推测骨硬化蛋白是通过调节p-β-catenin水平,进而参与调节骨折修复重建过程的。4、β-catenin表达的下降,可使骨钙素、PINP和β-CTX的mRNA水平显著降低,说明骨硬化蛋白是通过上调Wnt信号通路中β-catenin的磷酸化水平,来降低β-catenin表达,进而影响骨代谢标志物的表达水平。
[Abstract]:The research background of hyperglycemia induced nonunion of fracture is the main reason in the process of fracture repair, high blood glucose and promote the apoptosis of cartilage cells and osteoclast differentiation, reduce bone formation and endochondral ossification. The osteocalcin, type 1 collagen amino terminal peptide (PINP) and type I collagen carboxy terminal peptide sequence (beta beta special -CTX) the study is mainly aimed at the research of diabetic osteoporosis, yet sclerosin and the above indexes in type 2 diabetes mellitus (Type2DiabetesMellitus, T2DM) in the healing related changes of femoral fractures were reported. Have found that Wnt signaling pathway in bone marrow mesenchymal stem cells to differentiate into osteoblasts, and inhibit bone formation apoptosis plays a positive role, but the Wnt signal for patients with T2DM fracture repair does play a role and its mechanism of action has not been reported. The purpose of the study of 1 patients with fracture of femur with T2DM for patients without T2 In DM patients in the emergency admission blood bone metabolism index of fracture healing (sclerostin, osteocalcin, PINP and beta -CTX) change detection, through the analysis and comparison, to determine the key factor.2 candidate mainly involved in fracture repair and reconstruction, from the cellular level verification of sclerostin on bone function changes into by osteoblast hFOB 1.19 after interference, to determine whether the sclerostin regulates the other three markers of bone metabolism of.3 by Wnt agonists and sclerosin hFOB 1.19, to detect whether the sclerostin by regulating Wnt signaling pathway of beta catenin (beta -catenin). The regulation of bone remodeling.4 silencing cell hFOB 1.19 beta -catenin, detection of bone metabolic markers osteocalcin, changes of PINP and beta -CTX, further reveal the mechanism of regulation of bone remodeling, for research and treatment of diabetes Guide Provide a potential therapeutic target of drug induced bone nonunion. Methods 32 patients with T2DM from June 2014 to June 2015 as the experimental group in the Department of orthopedics of Hohhot First Hospital treatment of femoral fractures, the same period in the time period of femoral fracture is not associated with diabetes in 27 patients as control group, all patients in the emergency admission respectively. A venous blood sample. Using enzyme-linked immunosorbent assay (Elisa) measurement of fracture repair and reconstruction in the process of osteocalcin, PINP, content changes of beta -CTX and sclerostin. HFOB 1.19 cells were cultured, sclerostin interference hFOB 1.19 cells was detected by qRT-PCR PINP, osteocalcin, beta -CTX expression of mRNA in.Wnt agonist and sclerostin treatment hFOB 1.19 cells by immunoblotting (Western blot) method to detect beta -catenin and phosphorylated -catenin beta (p- beta -catenin) content changes. To hFOB 1.19 cells transfected with HiPerFect transfection kit siRNA- beta -catenin, Western detection of blot beta -catenin protein content and qRT-PCR detection of osteocalcin, PINP, expression of beta -CTX mRNA in serum of patients with femoral fractures. Results emergency admission, the content of T2DM of sclerosin compared with the control group significantly increased, content of osteocalcin, PINP and beta -CTX decreased significantly, and sclerostin and osteocalcin, PINP, between the content of beta -CTX is linear negative correlation. Sclerosin interference in hFOB 1.19 cells, osteocalcin, PINP and beta -CTX levels were mRNA with the concentration of sclerostin increased gradually decreased,.Wnt was negatively related to activation and bone sclerostin interference hFOB 1.19 cells, the cytoplasm of beta -catenin protein expression had no significant difference, but the Wnt activator can downregulate the p- beta -catenin in the cytoplasm, egg white can be bone sclerosis P- beta -catenin level, and sclerostin concentration of p- beta -catenin increases the higher the level of.SiRNA- beta -catenin transfected 1.19 cells successfully interfered with the expression of hFOB beta -catenin, and osteocalcin, PINP and beta -CTX level of mRNA was significantly reduced. Conclusion in 1, femoral fracture healing process, the content of T2DM sclerostin compared with the control group significantly increased osteocalcin, content of PINP and beta -CTX decreased significantly, presumably sclerostin may participate in the healing process of fracture T2DM.2, sclerostin can downregulate the level of osteocalcin, PINP and beta -CTX mRNA expression levels, effects of bone repair and reconstruction process of.3 and sclerostin can up regulate p- beta the level of -catenin, and the concentration of p- beta -catenin raised more high levels that sclerostin by regulating p- beta -catenin level, which is involved in the regulation of fracture repair and reconstruction of the beta.4, -catenin The expression decreased, the osteocalcin, PINP and beta -CTX level of mRNA was significantly decreased, indicating sclerostin is through upregulation of Wnt signaling pathway in beta -catenin phosphorylation, to reduce beta -catenin expression, thereby affecting bone metabolism markers expression.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.1;R683

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