本文選題：成骨細胞　切入點：2型糖尿病　出處：《南方醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：研究背景高血糖導致的骨折不愈合主要原因是在骨折修復的過程中,高血糖促進軟骨細胞凋亡和破骨細胞分化,減少成骨和軟骨內(nèi)骨化。以往對骨鈣素、1型膠原氨基端肽(PINP)和Ⅰ型膠原羧基端肽β特殊序列(β-CTX)的研究主要是針對于糖尿病骨質(zhì)疏松的研究,尚未有骨硬化蛋白及上述指標在2型糖尿病(Type2DiabetesMellitus,T2DM)股骨骨折愈合中相關(guān)變化規(guī)律的報道。有發(fā)現(xiàn),Wnt信號通路在骨髓間充質(zhì)干細胞向成骨細胞分化,及抑制成骨細胞凋亡方面起到積極的作用,但Wnt信號對于T2DM患者骨折修復是否發(fā)揮作用及其作用機制尚未見相關(guān)報道。研究目的1、股骨骨折合并T2DM患者相對于未合并T2DM患者在骨折愈合在急診入院時血液骨代謝指標(骨硬化蛋白、骨鈣素、PINP和β-CTX)的變化情況檢測,通過對結(jié)果的分析比較,以確定候選主要參與骨折修復重建的關(guān)鍵因子。2、從細胞水平驗證骨硬化蛋白對成骨細胞hFOB 1.19干擾后引起的成骨功能變化的規(guī)律,以確定骨硬化蛋白是否具有調(diào)節(jié)其余三個骨代謝標志物的作用。3、通過Wnt激動劑和骨硬化蛋白處理hFOB 1.19,來檢測骨硬化蛋白是否通過調(diào)節(jié)Wnt信號通路上的β-連環(huán)蛋白(β-catenin)來調(diào)節(jié)骨重建的。4、沉默hFOB 1.19細胞β-catenin的表達,檢測骨代謝指標骨鈣素、PINP和β-CTX的變化規(guī)律,進一步揭示骨重建可能的調(diào)節(jié)機制,為研制治療糖尿病導致的骨不愈合的藥物提供新的藥物治療靶點。方法隨機選取2014年6月到2015年6月在呼和浩特市第一醫(yī)院骨科就診的股骨骨折伴有T2DM患者32例作為實驗組,同期在該時間段內(nèi)股骨骨折未合并有糖尿病的患者27例作為對照組,全部患者在急診入院時分別抽取靜脈血液樣本。采用酶聯(lián)免疫吸附法(Elisa)測量骨折修復重建過程中骨鈣素、PINP、β-CTX和骨硬化蛋白的含量變化。對hFOB 1.19細胞進行培養(yǎng),骨硬化蛋白干擾hFOB 1.19細胞后,采用qRT-PCR檢測骨鈣素、PINP、β-CTX的mRNA表達量。Wnt激動劑和骨硬化蛋白處理hFOB 1.19細胞,采用免疫印跡(Western blot)法檢測β-catenin和磷酸化β-catenin(p-β-catenin)含量變化。利用HiPerFect轉(zhuǎn)染試劑盒轉(zhuǎn)染 siRNA-β-catenin 到 hFOB 1.19 細胞中,Western blot 檢測 β-catenin蛋白含量變化和qRT-PCR檢測骨鈣素、PINP、β-CTX的mRNA表達的變化。結(jié)果在股骨骨折急診入院患者血清中,T2DM骨硬化蛋白的含量相對于對照組顯著增高,骨鈣素、PINP和β-CTX的含量顯著降低,且骨硬化蛋白與骨鈣素、PINP、β-CTX含量之間為線性負相關(guān)。骨硬化蛋白干擾hFOB 1.19細胞后,骨鈣素、PINP和β-CTX的mRNA水平均隨著骨硬化蛋白的濃度增高而逐漸降低,呈負相關(guān)。Wnt激活劑和骨硬化蛋白干擾hFOB 1.19細胞后,胞質(zhì)中的β-catenin蛋白表達量無顯著性差異,而Wnt激活劑可以下調(diào)細胞質(zhì)中p-β-catenin的水平,骨硬化蛋白可以上調(diào)p-β-catenin的水平,而且骨硬化蛋白濃度越大p-β-catenin上調(diào)的水平越高。siRNA-β-catenin轉(zhuǎn)染后成功干擾hFOB 1.19細胞β-catenin的表達,同時使骨鈣素、PINP和β-CTX的mRNA水平顯著降低。結(jié)論1、在股骨骨折早期愈合過程中,T2DM骨硬化蛋白的含量相對于對照組顯著增高,骨鈣素、PINP和β-CTX的含量顯著降低,推測骨硬化蛋白可能參與T2DM骨折愈合過程。2、骨硬化蛋白可以下調(diào)骨鈣素、PINP和β-CTX的mRNA表達水平,影響骨修復重建的進程。3、骨硬化蛋白可以上調(diào)p-β-catenin的水平,而且其濃度越大p-β-catenin上調(diào)的水平越高。推測骨硬化蛋白是通過調(diào)節(jié)p-β-catenin水平,進而參與調(diào)節(jié)骨折修復重建過程的。4、β-catenin表達的下降,可使骨鈣素、PINP和β-CTX的mRNA水平顯著降低,說明骨硬化蛋白是通過上調(diào)Wnt信號通路中β-catenin的磷酸化水平,來降低β-catenin表達,進而影響骨代謝標志物的表達水平。
[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.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R587.1;R683

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