高糖及PTHrp对成骨细胞及细胞内经典Wnt信号通路影响的研究

发布时间：2018-04-16 18:41

本文选题：糖尿病 + 骨质疏松　；参考：《南京医科大学》2015年硕士论文

【摘要】：[背景]:在糖尿病患者中,骨质疏松症的发生率明显高于同龄同性别无糖尿病患者,且此类骨质疏松症患者可明显增加骨折不愈合或者延迟的风险。在这些患者中,常常是因为骨的形成减少而导致骨量丢失和骨折愈合减缓;有研究表明甲状旁腺素相关蛋白(PTHrP)可通过解除高糖对成骨细胞的抑制而促进糖尿病病体的骨形成;但关于这方面的潜在机制目前尚不清楚。[目的]:在体外探讨高糖和(或)PTHrp对成骨细胞功能及细胞内经典Wnt信号通路相关组件蛋白的影响。[方法]:取出生2-3天内的PTHrP野生型(WT)和PTHrp杂合子(PTHrp+/-)小鼠的颅骨成骨细胞作为实验对象,用含5mM葡萄糖DMEM培养基培养两种基因型细胞,待第二代长至70%后分别采用含5mM葡萄糖(正常浓度)和25mM葡萄糖(高糖浓度)的DMEM培养基培养成骨细胞;同时此基础上予经典Wnt信号通路抑制剂XAV939和不同浓度的外源性PTHrp干预成骨细胞。用CCK-8检测各组成骨细胞的增殖、茜素红染色法检测成骨细胞体外矿化、免疫荧光检测成骨细胞内β-catenin的降解程度;用RT-PCR、Western blot方法分析经典Wnt信号通路相关基因和蛋白表达水平。[结果]:相对于正常糖浓度WT组,高糖及高糖+PTHrp+XAV939(2μmol/L)组WT成骨细胞内的β-catenin、Lef-1、ALP、OPG、Runx2等基因或蛋白的相对表达水平均出现下调(p0.05);而高糖+PTHrp处理组的相关基因蛋白表达水平则明显上调,差异有统计学意义(P0.05)。与正常糖浓度WT组相比,高糖浓度WT组成骨细胞的增殖、钙化结节数目都明显减少(P0.05);此外内源性的PTHrp不足,成骨细胞的增殖及矿化能力将进一步受阻(P0.05)。与正常糖浓度WT组相比,培养在高糖浓度中两种基因型成骨细胞组内Wnt信号通路中的p-GSK3β、β-catenin、Lef-1因子以及RunX2、OPG等下游细胞因子明显下调(p0.05)。[结论]:以上结果数据说明高糖可通过抑制成骨细胞内经典Wnt信号通路,导致成骨细胞内经典Wnt信号通路受阻而影响细胞正常功能。外源性的PTHrp具有抵消高糖对成骨正常功能的抑制作用;而内源性的PTHrp分泌不足可导致经典Wnt信号通路受阻情况进一步恶化。这为运用PTHrp治疗糖尿病性骨质疏松症或糖尿病性骨质疏松继发骨折提供了新理论基础。
[Abstract]:Background: the incidence of osteoporosis in diabetic patients is significantly higher than that in nondiabetic patients of the same age, and the risk of nonunion or delay in this type of osteoporosis is significantly increased.In these patients, bone mass loss and fracture healing are often slowed down by reduced bone formation; PTHrPs have been shown to promote bone formation in diabetic bodies by removing the inhibition of high glucose on osteoblasts;But the potential mechanism for this is unclear.Objective: to investigate the effects of high glucose and / or PTHrp on the function of osteoblasts and the component proteins associated with classical Wnt signaling pathway in vitro.[methods] the osteoblasts of PTHrP wild-type WTand PTHrp heterozygotes (PTHrp / -) mice were taken as experimental objects. The two genotypes were cultured in DMEM medium containing 5mM glucose.After the second generation reached 70%, osteoblasts were cultured on DMEM medium containing 5mM glucose (normal concentration) and 25mM glucose (high glucose concentration).At the same time, XAV939, a classical Wnt signal pathway inhibitor, and exogenous PTHrp of different concentrations were used to interfere with osteoblasts.The proliferation of osteoblasts in each group was detected by CCK-8, mineralization of osteoblasts in vitro was detected by alizarin red staining, the degradation of 尾 -catenin in osteoblasts was detected by immunofluorescence, and the expression levels of genes and proteins related to classical Wnt signaling pathway were analyzed by RT-PCR- Western blot.[results]: the relative expression levels of 尾 -cateninine PTHrp XAV939(2 Runx2 and other genes or proteins in WT osteoblasts were down-regulated in high glucose and high glucose PTHrp XAV939(2 渭 mol / L groups compared with normal glucose concentration WT group, while the relative expression level of related gene proteins in high glucose PTHrp treatment group was significantly up-regulated.The difference was statistically significant (P 0.05).Compared with normal glucose concentration WT group, the proliferation of osteoblasts and the number of calcified nodules in high glucose concentration WT group decreased significantly (P 0.05), in addition, the endogenous PTHrp deficiency further hindered the proliferation and mineralization ability of osteoblasts.Compared with normal glucose concentration WT group, p-GSK3 尾, 尾 -catenin Lef-1 factor and RunX2OPG down-regulated p0.05 in the Wnt signaling pathway of two genotypic osteoblasts cultured in high glucose concentration.[conclusion] the above results suggest that high glucose may affect the normal function of osteoblasts by inhibiting the classical Wnt signaling pathway in osteoblasts and resulting in the blockage of classical Wnt signaling pathway in osteoblasts.Exogenous PTHrp can counteract the inhibitory effect of high glucose on the normal function of osteogenesis, while the lack of endogenous PTHrp secretion can further worsen the obstruction of classical Wnt signaling pathway.This provides a new theoretical basis for the application of PTHrp in the treatment of diabetic osteoporosis or secondary fractures of diabetic osteoporosis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580;R587.1

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