Exendin-4改善成骨细胞糖毒性损伤与内质网应激及Toll样受体4基因表达的关系

发布时间：2018-06-06 15:23

本文选题：Exendin-4 + Toll样受体4　；参考：《福建医科大学》2016年硕士论文

【摘要】：目的:(1)从增殖、凋亡和成骨矿化等方向研究高糖环境是否对成骨细胞的增殖分化发挥损伤作用,建立成骨细胞糖毒性损伤模型;(2)观察新型降糖药物内源性胰高血糖素样肽-1(GLP-1)类似物Exendin-4能否在体外发挥对成骨细胞糖毒性损伤的保护作用;(3)初步探讨Exendin-4改善成骨细胞糖毒性损伤与TLR-4/NF-κB信号及内质网应激的关系。方法:(1)以小鼠颅顶前骨细胞亚克隆14(MC3T3-E1 subclone 14)作为研究对象,应用含33mmol/L葡萄糖的α-MEM完全培养基干预成骨细胞24小时,建立成骨细胞糖毒性损伤细胞模型,以含不同浓度(0、25、50、100nmol/L)的α-MEM完全培养基在不同时间段(0、24、48、72h)干预成骨细胞;验证糖毒性损伤模型的建立是否成功,体外观察Exendin-4对成骨细胞糖毒性损伤的保护作用以及对TLR-4/NF-κB信号和内质网应激相关基因表达的影响。(2)MTT法检测成骨细胞的增殖率,流式细胞术和TUNEL法检测成骨细胞的凋亡率;茜素红染色法用来检测成骨细胞矿化结节的形成;RT-PCR及Western blot法检测成骨细胞TLR4/NF-κB基因、成骨相关基因BMP-2和内质网应激相关蛋白(p ERK-1/2,CHOP,Caspase12)的表达,ELISA法检测成骨细胞分泌蛋白OCN及ALP的表达。结果:(1)高糖环境中(33mmol/L)成骨细胞的增殖率显著下降(p0.05),凋亡率显著增加(p0.05)。Exendin-4在干预时间内可呈时间和剂量依赖性改善成骨细胞糖毒性损伤导致的增殖率的下降及凋亡率的升高(均p0.05);(2)高糖环境中(33mmol/L)成骨细胞的矿化速率显著降低(p0.05),上清液中成骨细胞分泌蛋白OCN和ALP的表达显著降低(均p0.05)。Exendin-4在干预时间内可改善成骨细胞糖毒性损伤导致的成骨矿化速率的下降及分泌蛋白(OCN,ALP)表达的下降(均p0.05);(3)高糖环境中(33mmol/L)成骨细胞内慢性低度炎症相关基因TLR4/NF-κB及内质网应激相关基因(p ERK-1/2,CHOP,Caspase12)m RNA和蛋白的表达均显著增加(均p0.05);成骨相关基因BMP2 m RNA和蛋白的表达显著降低(p0.05)。Exendin-4下调糖毒性损伤导致的TLR4/NF-κB,内质网应激相关蛋白(p ERK-1/2,CHOP,Caspase12)表达的增加,并且可上调成骨相关基因BMP-2m RNA和蛋白表达的下降(均p0.05)。结论:高糖环境可干扰成骨细胞的增殖和分化,并且通过激活成骨细胞内的多种炎症通路对成骨细胞造成损伤。而Exendin-4可能通过抑制TLR4/NF-κB信号和拮抗内置网应激从而发挥对成骨细胞糖毒性损伤的保护作用。
[Abstract]:Objective: to study whether high glucose environment may damage the proliferation and differentiation of osteoblasts from the aspects of proliferation, apoptosis and osteogenesis mineralization. To observe whether Exendin-4 (Exendin-4), an endogenous glucagon like peptide, can protect osteoblast from glucose toxicity injury in vitro and to explore the effect of Exendin-4 on the improvement of osteoblasts. Relationship between cytotoxic injury and TLR-4 / NF- 魏 B signal and endoplasmic reticulum stress. Methods subclone 14 MC3T3-E1 subclone 14 of mouse precranial bone cells was used as the study object. The osteoblasts were treated with 伪 -MEM complete medium containing 33 mmol / L glucose for 24 hours to establish the model of glucose toxicity injury of osteoblasts. Osteoblasts were interfered with 伪 -MEM complete medium containing different concentrations of 25, 50, 100 nmol / L at different times for 72 h) to verify whether the model of glucose toxicity injury was successful or not. To observe the protective effect of Exendin-4 on glucose toxicity of osteoblasts in vitro and the effect on TLR-4 / NF- 魏 B signal and endoplasmic reticulum stress-related gene expression. The proliferation rate of osteoblasts was detected by MTT, the apoptosis rate of osteoblasts was detected by flow cytometry and Tunel. Alizarin red staining was used to detect the formation of mineralized nodules in osteoblasts by RT-PCR, and the expression of osteoblast TLR4 / NF- 魏 B gene, osteoblast-associated gene BMP-2 and endoplasmic reticulum stress-related protein (ERK-1 / 2CHOPS-Caspase12) was detected by Western blot. The expression of OCN and ALP in osteoblasts was detected by Elisa. Results (1) the proliferation rate of osteoblasts decreased significantly in high glucose environment (P < 0.05). The apoptosis rate of osteoblasts was significantly increased in a time-and dose-dependent manner. The apoptosis rate of osteoblasts induced by glucose toxicity injury was improved in a time-and dose-dependent manner. The mineralization rate of osteoblasts in high glucose environment decreased significantly, while the expression of OCN and ALP in supernatant decreased significantly. Decreased osteogenic mineralization rate and decreased expression of secretory protein (OCN-ALP) in osteoblasts in high glucose environment (p0.05 / L). The expression of TLR4 / NF- 魏 B and ERK-1 / 2CHOPCaspase12m mRNA and protein in osteoblasts were significantly increased in high glucose environment. The expression of TLR4 / NF- 魏 B and ERK-1 / 2CHOPCaspase12m mRNA and protein were significantly increased in osteoblasts. The expression of BMP2 mRNA and protein significantly decreased the increase of TLR4 / NF- 魏 B, endoplasmic reticulum stress-related protein (ERK-1 / 2CHOPN Caspase12) induced by glucose toxicity injury. The expression of BMP-2m mRNA and protein was up-regulated (P 0.05). Conclusion: high glucose environment can interfere with the proliferation and differentiation of osteoblasts and damage osteoblasts by activating many inflammatory pathways in osteoblasts. Exendin-4 may protect osteoblast from glucose toxicity by inhibiting TLR4 / NF- 魏 B signal and antagonizing built-in reticulum stress.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R587.1

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