不同浓度葡萄糖对RANKL诱导RAW264.7细胞向破骨细胞分化的影响

发布时间：2018-06-20 15:24

本文选题：葡萄糖 + 破骨细胞　；参考：《吉林大学》2014年硕士论文

【摘要】：糖尿病是一种与高血糖密切相关的慢性内分泌性疾病，可以导致包括骨量减少、骨折及骨质疏松症在内的多种并发症。但糖尿病性骨质疏松相关的生物学机制仍不十分明确。近年来针对糖尿病性骨质疏松的研究已经成为国内外的研究热点。高血糖是引起骨稳态平衡失调的主要因素之一,长期高血糖可产生大量糖基化终末产物(Advanced Glycosylate End Products,AGE)。高浓度的AGE可使破骨细胞酸性磷酸酶(Acid phosphatase, ACP)与抗酒石酸酸性磷酸酶(TartrateResistant Acid Phosphatase, TRAP)活性显著增高,但葡萄糖直接对破骨细胞的影响目前尚未见报道。破骨细胞是参与骨稳态调节的主要细胞,破骨细胞的骨吸收功能与成骨细胞的骨形成功能相互作用调节,形成一种特殊的耦联机制,维持骨稳态。因此,该耦联的分子机制成为近年来骨生理和骨疾病研究治疗及药物研发的重点对象。当骨稳态失调时，可以导致包括骨硬化、骨质疏松等骨量异常所引发的疾病。早期关于该藕联的分子机制研究最多的就是1997年由美日学者同时发现的RANKL/RANK/OPG信号系统。近几年,研究者发现Eph/ephrin双向信号转导通路通过调节成骨细胞和破骨细胞的功能,可以同时促进骨吸收抑制骨形成，，也可以同时促进骨形成抑制骨吸收。这是由于ephrin配体同时兼具受体样功能，从而产生双向信号转导。其中,膜蛋白Eph与配体ephrin结合，向细胞胞内转导的信号,称之为正向信号; ephrin自身表达细胞胞内转导的信号,称之为逆向信号。该通路凭借对骨稳态调节功能中关键问题的成功诠释,在骨组织领域受到越来越多的关注。目的：本实验用RANKL诱导小鼠单核巨噬细胞RAW264.7分化为破骨细胞,同时给予不同浓度的葡萄糖干预,观察葡萄糖对破骨细胞分化及功能的影响,为糖尿病性骨病的发病机制提供进一步的理论依据。方法：不同葡萄糖浓度（5mmol/L、15mmol/L、30mmol/L）条件下，RANKL刺激小鼠单核巨噬细胞RAW264.7细胞，诱导其向破骨细胞分化。以5mmol/L为低糖对照组,15mmol/L、30mmol/L为实验组，以高浓度甘露醇为高渗对照组,诱导培养5天。用TRAP染色试剂盒分析各组细胞的抗酒石酸酸性磷酸酶活性变化；用Real-time PCR技术分析不同时间点各组细胞中破骨细胞标志蛋白ACP5、ctsk基因的表达的变化及破骨细胞Eph/ephrin信号通路中EphA2基因及EphA2/ephrinA2信号通路下游转录因子c-Fos、NFATc1基因的表达变化；RAW264.7细胞与骨片共培养，甲苯胺蓝染色后计算骨吸收陷窝数，根据骨陷窝数评估破骨细胞骨吸收功能。结果：培养1天、3天、5天后采用Real-time PCR检测ACP5、ctsk、c-Fos、NFATc1、EphA2等基因表达变化。第1、3天时，c-Fos、NFATc1表达量实验组明显高于低糖对照组（P＜0.05）与高渗对照组（P＜0.05），第5天时，c-Fos、NFATc1表达量实验组明显低于低糖对照组（P＜0.05）与高渗对照组（P＜0.05），且表达量随葡萄糖浓度增加逐渐降低。第1、3、5天时，EphA2表达量实验组明显低于低糖对照组（P＜0.05）与高渗对照组（P＜0.05），且表达量随葡萄糖浓度增加逐渐降低。第1、3天时，ACP5、ctsk表达量实验组与对照组相比均无明显变化，第5天时，ACP5、ctsk表达量实验组明显低于低糖对照组（P＜0.05）与高渗对照组（P＜0.05），且表达量随葡萄糖浓度增加逐渐降低。骨吸收陷窝经甲苯胺蓝染色后呈蓝色,骨吸收陷窝呈圆形、椭圆形或不规则形。培养5天时,骨吸收陷窝数实验组明显低于低糖对照组（P＜0.05）与高渗对照组（P＜0.05），且骨吸收陷窝数随葡萄糖浓度增加逐渐降低。结论：高浓度葡萄糖条件下，RANKL诱导的RAW264.7细胞分化能力下降，破骨细胞骨吸收能力降低。
[Abstract]:Diabetes is a chronic endocrine disease closely related to hyperglycemia, which can lead to a variety of complications, including bone loss, fracture and osteoporosis. However, the biological mechanism of diabetes related osteoporosis is still not very clear. Research on diabetes osteoporosis has become a domestic and foreign research in recent years. Hyperglycemia is one of the main factors causing the imbalance of homeostasis of bone. Long term hyperglycemia can produce a large number of Advanced Glycosylate End Products, AGE. High concentration of AGE can make osteoclast acid phosphatase (Acid phosphatase, ACP) and anti alcohol acid acid phosphatase (TartrateResistant Acid Phosphatase). AP) activity significantly increased, but the effect of glucose directly on osteoclasts has not yet been reported. Osteoclasts are the main cells involved in the regulation of bone homeostasis. The bone resorption function of osteoclasts can interact with the bone formation of osteoblasts, forming a special coupling mechanism to maintain bone homeostasis. Therefore, the coupled molecular machine is used. The system has become a key object in the research and development of bone physiology and bone disease in recent years. When the bone homeostasis is dysfunctional, it can lead to diseases such as bone sclerosis and osteoporosis, and the molecular mechanism of this coupling is the most important in the RANKL/RANK/OPG signal system found at the same time in 1997 by American and Japanese scholars. In recent years, researchers have found that the Eph/ephrin bi-directional signal transduction pathway can promote bone absorption and bone formation by regulating osteoblast and osteoclast, which can also promote bone formation and inhibit bone absorption. This is due to the simultaneous receptor like function of the ephrin ligand, which produces bi-directional signal transduction, including membrane protein Eph. In conjunction with the ligand ephrin, signals transduced into cell cells are called positive signals; ephrin itself expresses cell intracellular transduction signals, known as reverse signals. This pathway has attracted more and more attention in the field of bone tissue by the successful interpretation of key problems in the regulation of bone homeostasis.
Objective: in this experiment, the mouse monocyte macrophage RAW264.7 was induced to differentiate into osteoclast by RANKL, and the effects of glucose on the differentiation and function of osteoclast were observed at the same time, and further theoretical basis was provided for the pathogenesis of diabetic osteopathy.
Methods: under the condition of different glucose concentrations (5mmol/L, 15mmol/L, 30mmol/L), RANKL stimulated the mouse mononuclear macrophage RAW264.7 cells and induced their differentiation into osteoclasts. 5mmol/L was the low sugar control group, 15mmol/L, 30mmol/L as the experimental group, and the high concentration mannitol was used as hypertonic control group for 5 days. The groups were analyzed with TRAP staining kit. Changes in the activity of anti tartaric acid acid phosphatase in cells; the changes in the expression of the osteoclast marker protein ACP5, the ctsk gene in different time points and the EphA2 gene and the downstream transcription factor c-Fos of the EphA2/ephrinA2 signal pathway in the Eph/ephrin signaling pathway of osteoclasts and the expression changes of the NFATc1 gene in the cells of different time points using Real-time PCR technique RAW264.7 cells were co cultured with bone fragments, toluidine blue staining was used to calculate the number of bone resorption lacunae, and the osteoclastic bone resorption function was assessed according to the number of bone lacuna.
Results: the expression of ACP5, ctsk, c-Fos, NFATc1, EphA2 was detected by Real-time PCR for 1 days, 3 days and 5 days. At day 1,3, the experimental group of c-Fos and NFATc1 expression was significantly higher than that in the low sugar control group (P < 0.05) and the hypertonic control group (P < 0.05), and the experimental group was significantly lower than the low sugar control group at the time of fifth days. In the hypertonic control group (P < 0.05), the expression level gradually decreased with the increase of glucose concentration. At the 1,3,5 day, the expression of EphA2 in the experimental group was significantly lower than that in the low sugar control group (P < 0.05) and the hypertonic control group (P < 0.05), and the expression amount decreased gradually with the increase of glucose concentration. At the time of 1,3, the experimental group of ACP5 and ctsk expression was not obvious compared with the control group. At fifth days, the expression of ACP5 and ctsk in the experimental group was significantly lower than that in the low sugar control group (P < 0.05) and the hypertonic control group (P < 0.05), and the expression amount decreased gradually with the increase of glucose concentration. The bone resorption lacuna was blue after toluidine blue staining, and the bone resorption lacuna was round, oval or irregular. The number of resorption lacunae in the 5 days of culture was tested. The group was significantly lower than that of the low sugar control group (P < 0.05) and hypertonic control group (P < 0.05), and the number of bone resorption lacunae gradually decreased with the increase of glucose concentration.
Conclusion: under high glucose condition, the differentiation ability of RAW264.7 cells induced by RANKL decreased, and the osteoclastic bone resorption ability of osteoclasts decreased.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.1;R783.6

【共引文献】