CGRP作用下的MG63蛋白质差异表达的实验研究

发布时间：2018-05-11 18:43

本文选题：CGRP + MG-63　；参考：《第三军医大学》2011年硕士论文

【摘要】：降钙素基因相关肽(calcitonin gene-related peptide,CGRP)是一种感觉神经肽,在骨组织中的分布广泛,主要分布于骨代谢活跃的部位,直接或间接地调节成骨细胞和破骨细胞分化及活性。目前的研究表明CGRP在骨折愈合中发挥重要作用。有关CGRP在成骨细胞正常代谢中的具体机制国内外的研究较多,但对细胞蛋白表达的研究较少。本研究寻找与CGRP相互作用、且在成骨愈合过程中起重要作用的蛋白,并且通过免疫荧光等技术确定它们在信号通路中的上下游关系,从而探讨CGRP在骨创伤愈合过程中的可能作用机制。目的: 探讨CGRP作用下成骨样细胞MG-63内蛋白质表达谱的变化。方法: 传代培养MG-63成骨样细胞,随机分为CGRP组和对照组。通过MTT比色法检测细胞生长曲线并进行统计分析。提取各组MG-63内蛋白质,经双向电泳分离、考马斯亮蓝染色后扫描分析蛋白质表达变化,选取6个差异显著的蛋白点进行胶内酶切、肽质量指纹图谱分析和IPI数据库检索。并对检索出的差异蛋白HRNR Hornerin通过western blot及免疫荧光方法进行验证。结果: 1、MTT比色法检测表明,CGRP实验组和对照组细胞生长曲线有显著性差异。 2、双向电泳可分离(967±17)个蛋白质,点匹配率为(85.1±1.4)% ,成骨样细胞6种蛋白点出现明显差异表达,与对照组比较,CGRP组6种蛋白质表达下调。 3、质谱分析鉴定出核糖体结合蛋白p180(p180/ribosome receptor)及钙结合蛋白HRNR Hornerin等5种蛋白质,前者与核糖体定位和合成的新蛋白转位有关,而后者可影响细胞内钙浓度的。 4、通过对HRNR Hornerin的western blot及免疫荧光分析,发现HRNR Hornerin在CGRP实验组中表达显著下降。结论: 1、CGRP作用于成骨样细胞(MG-63)后,显著促进MG-63生长,实验组与对照组MTT细胞生长曲线差异显著。 2、通过双向电泳,发现CGRP可诱导成骨样细胞(MG-63)6个蛋白点发生表达的差异性明显,说明CGRP可能通过调节蛋白质含量或传导通路的途径发挥细胞调节作用。 3、质谱分析技术鉴定出的差异显著的蛋白质可能在CGRP作用下成骨样细胞(MG-63)中发挥着直接或间接的作用。CGRP可能通过这几种蛋白参与第二信使及细胞因子的调节并发挥功能。 4、采用western blot及免疫荧光染色方法分析,MG-63培养液加入CGRP作用24h后,细胞质中HRNR Honerin表达降低,证实了质谱分析结果。分析钙离子结合蛋白HRNR Honerin在CGRP作用于成骨样细胞MG-63中发挥着重要作用。CGRP可能通过调节钙离子浓度这一途径促进成骨样细胞MG-63的生长,从而最终在骨折愈合中发挥作用。
[Abstract]:Calcitonin gene-related peptidine (CGRP) is a kind of sensory neuropeptide, which is widely distributed in bone tissue and mainly distributes in the active site of bone metabolism, directly or indirectly regulates the differentiation and activity of osteoblasts and osteoclasts. Current studies have shown that CGRP plays an important role in fracture healing. The specific mechanism of CGRP in the normal metabolism of osteoblasts has been studied more at home and abroad, but the expression of cell protein is less studied. In this study, we looked for proteins that interact with CGRP and play an important role in osteogenesis healing, and used immunofluorescence techniques to determine their upstream and downstream relationship in signal pathway. To explore the possible mechanism of CGRP in bone wound healing. Objective: To investigate the changes of protein expression profile in MG-63 of osteoblast like cells treated with CGRP. Methods: MG-63 osteoblast-like cells were subcultured and randomly divided into CGRP group and control group. Cell growth curves were measured by MTT colorimetry and analyzed statistically. The proteins in MG-63 were extracted and separated by two-dimensional electrophoresis. After Coomassie brilliant blue staining, protein expression was scanned and analyzed. Six protein spots with significant difference were selected for endonuclease digestion, peptide mass fingerprint analysis and IPI database retrieval. The identified differential protein HRNR Hornerin was verified by western blot and immunofluorescence. Results: 1MTT colorimetric assay showed that the growth curve of CGRP group was significantly different from that of control group. 2, 967 卤17) proteins were separated by two dimensional electrophoresis, and the point matching rate was 85.1 卤1.4%. The expression of six protein spots in osteoblast-like cells was significantly different, and the expression of six proteins in CGRP group was down-regulated compared with the control group. 3. Five proteins, ribosomal binding protein (p180(p180/ribosome receptor) and calcium-binding protein (HRNR Hornerin), were identified by mass spectrometry. The former was related to ribosomal localization and translocation of synthesized new proteins, while the latter could affect intracellular calcium concentration. 4. By western blot and immunofluorescence analysis of HRNR Hornerin, it was found that the expression of HRNR Hornerin decreased significantly in CGRP experimental group. Conclusion: (1) the growth curve of MTT cells in the experimental group and the control group was significantly different from that in the control group and the osteoblast like cell line MG-63 (P < 0.01). 2. By two dimensional electrophoresis, it was found that CGRP could induce the expression of MG-63 protein in osteoblast like cells, which indicated that CGRP might play a regulating role by regulating protein content or conduction pathway. 3. The differential proteins identified by mass spectrometry may play a direct or indirect role in osteoblast like cells (MG-63) under the action of CGRP. CGRP may participate in the regulation and function of the second messenger and cytokines through these proteins. (4) western blot and immunofluorescence staining were used to analyze the decrease of HRNR Honerin expression in the cytoplasm after 24 h exposure to MG-63 medium, which confirmed the results of mass spectrometry. It is analyzed that calcium binding protein (HRNR Honerin) plays an important role in MG-63 of osteoblast like cells acting on CGRP. CGRP may promote the growth of osteoblast like MG-63 by regulating the concentration of calcium ion, and finally play a role in fracture healing.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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