NGF通过MAPK信号通路调控CGRP表达影响成骨细胞增殖分化作用机制的研究

Nervous system participates in bone repair and reconstruction at certain degree.Research of neurotransmitter signal transduction pathways in osteoblasts is a key step of nervous bone physiology in bone trauma healing process. .Calcitonin gene related protein(CGRP)is a sensory neural polypeptide that distributes in the osteogenic activity regions. our previous studies found that CGRP was highly expressed in the mandibular bone defect healing process. at the same time, high expression of CGRP promoted callus healing and mineralization level. Our research in vitro confirmed that CGRP upregulated the Induction type Nitric oxide synthase(iNOS) expression and enzyme activity in osteoblast. However, which factor(s) influences CGRP and promotes bone trauma healing remains to be clarified. Accumulating evidence suggests that nerve growth factor(NGF)promotes fracture healing. However, the effects of NGF and CGRP in the bone trauma healing process and their corelationship remains unknown. .In this study, we would like to investigate the effects of NGF on CGRP on osteoblasts, both in vitro and in vivo. The effects of low and high expression of NGF on the expression and activity of CGRP in osteoblasts,consequently promoting proliferation and differentiation of osteoblast will be studied.An experiment of their molecular mechanism of NGF on CGRP through MAPK signal transduction pathways (including JNK,ERK,P38 signal transduction pathways)will be designed in vitro.Combined with the preliminary NGF PLGA sustained release microsphere（NGF-PLGA-Ms）gel preparation, NGF-PLGA-Ms gel would be further optimized to acquire sustained release nanometers (NGF-PLGA-Ns),and be used in defect area of rabbit mandibular defect model in vivo.The effects of NGF-PLGA-Ns on promoting defect healing will be detected. At the same time, the effects of NGF on the expression of CGRP would be studied in vivo. .From above studies in vitro and in vivo, we expect that this study will provide valuable information for bone trauma repair and reconstruction mechanisms,and can greatly benefit the efficacy of clinical treatment of bone trauma.

临床已证实感觉神经损伤能影响颌骨创伤愈合的速度和质量，CGRP是分布于成骨活性较高区域的一种感觉神经源性神经多肽。前期研究发现在下颌骨骨缺损愈合过程中，CGRP高表达与骨痂的愈合及矿化水平正相关。但何种因子通过何种途径影响CGRP，进而影响骨创伤愈合仍未可知。本研究根据神经生长因子（NGF）有促进骨折愈合的作用，通过对体外培养的兔成骨细胞，采用慢病毒转染和siRNA等技术，探讨NGF在成骨细胞内过表达和低表达时对CGRP的调控作用，以及对成骨细胞增殖和分化的影响，同时研究NGF在JNK，ERK和P38信号通路上对CGRP的调控作用；并结合初步制备成功的NGF缓释微球凝胶，置于兔下颌骨缺损处进行缓释，从体内实验验证NGF对CGRP的调控机理。从分子生物层面揭示神经骨效应的机制，从而为临床进行骨创伤修复重建提供新的思路和理论依据。

临床已证实感觉神经的损伤能影响骨创伤愈合的速度和质量。CGRP是分布于成骨活性较高区域的一种感觉神经源性神经多肽。前期研究发现在下颌骨骨缺损愈合过程中，CGRP高表达与骨痂的愈合及矿化水平正相关。但何种因子通过何种途径影响CGRP，进而影响骨创伤愈合仍未可知。.主要研究内容：本课题根据神经生长因子（NGF）有促进骨折愈合的作用，通过对体外培养的兔成骨细胞，采用慢病毒转染和siRNA等技术，探讨NGF在成骨细胞内过表达和低表达时对CGRP的调控作用，以及对成骨细胞增殖和分化的影响，同时研究NGF在JNK，ERK和P38信号通路上对CGRP的调控作用；并结合初步制备成功的NGF缓释微球凝胶，置于兔下颌骨缺损处进行缓释，从体内实验验证NGF对CGRP的调控机理。从分子生物层面揭示神经骨效应的机制。.1. 在MG63细胞中加入不同浓度NGF，通过ELISA检测NGF和CGRP mRNA和蛋白表达显示MG63细胞自身可以产生少量的CGRP，在NGF作用下，可以明显上调MG63细胞分泌的CGRP(p＜0.05)，而且随NGF浓度升高，此作用也相应增强。.2. 通过复乳法制备mPEG-PLA-NGF缓释微球，用ELISA法测定微球包封率及载药量，用动态透析释药法测定体外释放率，结果显示mPEG-PLA-NGF缓释微球具有良好的物理特性和很好的缓释效果。.3. 体内实验表明骨缺损区域的NGF能够促进大鼠下颌骨骨缺损的愈合，促进大鼠的三叉神经节细胞合成CGRP。.4. 体外研究表明CGRP对成骨细胞NGF的表达有促进作用，并与剂量和时间呈正相关。.5. CGRP 促进 MG63 细胞的增殖与分化，并且具有时间以及剂量依赖性，优势浓度为 10-8M，优势时段为 48h。 .6. CGRP 可能通过 BMP-2 途径促进 MG63 细胞的分化，而不通过 BMP-2 途径促进 MG63 细胞的增殖。 .结果的科学意义：相关结果从细胞生物学和分子生物学层面证明了NGF能够通过调节MG-63细胞自身分泌的CGRP表达量从而调控MG-63细胞的增殖分化。而通过对NGF的受体阻断发现NGF对MG-63细胞的促增殖作用明显降低，从反面验证了NGF对MG-63细胞的促增殖作用。初步验证了NGF在骨创伤修复和重建过程中的调控作用和机制，探讨了CGRP在骨创伤愈合中的作用及其作用机制。