感觉神经递质CGRP和SP对颌骨骨质疏松的影响及其信号传导机制的实验研究

It is of great significance to prevent and control osteoporosis of jaw in the field of stomatology. The current hot topic of research is focused on the neural modulation to bone metabolism. The denervation of sensory nerve can result in mandibular osteoporosis. CGRP and SP plays a key role in this process, but the mechanism still remains unknown. Our subject purposed to establish the model of jaw osteoporosis in adult rabbits via the methods of ovariectomy and low-calcium diet, use radioimmunoassay and immunohistochemistry to examine the changes in the quantity and distribution of both sensory and sympathetic fibers and neurotransmitters, and to validate if the jaw osteoporosis is related to the weakened sensory innervation and the augmented sympathetic innervation. We planned to culture the bone marrow stem cells (BMSCs) separated from the osteoporotic rabbits' femur in vitro and target to observe and evaluate the influences to proliferation and osteoblastic ability to BMSCs under different concentrations of CGRP and SP, as well as the changes in the activity of calcium channels and second messengers, and further more to clarify the molecular mechanism of signal transduction in neurotransmitter modulation to local bone metabolism. The water/oil/water (W/O/W) double-emulsion solvent evaporation method was used in our subject to prepare different concentrations of CGRP and SP-loaded [poly (lactic-co-glycolic acid, PGLA)] microspheres. These microspheres were locally used in the extraction socket of osteoporosis rabbits and the height and width of the alveolar bone and the quality of osteogenesis was valuated during the healing process of extraction sockets. We expected to conclude the minimal effective concentration and the optimum effective concentration in order to provide enough experimental supports for clinical use of neuropeptide as medication to facilitate osteogenesis and to cure jaw osteoporosis.

口腔领域中颌骨骨质疏松的防治意义重大，神经系统对骨代谢的调控是近年来国内外研究的热点，丧失感觉神经的支配可导致下颌骨骨质疏松，CGRP和SP在其中起着重要作用，但其机制尚不清楚。本课题拟利用去势加低钙饮食法建立家兔颌骨骨质疏松模型，检测颌骨内感觉和交感神经纤维及血清中神经递质数量和分布的变化，验证颌骨骨质疏松是否与感觉神经支配减弱而交感神经支配增强有关。体外培养骨质疏松状况下骨髓间充质干细胞(BMSCs)，观察不同剂量浓度CGRP、SP对BMSCs向成骨细胞分化和成骨活性的作用，钙离子通道及第二信使活性的变化，阐明感觉神经递质调节局部骨代谢的分子信号机制。采用W/O/W双层乳液法制备PGLA负载不同浓度的CGRP、SP微球，放入骨质疏松颌骨拔牙窝内，观察对牙槽骨高度、宽度变化及成骨质量的作用，掌握最低有效浓度、最佳效应浓度，为研究将神经肽类成骨因子作为药物在临床中促进颌骨生成提供实验依据。

神经系统对骨代谢的调控是近来国内外研究的热点，丧失感觉神经的支配可导致颌骨骨质疏松，感觉神经递质降钙素基因相关肽(CGRP)、P物质(SP)对局部调控骨代谢起着重要作用，但其机制尚不清楚。骨质疏松患者是否与感觉神经纤维数量减少，局部CGRP、SP分泌降低有关尚无报道，需要实验验证。骨质疏松症有很多治疗方法，包括骨吸收抑制剂、骨矿化剂等，全身用药有如此严重的并发症，局部应用成骨诱导因子如CGRP、SP不失为新的思路和治疗手段。本课题成功建立了成年大鼠和家兔颌骨骨质疏松模型，检测到其血浆中及骨组织中的CGRP和SP含量均较正常水平明显减少，验证了骨质疏松伴随着感觉神经支配的减弱。体外培养骨质疏松状况下大鼠骨髓间充质干细胞(BMSCs)，培养液中加入不同浓度的CGRP，结果BMSCs的增殖能力增强，成骨能力增强，而成脂能力减弱，并具有一定的剂量依赖性，高浓度的CGRP可以使骨质疏松条件下的BMSCs的成骨功能接近正常水平。同时进行了体内实验，将加入不同浓度CGRP培养的BMSCs混合Bio-Oss骨粉，植入裸鼠体内，结果在体内CGRP能够促使BMSCs的骨形成能力增强。骨质疏松兔制造股骨髁部的骨质缺损区域，植入负载不同浓度的CGRP和SP明胶缓释微球，发现不同浓度的CGRP/SP对于提高成骨效果有着积极的意义，SP在浓度为10-6mol/l时效果最佳，而CGRP在浓度为10-8mol/l时即达到最佳效果。.通过此研究，我们可以进一步了解绝经后骨质疏松症与感觉神经支配减弱的联系，这可能导致新的治疗绝经后骨质疏松症的方案，即通过上调感觉神经或抑制交感神经支配。成功验证了负载CGRP和SP的明胶缓释微球能有效地促进骨质疏松兔骨质缺损区的成骨，对于是否更高浓度的缓释微球效果更好以及是否能有效应用于人类牙槽嵴的缺损，尚需后期更深入的研究。