质子感知受体对成骨细胞破骨因子表达中的作用及其分子机理研究

发布时间：2018-08-14 18:44

【摘要】：骨的代谢是由成骨细胞(Osteoblast)和破骨细胞(Osteoclast)共同调节。生理或病理的条件下细胞外或细胞间会产生酸性的环境,酸性条件会促进破骨胞的增殖并提高其活性。RANKL是破骨细胞增殖和激活的一个重要因子,这个因子表达于成骨细胞表面。相关研究证明,成骨细胞在骨形成过程中除了发挥形成骨基质的作用外,还有促进破骨细胞分化的功能。一些文章也证明酸性不仅增强了破骨细胞的活力,抑制了成骨细胞的分化与矿物化,同时也使的成骨细胞促进破骨细胞的进一步分化成熟。质子感知受体GPCR可以感知脂质分子以及质子,所以在酸性条件下成骨细胞生理活性的改变与GPCR有一定的关系。GPCR家族中包含OGR1、GPR4、TDAG8、G2A四个受体,酸性条件下究竟哪个受体在发挥作用以及它的调控方式是一个新的研究点。同时通过对其信号通路的研究不仅在基础理论方面对骨代谢途径提供了新的理论支持,同时为骨质疏松等骨病提供了新的治疗靶位点。因此,该研究在理论和实践方面都具有重要的意义。实验过程中,细胞在酸性条件下分别对其用CCK法测定细胞增殖情况,用Micro chemotaxis chamber法测定成骨细胞(hFO8)细胞的迁移能力。我们发现酸性会抑制成骨细胞的增殖和迁移。用Real Time PCR技术检测hFOB的成骨性子CollagenI、OPG、BMP2、ALP进行了检测,用ALP试剂盒对细胞ALP的表达情况进行了检测。结果显示,酸性条件抑制成骨细胞的分化和骨化。对TGF、RANKL、M-CSF几个重要的破骨因子也进行了 qPCR,发现酸性条件对破骨性因子的表达有一定的促进作用。通过对hFOB进行qPCR检测,发现TDAG8和GPR4在GPCR中高表达。分别对两个基因用质粒过表达之后再对hFOB的成骨性因子与破骨性因子进行了检测,我们发现在整个调控过程中TDAG8发挥的作用更大,GPR4作为一种辅助作用。同样用ELISA结果测得PGE2也参与酸性条件下促进破骨细胞分化的调控。结论1.酸性条件下成骨细胞的增殖和迁移能力都受到了抑制,同时形成骨的能力减弱,而促进破骨细胞分化成熟的能力提高。2.TDAG8和GPR4都参与调控酸性条件下成骨细胞的成骨因子和破骨因子的调节,分别转染TDAG8和GPR4之后,BMP2和OPG的表达量会下降,OPG的表达量受酸性影响更显著,M-CSF的表达情况也是如此。过表达TDAG8和GPR4之后使RANKLL的表达量显著升高,其中TDAG8起主要作用。同时表达AG8使TGF-β1的表达量上升。TDAG8可以作为治疗骨质疏松症的一个新的药物靶位点。
[Abstract]:Bone metabolism is regulated by both osteoblasts and osteoclasts. Under physiological or pathological conditions, there is an acidic environment between cells or extracellular. Acidic conditions promote the proliferation and activity of osteoclasts. RANKL is an important factor in the proliferation and activation of osteoclasts, and this factor is expressed in osteoblasts. Cell surface. Studies have shown that osteoblasts not only play a role in the formation of bone matrix, but also in the differentiation of osteoclasts. Some papers have also shown that acidity not only enhances the activity of osteoclasts, inhibits the differentiation and mineralization of osteoblasts, but also promotes osteoclasts. The GPCR family contains four receptors: OGR1, GPR4, TDAG8 and G2A. Under acidic conditions, which receptor plays a role and its regulation mode is one. The study of its signaling pathway not only provides a new theoretical support for bone metabolism pathway, but also provides a new therapeutic target for osteoporosis and other bone diseases. Therefore, this study has important theoretical and practical significance. We found that acidity inhibited the proliferation and migration of osteoblasts. Collagen I, OPG, BMP2 and ALP were detected by Real Time PCR, and the expression of ALP was detected by ALP kit. The results showed that acidic conditions inhibited the differentiation and ossification of osteoblasts. Several important osteoclast factors such as TGF, RANKL and M-CSF were also detected by qPCR. It was found that acidic conditions could promote the expression of osteoclast factors to some extent. We found that TDAG8 played a more important role in the whole regulation process, and GPR4 acted as a supplementary role. PGE2 also participated in the regulation of osteoclast differentiation under acidic conditions. Conclusion 1. Both TDAG8 and GPR4 are involved in the regulation of osteogenic factors and osteoclasts in acidic conditions. After transfection of TDAG8 and GPR4, the expression of BMP2 and OPG decreases. Overexpression of TDAG8 and GPR4 significantly increased the expression of RANKLL, in which TDAG8 played a major role. Expression of AG8 increased the expression of TGF-beta 1. TDAG8 could be used as a new drug target for osteoporosis.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R68

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