【作者】 文巍；

【导师】 冯涛； 罗进勇；

【作者基本信息】 重庆医科大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 BMPs属于TGFβ超家族,最初是作为骨生长因子被发现的,目前共分离和鉴定了20余种BMPs。骨形态发生蛋白(BMPs)在器官发育和组织损伤再生等过程中发挥着重要作用。目前,已报道的具有诱导成骨活性的BMPs主要是BMP2、4、7等。近年来的研究发现BMP9具有强大的诱导成骨能力,但是由于其被关注的时间较短,所以对BMP9信号转导过程以及诱导成骨的分子机制还缺乏了解。BMPs是通过跨膜丝氨酸/苏氨酸激酶受体传递信号的。TGFβ受体分为Ⅰ型受体与Ⅱ型受体,其中Ⅱ型受体处于持续磷酸化状态,即持续激活的状态,而Ⅰ型受体则需要Ⅱ型受体磷酸化相应位点后才能被激活,Ⅰ型受体激活后,再磷酸化激活下游的转录因子Smads。Ⅰ型受体和Ⅱ型受体复合物是BMPs信号途径的必要分子。其中Ⅰ型受体,在BMPs信号转导过程和功能发挥中起着承上启下的关键作用。我们实验室前期工作,利用显性负性突变型TGFβⅠ型受体的腺病毒和BMP9共同作用发现,ALK1、ALK2这两种Ⅰ型受体很可能与BMP9诱导成骨有关。基于此,本研究拟对Ⅰ型受体ALK1和ALK2参与BMP9诱导成骨的作用进行进一步验证。本研究利用T-C He等设计的一种siRNA筛选系统(pSOS系统)成功构建和筛选了能有效干扰ALK1和ALK2表达的siRNA的腺病毒,在体内体外水平验证了ALK1和ALK2对于BMP9诱导成骨的影响。在体外实验中,siALK1、siALK2可以有效抑制碱性磷酸酶、钙盐、荧光素酶的活性,而这三项是验证BMP9信号传导和体外成骨的重要指标。实验结果说明,siALK1、siALK2抑制了BMP9的信号转导,并抑制了BMP9诱导骨髓间充质干细胞向成骨细胞分化。在体内实验中,siALK1、siALK2有效抑制了裸鼠皮下包块形成的大小,H&E染色、Alcian blue染色的组织学评价说明包块中骨基质较少,软骨基质较多,表明其成骨过程不活跃且较滞后。体内实验说明siALK1和siALK2在动物水平抑制了BMP9的诱导成骨效应。综上,通过RNAi抑制ALK1、ALK2后的体内体外实验,,鉴定和验证了ALK1、ALK2是参与BMP9信号转导和诱导成骨过程的Ⅰ型受体,发挥着承上启下的重要作用,本研究将有助于进一步阐明BMP9诱导成骨的机制。更多还原

【Abstract】 BMPs(Bone Morphogenetic Proteins)belong to the transforming growth factorb (TGFβ) superfamily, Originally isolated as proteins that induce bone and cartilage formation in vivo. More than 20 BMPs have been identified. Bone Morphogenetic Proteins (BMPs) play an important role during organ development and during regeneration after tissue damage. Prior works show that BMP2, BMP4, BMP7 has potent osteogenic activity. Presently, it has been found that BMP9 is the strongest factor to induce bone formation. But the mechanism under the bone formation induced by BMP9 reamins unclear.BMPs signal via transmembrane serine/threonine kinase receptors, termed TGFβtype I and type II receptor,and then, the activated receptor subsequently phosphorylates transcriptional factors, called Smads, which activate the expression of target genes in concert with coactivators. Therefore, in this signal pathway, TGFβreceptors act as a key point which can bind BMPs and activate Smads.TGFβreceptors are the most important molecular in early signal transduction of BMPs, and involve in osteogenic activity of BMPs. Among, TGFβtype I receptor, which is the connecting link of BMPs pathway, plays an important role.In our lab’s previous study, we use TGFβtype I dn-receptor virus and BMP9 to co-stimulate stem cell and nude mouse, preliminary screening TGFβtypeⅠreceptor ALK1 and ALK2 which can inhibit the osteogenesis of BMP9. According to the experimental result, it is very probable for ALK1 and ALK2 to concern with osteogenesis of BMP9. Based on this background, our study further identify and validate the effect of ALK1 and ALK2 on the osteogenesis of BMP9.We apply a special siRNA screening system—pSOS designed by T-C He to successfully construct and screen siALK1 and siALK2 adenovirus which have potent effect to interfere the expression of ALK1 and ALK2, validating the impact of ALK1 and ALK2 on osteogenesis of BMP9 in vitro and vivo. In vitro, we have found siALK1 and siALK2 can inhibit the activity of alkaline phosphatase, luciferase and calcium salts which are the most important signaling and osteoblast markers. The experimental result shows that siALK1 and siALK2 inhibit osteogenesis of BMP9 in vitro. In vivo, siALK1 and siALK2 inhibit the subcutaneous bulb’s size in nude. Bone matirx exhibits less while cartilage matrix more, indicating inactive and hysteretic osteogenesis. These show siALK1 and siALK2 inhibit osteogenesis of BMP9 in vivo. From the above, we identify and validate the potent effect of ALK1 and ALK2 on the osteogenesis of BMP9, which can provide virtual evidence to elucidate the mechanism under BMP9 induced osteogenesis.更多还原