生长因子对成骨细胞特异转录因子Cbfal基因表达的影响以及Cbfal基因多态性与骨质疏松的关系

【作者】 裴育；

【导师】 孟迅吾； 周学瀛； 邢小平； 夏维波；

【作者基本信息】 中国协和医科大学 ， 内科学内分泌， 2003， 博士

【摘要】 实验目的 成骨细胞由间充质的前体细胞分化而来，许多激素和细胞因子参与了此分化过程的调节，但其确切机制尚不清楚。目前人们正致力于研究局部因子与循环激素，乃至局部因子与细胞内介质的相互作用在成骨细胞分化过程中的重要意义。成骨细胞特异转录因子Cbfal(Core binding factor α1，Cbfal)是近年来发现的调节成骨细胞分化和骨形成过程的关键转录因子。Cbfal缺乏的小鼠缺乏成熟的成骨细胞，且骨形成完全受阻。许多成骨细胞分化相关基因如Ⅰ型胶原、骨钙素、骨桥素等基因的启动子区域存在Cbfal的结合位点。而将Cbfal基因转染至非成骨细胞如C3H10T1／2和原代培养皮肤成纤维细胞中，导致上述细胞向成骨细胞分化，显示了其对成骨趋向的强力调控。已有研究证实Cbfal基因突变直接导致人类一种单基因显性遗传病，即颅骨锁骨发育不全。 自Cbfal作为成骨细胞特异转录因子被发现以来，各种影响因素及不同信号传导通路对其表达及活性的影响一直受到广泛地关注。有研究证实Cbfal被MAPK信号传导通路磷酸化并激活。由于MAPK信号传导通路是许多跨细胞信号，如细胞外基质—整合素相互作用、机械作用的信号以及某些生长因子的作用等的交叉点。因此本研究的目的在于：探讨生长因子IGF-Ⅰ、GM-CSF、EGF、VEGF和FGF-9是否通过MAPK信号传导通路影响成骨细胞特异转录因子Cbfal基因的表达。 实验方法 1、含小鼠Cbfal基因调控序列的荧光素酶报告基因表达质粒(p696-Luc)的构建：首先通过PCR扩增获得小鼠Cbfal基因调控序列的基因片段(-641bp—+55bp)，连接dATP后插入pGEM-T Easy Vector过渡载体中。然后设计分别含MluⅠ和XhoⅠ酶切位点的上下游引物再次PCR扩增小鼠Cbfal基因调控序列的基因片段(-641bp—+55bp)，并将其最终克隆至PGL3-Basic荧光素酶报告基因表达质粒中。 2、p696-Luc活性的检测：利用脂质体瞬时转染技术将构建成功的p696-Luc转染至MC3T3-E1和C2C12细胞中，于转染后16hr分别将不同浓度的生长因子IGF-Ⅰ、GM-CSF、更多还原

【Abstract】 ObjectiveOsteoblasts originate from common progenitors, which are capable of differentiating into other mesenchymal cell lineages such as chondrocytes, myoblasts, and bone marrow stromal cells including adipocytes. During the differentiation process from mesenchymal progenitors, various hormones and cytokines regulate osteoblast differentiation. Recently, an osteoblast specific transcription factor Cbfal was coloned. It is essential but not sufficient for osteoblast differentiation and bone formation. Cbfal- deficient mice completely lacked bone formation due to maturational arrest of osteoblasts. The phenotype of the heterozygous Cbfal mutation is similar to that of CCD. Patients suffering from this disease exhibit Cbfal mutations. Cleidocranial dysplasia (CCD) is an autosomal-dominant disease.Cbfal is an essential transcription factor for osteoblast differentiation and bone formation, however, the sigaling pathways regulating Cbfal has not been clarified. Xiao et al showed that Cbfal can be phosphorylated and activaled by the mitogen-activated protein kinase (MAPK) pathway. This pathway can be stimulated by a variety of signals including those initiated by extracellular matrix (ECM), mechanical loading and osteogenic growth factors. So, the present study was undertaken to further explore the involvement of the MAPK pathway in Cbfal gene expression affected by growth factors IGF-I, GM-CSF, EGF, VEGF and FGF-9. Methods1. Construction of reported Cbfal promoter (p696-Luc): To generate a targeting vector, genomic fragment containing Cbfal promoter (-641bp—+55bp) region were isolated from the genome of C57BJ mouse white blood cells by PCR. The A-tailing PCR fragment was ligated into pGEM-T Easy Vector, named p696-Teasy. Then we got the Cbfal promoter (-641bp— +55bp) region from the p696-Teasy using a 5’primer built in the MluI site and a 3’primer with a XhoI site and digested the product with MluI and XhoI. The gel-purified fragment was ligated into MluI/XhoI sites of pGL3-Basic vector. The resulting product contains nucleotides -641— +55 (696bp) of the reported Cbfal promoter, named p696-Luc.2. Determination of luciferase activity of p696-Luc: MC3T3-E1 and C2C12 cells were更多还原