Functional analysis of proteins belongs to SIBLING family in physiological and pathological calcification

1. 响应环境变化的骨桥蛋白启动子区鉴定。转染GFP cDNA连接远端启动子（-5505 bp至mRNA起始位点）（GFP5505 OPN）的转基因小鼠在骨负荷机械应力时，骨桥蛋白表达上调。在去卵巢的转基因小鼠中也观察到类似的效果。进一步研究发现，机械应力响应元件和雌激素响应元件位于转录起始位点-3156 ~ -5505之间。转基因小鼠原代培养细胞的分析。培养含成骨细胞和骨细胞的转基因小鼠骨细胞。当机械应力作用于培养细胞时，GFP表达水平升高。利用荧光素酶cDNA连接的启动子区域的不同区域，采用荧光素酶法对机械应力响应元件进行鉴定。最后，我们确定了骨桥蛋白基因mRNA起始位点在-3983和-315G之间的机械应力响应元件。截短骨桥蛋白对骨组织发育的影响。产生了携带钙结合位点或骨桥蛋白控制的-5505启动子细胞附着位点的转基因小鼠。将转基因小鼠引入骨桥蛋白基因敲除小鼠。生成的小鼠骨骼结构出现异常。结果表明骨桥蛋白的钙结合位点和细胞附着位点在体内起作用。

1. Identification of promoter region of Osteopontin in response to environmental change.Up-regulation of osteopontin was observed in transgenic mice carrying GFP cDNA ligated with distal promoter (-5505 bp to mRNA start site) (GFP5505 OPN) when mechanical stress was loaded to bone. Similar effect was observed in the ovaryextomized transgenic mice. Further investigation revealed that mechanical stress response element and estrogen respose element lies between -3156 and -5505 in the start site of transcription.2. Analysis of primary cultured cells derived from transgenic mice.Bone cells containing osteoblasts and osteocytes of the transgenic mice were cultured. Expression level of GFP was elevated when mechanical stress was loaded to cultured cells. Identification of mechanical stress-response element was performed by luciferase assay using various region of promoter region joined to luciferase cDNA. Finally, we identified the mechanical stress-response element between-3983 and -315G to the mRNA start site of osteopontin gene.3. Effects of truncated osteopontin on development of bone tissue.Transgenic mice carrying either calcium binding site or cell attachment site of osteopontin controlled -5505 promoter were generated. Transgenic mice were introduced to knockout mice of osteopontin gene. Generated mice showed abnormal structure in bone. The result indicate that calcium binding site and cell attachment site of osteopontin functioning in vivo.