Isolation of novel candidate genes involved in bovine adipocyte differentiation and marbling using a clonal bovine intramuscular preadinocyte (BIP) cell line

为了分离与牛脂肪细胞分化相关的候选基因，我们利用抑制消减杂交的方法，从克隆的牛肌肉前脂肪细胞(BIP)细胞系中构建了一个消减文库。我们分离了一组在BIP细胞成脂分化过程中相应表达上调的消减cDNA片段，并从分化的BIP-lambda ZAP II cDNA文库中克隆了全长cDNA。克隆4-3与人、小鼠和大鼠胶原蛋白4羟基酶αIII亚单位(C-P4HIII)高度同源。已知C-P4HIII可折叠胶原蛋白和胶原样蛋白，并作为折叠胶原蛋白三螺旋结构所必需的。从BIP细胞成脂过程中C-P4HIIImRNA和蛋白表达的分析可知，C-P4HIIImRNA和蛋白的表达均升高。众所周知，细胞表面的细胞外基质(ECM)在成脂过程中发生了重构。细胞外基质通过成脂诱导分解，表达上调的成分，包括脂肪细胞的胶原蛋白。这些结果表明，C-P4HIII可能与牛脂肪细胞分化和脂肪形成有关，是一种潜在的脂肪生成标记。

To order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intramuscular preadipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up regulated during the adipogenic differentiation of BIP cells, and cloned a full-length cDNA from differentiated-BIP-lambda ZAP II cDNA library. The clone 4-3 was isolated and was highly homologous to the sequence of human, mouse and rat collagen prolyl 4 hydroxylase alpha III subunit (C-P4HIII). C-P4HIII is known to refold collagens and collagen like proteins, and as necessary for folding collagen triple helices structure. From the expression analysis of C-P4HIII mRNA and protein during adipogenesis in BIP cells, C-P4HIII expression was elevated both mRNA and protein. It is known that extracellular matrix (ECM) of cell surface has been restructured during adipogenesis. The ECM was decomposed by adipogenic induction, and up-regulated expression of components, included collagens for the adipocyte. These results suggest that C-P4HIII may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.