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，我们采用抑制性消减杂交方法从克隆的牛肌内前脂肪细胞（BIP）细胞系中构建了消减文库。我们分离了一组在BIP细胞成脂分化过程中mRNA水平上调的消减cDNA片段，并从分化的BIP-λ ZAP II cDNA文库中克隆了全长cDNA。克隆4-3是分离的，并且与人、小鼠和大鼠胶原蛋白脯氨酰4羟化酶α III亚基（C-P4 HIII）的序列高度同源。已知C-P4 HIII可重折叠胶原和胶原样蛋白，并且是折叠胶原三螺旋结构所必需的。通过对BIP细胞成脂过程中C-P4 HIII mRNA和蛋白表达的分析，发现C-P4 HIII mRNA和蛋白表达均升高。脂肪形成过程中细胞表面的细胞外基质（extracellular matrix，ECM）发生了结构性改变。ECM通过成脂诱导被分解，并且上调组分的表达，包括脂肪细胞的胶原。这些结果表明，C-P4 HIII可能与脂肪细胞分化和脂肪形成牛，是一个潜在的有用的标志物脂肪形成。

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.