在奶牛乳腺上皮细胞原代培养条件下研究了催乳素( prolactin )和瘦素( leptin )对主要乳蛋白与乳蛋白合成信号通路关键因子基因表达的影响. 采用胶原酶消化法进行乳腺上皮细胞原代培养,并通过细胞形态观察、生长曲线测定及特异性基因表达对所培养细胞进行鉴定.试验分为4个处理,瘦素浓度均为100 ng/mL,催乳素浓度分别为0、0.1、1.0、10.0 μg/mL,每个处理6个重复. 首先采用噻唑蓝( MTT)法测定了催乳素和瘦素对奶牛乳腺上皮细胞增殖的影响,然后采用实时定量PCR测定了主要乳蛋白[α-酪蛋白、β-酪蛋白、κ-酪蛋白、β-乳球蛋白(β?LGB) ]及Janus激酶2( JAK2)、信号转导和转录激活子5( STAT5)、哺乳动物雷帕霉素靶分子( mTOR)信号分子基因表达量. 结果表明,瘦素一定浓度( 100 ng/mL)基础上,与0 μg/mL 催乳素处理相比:0.1和1.0 μg/mL催乳素对乳腺上皮细胞有显著的促增殖作用( P<0.05);0.1与10.0 μg/mL的催乳素均显著降低了αs1-酪蛋白、αs2-酪蛋白、β-酪蛋白、β?LGB的基因表达量( P<0.05);而1.0 μg/mL催乳素处理对αs1-酪蛋白、κ-酪蛋白、β?LGB均有显著的促进表达效应(P<0.05);各催乳素添加处理均显著促进了 JAK2 基因的表达(P<0.05),而对 STAT5 和mTOR基因表达只有1.0 μg/mL催乳素处理有显著促进效应( P<0.05). 结果提示,培养液含瘦素100 ng/mL基础上,催乳素对乳蛋白及信号通路关键因子基因表达有促进作用,但浓度限制在一定范围(0.1~1.0 μg/mL),过高或过低都会呈现抑制效应. 瘦素100 ng/mL 与催乳素1.0 μg/mL对部分乳蛋白基因及乳蛋白合成相关基因表达有显著的促进作用,且通过调控信号转导因子JAK2、STAT5与mTOR基因表达,从而影响乳蛋白基因的表达.%This experiment was conducted to investigate the effects of prolactin and leptin on gene expressions of milk proteins and key factors related to milk protein synthesis of bovine mammary epithelial cells under pri-mary culture condition. The primary bovine mammary epithelial cells ( pBMECs) were cultured by collagenase digestion. The epithelial origin of pBMECs was identified by morphological observation, growth curve assay and specific milk protein gene expressions detection. There were four treatments with four concentrations of prolactin (0, 0.1, 1.0 and 10.0μg/mL, respectively), leptin concentration was the same (100 ng/mL), and each treatment had six replicates. Thiazole blue ( MTT) assay was used to detected the effects of prolactin and leptin on cells proliferation, and real time PCR was used to assay the effects of prolactin and leptin on gene ex-pressions of main milk proteins [α-casein,β-casein,κ-casein andβ-lactoglobulin (β-LGB) ] , Janus kinase 2 ( JAK2) , signal transduction and transcription activator 5 ( STAT5 ) and mammalian target of rapamycin ( mTOR) . The results showed that based on certain concentration of leptin ( 100 ng/mL ) , compared with 0 μg/mL prolactin treatment: the treatments of 0.1 and 1.0 μg/mL prolactin significantly promoted pBMECs proliferation (P<0.05); the treatments of 0.1 and 10.0 μg/mL prolactin significantly decreased the gene ex-pressions of αs1-casein, αs2-casein, β-casein and β-LGB(P<0.05); the treatment of 1.0 μg/mL prolactin significantly increased the gene expressions ofαs1-casein,κ-casein,β-LGB ( P<0.05);all supplemental treat-ments of prolactin exerted promotion effects on JAK2 gene expression ( P<0. 05 ); only the treatment of 1.0 μg/mL prolactin significantly increased STAT5 and mTOR gene expressions ( P<0. 05 ) . In conclusion, based on 100 ng/mL leptin in culture medium, positive effects of prolactin on gene expressions of milk pro-teins and key factors related to milk protein synthesis are observed, but the concentration should be limited in a certain range (0.1 to 1.0 μg/mL), and reverse inhibition effects emerge with high or low concentration. Our finding suggests that 100 ng/mL leptin and 1.0μg/mL prolactin show significant promotion effects on gene ex-pressions of milk proteins and key factors related to milk protein synthesis by regulating the gene expressions of signal factors of JAK2, STAT5 and mTOR.
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