初代乳腺上皮細胞，將細胞培養在類似基底膜的環境下並給予胰島素，氫皮酮，泌乳荷爾蒙刺激是會合成和分泌乳蛋白。乙型酪蛋白是乳蛋白中主要的成分。乙型酪蛋白的重要性除了可以提供營養之外，也提供當作一個研究乳腺分化的指標。泌乳荷爾蒙結合上為細胞外蛋白的接受器因而起始JAK2/STAT5訊號傳遞。早期研究指出豐富的乙型轉形生長因子轉錄產物表現在懷孕時期乳腺，但是只有非常少量的表現在泌乳時期的老鼠。乙型轉形生長因子是分泌性雙聚體蛋白質，結合到接受器上再活化由接受器調節的Smad分子，這分子與Smad4形成聚合物並聚集在細胞核內調節標的基因轉錄作用進行。將細胞養在單只有泌乳荷爾蒙的環境下，或者再加上第一型乙型轉形生長因子，利用抽取RNA做RT-PCR，或者收細胞溶胞產物以西方墨點法就可檢測乙型酪蛋白的表現情形。我們的研究證實第一型乙型轉形生長因子存在下可抑制乙型酪蛋白表現，但是調控機轉主要不是影響在mRNA的地方，主要應該在轉譯的層次。外加給予乳腺細胞第一型乙型轉形生長因子可以誘發Smad的磷酸化。於是利用腺病毒感染的方式大量表現不會活化的Smad，結果發現可以反轉第一型乙型轉形生長因子抑制乙型酪蛋白的表現。也證實第一型乙型轉形生長因子透過Smad路徑抑制乙型酪蛋白的表現。 Primary epithelial cells culture on basement membrane synthesize and secrete milk proteins in culture in response to insulin, hydrocortisone,prolactin. b-casein is a major protein component of milk. In addition providing nutrients,b-casein serves as a molecular maker for functional differentiation in the mammary gland. Prolactin binds to the prolactin receptor and initiates the JAK2/STAT5 signal transduction cascade. Other studies demonstrated abundant expression of TGF-btranscripts in the mammary glands of pregnant mice, but very low levels of expression of TGF-b in the glands of lactating mice. Transforming growth factor TGF-bis a secreted homodimeric protein, by binding to the receptor it then activated receptor regulated Smad protein, which in turn from
complexes with Smad4 that accumulate in the nucleus and regulate the transcription of target gene. The nature of synthesis b-casein was determined by RT-PCR and western blot analysis of the RNA and total cell lysatesWe demonstrate that TGF-b1 treatment can completely
abolish the b-casein protein expression but partially affect the level of casein mRNA. In mammary epithelial cells, exogenous TGF-b1 induced phosphorylation of Smad. Overexpression of dominant-negative Smad3 deliuered by adenoviral vector restore b-casein expression. These result
sindicateinhibition of b-casein expression by TGF-b is through the Smad pathway.
