山羊disabled-2基因之功能性區域於不朽化山羊乳腺上皮細胞之功能性研究

Abstract

初級培養之山羊乳腺上皮細胞 (CMECs)具有近似活體乳腺上皮之功能性，受到泌乳激素與胞外基質的刺激，可歷經形態及功能之分化，適合應用於乳腺發育、泌乳機制調節、乳癌發生等領域之研究，亦可做為測試重組基因表現效率之平台，提升產製轉基因動物之成功率。惟初級培養CMECs之應用受限於有限的繼代次數，主要肇因為染色體末端之端粒，逐漸耗損於每一回細胞分裂，終至喪失保護染色體的功用而引發細胞生長休止。據此，可長期繼代培養且保有乳腺上皮特性之不朽化山羊乳腺上皮細胞株 (CMCs)，乃本研究之主要目標。 藉由表現外源性人類端粒酶基因 (hTERT)，業已成功建立五株CMCs，以特定細胞系骨架蛋白 (cell lineage specific cytoskeleton)抗體，經西方印漬術 (western blotting)證實分別為二株肌上皮細胞 (myoepithelial cell lineage)，與三株腔上皮細胞系 (luminal epithelial cell lineage)。Telomeric repeat amplification protocol (TRAP)顯示CMCs與初級培養CMECs相較，具有較高端粒酶活性。核型分析試驗發現二株肌上皮細胞與一株腔上皮細胞，保有完整染色體數目 (2n = 60)與結構，另二株腔上皮細胞之染色體數明顯缺失。染色體異常多與細胞癌化相關，檢測抑癌因子p53蛋白質於DNA受損狀態下的表現，顯示二株染色體缺失之腔上皮細胞，缺乏p53蛋白質表現，同時具有癌化細胞anchorage-independent之生長特性，說明其潛在性之癌化傾向。關於CMCs功能性分化之能力，三株腔上皮細胞皆可在Matrigel胞外基質形成類乳泡構造，受到泌乳激素誘導，其中一株腔上皮細胞更可測得乳蛋白基因 (αs1- and β-casein genes)之表現。此結果證實，表現外源性人類端粒酶基因可有效不朽化初級培養之山羊乳腺上皮細胞，惟具備正常染色體數目與結構，對於保有乳腺上皮細胞之重要分化特性甚為重要。 為探討乳腺上皮細胞分化過程之分子調節機制，研究中選定細胞溶質之承接蛋白質 (cytosolic adaptor protein) Dab2，進行其功能性探討。比較不同發展階段之山羊乳腺組織，顯示Dab2蛋白質含量在懷孕末期與離乳期較高於泌乳階段，且表現位置主要位在乳泡構造。將初級培養CMECs誘導生成類乳泡構造，模擬乳腺上皮細胞之分化，顯示Dab2蛋白質表現量隨類乳泡構造之形成而有增加的趨勢。據此，推測Dab2之功能與乳泡構造的形成有關。結構上，Dab2蛋白質具有二個功能領域 (functional domains)，分別名為phosphotyrosine-binding domain (PTB)及proline-rich domain (PRD)，主宰Dab2的功能。因此，進一步將PTB domain與PRD domain架接於慢病毒表現載體 (lentivector)，並產製具感染能力之慢病毒粒子 (lentivirus particles)。經由慢病毒感染，選殖具穩定PTB domain或PRD domain表現之山羊乳腺上皮細胞株 (CMC-PTB or CMC-PRD)。已知Dab2會抑制Grb2/Sos/MAPK訊號途徑，血清饑餓試驗證實在CMC-PTB與CMC-PRD細胞內，Erk分子的磷酸化明顯受到干擾。因乳泡形成過程涉及細胞移動，以創傷癒合試驗得知CMC-PTB與CMC-PRD細胞之移動能力顯著高於未表現外源基因之控制組別。免疫細胞化學法顯示CMC-PTB與CMC-PRD細胞缺乏磷酸化focal adhesion kinase (FAK)分布於focal adhesion構造，推測此結果與其較高之細胞移動能力有關。比較PTB domain與PRD domain對山羊乳腺上皮細胞形成類乳泡構造的影響，不同於CMC-PTB細胞呈現具完整空腔之類乳泡構造，CMC-PRD細胞所形成之類乳泡構造周圍具有明顯凸起結構。此結果表明PTB domain與PRD domain對於乳腺上皮細胞之形態分化具有不同作用，異常表現PRD domain會影響類乳泡構造的完整性。 綜合上述研究結果，本論文主要利用人類端粒酶基因建構穩定且可完整定性之不朽化山羊乳腺上皮細胞株，可廣泛應用於乳腺發育、乳癌生成與產製重組蛋白質之體外測試平台等研究領域。另外，以不朽化山羊乳腺上皮細胞作為研究模式，發現Dab2蛋白質之PTB domain與PRD domain，對於乳腺上皮細胞形態分化成完整之乳泡構造，扮演重要的調節作用。

Finite lifespan limited the application of primary cultured mammary epithelial cells (MECs), whereas immortal cell lines retaining major characteristics of primary cultured MECs were more desirable. For the purpose of obtaining immortal caprine mammary epithelial cells (CMCs), human telomerase reverse transcriptase (hTERT) gene was introduced into primary cultured caprine-MECs (CMECs). Both luminal and myoepithelial cells were successfully immortalized and expressed their cell-lineage specific cytoskeleton markers. Activated telomerase in obtained immortal CMCs was confirmed by telomeric repeat amplification protocol (TRAP). The integrity of chromosomal structure and Capra hircus origin of CMCs was examined by karyotypic analysis. For morphologic differentiation, CMCs of luminal group, but not myoepithelial group, formed well-organized alveolar structures (acinus) when grown in Matrigel extracellular matrix (ECM). Furthermore, one luminal CMC clone expressed αs1- or β-casein gene in response to lactohormone stimulation. These results demonstrated that hTERT-immortalized CMCs do reserve important functional characteristics of primary cultured CMECs. As well-organized acinus is essential to full differentiation of MECs, the function of a cytosolic adaptor protein disabled-2 (Dab2), which was measured with a moderate level in pregnancy, followed by evident decrease during lactation and peaked in wean of normal caprine mammary epithelium, was explored. Immunohistochemistry (IHC) further showed Dab2 majorly distributed in alveolar structures. Thus, Dab2 was hypothesized to engage in process of alveologenesis, which was supported by evidence of elevated level of Dab2 during in vitro acinus formation of primary cultured CMECs. Dab2 molecule hold two functional domains, the amino-terminus phosphotyrosine-binding domain (PTB) and the carboxyl-terminus proline-rich domain (PRD), CMC clones stably expressed each domain were established for exploring their contributions on alveologenesis. The effect of PTB or PRD domain in CMC clones was confirmed by significant reduction of Erk phosphorylation after growth factor stimulation. As alveologenesis was associated with cell migration, wound healing assay showed enhanced cell motility of both PTB and PRD CMC clones. To examine the effect of PTB or PRD domain on alveolar structure formation, in vitro acinus culture exhibited CMC clone bearing PRD domain, but not PTB domain, represented obvious protrusions of the acinus. These data indicated both PTB and PRD domains of Dab2 were involved in regulating alveologenesis of MECs, but different mechanisms might be employed by different domains. In this study, the establishment and elaborate characterization of hTERT-immortalized CMC cell lines was demonstrated. These immortal CMC cell lines were valuable models for exploring the molecular mechanisms regulating the development of caprine mammary gland and provided a platform to examine the expression of recombined plasmids used to generate transgenic livestock. In addition, the findings of PTB and PRD domains in mediating alveologenesis of immortal CMC cells, which might claim the importance of Dab2 during the development of caprine mammary gland.