- 學位論文
選殖並表現山羊第一型類胰島素生長因子於乳腺上皮細胞
Cloning and expression of type 1 insulin-like growth factor in caprine mammary gland epithelial cells
摘要
第一型類胰島素生長因子（type 1 insulin-like growth factor, IGF-1）是一具有多功能之生長因子，包括與細胞生長、細胞分化調控及抑制凋亡等功能。IGF-1主要藉由其受器（insulin-like growth factor receptor, IGF-1R）活化來調控細胞功能。IGF-1R與胰島素受器構造相似，皆由四聚體構造所組成，胞質內脂質蛋白質片段具有酪胺酸激酶活性區域。IGF-1之活性受許多因子調控，其包含了六種具有高親和力之結合蛋白（type 1 insulin-like growth factor binding protein 1 to 6, IGFBP-1 to -6）以及可與IGFBPs作用之分子。於小鼠早期發身期時，IGF-1表現於乳腺組織中非上皮細胞部分，並於發身期中乳管發育時，IGF-1會表現於終端乳腺芽（terminal end bud, TEB）之上皮細胞及乳腺周圍基質中;當懷孕晚期時，IGF-1則會表現於乳泡（alveolar）及乳管上皮細胞中。上述於特定時期對於乳腺中特定部位IGF-1表現之調控，推測其應於小鼠乳腺發育中具有重要之功能，但對於山羊此一經濟動物之功能仍尚未釐清。 從山羊耳朵組織中選殖之IGF-1（gIGF-1）cDNA，其長度約為465個核苷酸。gIGF-1 cDNA與不同物種間之胺基酸序列相似度為70%至90%。繼之將gIGF-1 cDNA架接於帶有人類巨細胞病毒啟動子（cytomegalovirus promotor, CMV promotor）之後，構築可將gIGF-1於哺乳動物細胞中表現之質體，經由轉染將gIGF-1暫時表現於不朽化山羊乳腺上皮細胞株（immortalized caprine mammary epithelial cell line, CMC）中，再進一步利用細胞免疫螢光染色觀察gIGF-1於細胞株中是否能表現。結果確定gIGF-1能表現於細胞中，繼之檢測細胞株是否具有能力可將重組gIGF-1蛋白質分泌至細胞外。另外為了解決在CMC中細胞株短暫表現gIGF-1轉型效率不一而導致細胞分泌重組gIGF-1蛋白質效率降低之疑慮，本研究亦用大腸桿菌表現重組蛋白質系統（pET/ E. coli expression system）產製成熟態、訊息胜肽保留態兩種重組gIGF-1蛋白質，可藉由外源性添加一定濃度之gIGF-1來探討其與CMC細胞株生長之關係。本重組質體已架構完成，未來gIGF-1之重組蛋白質，於大腸桿菌中可經由外源性添加異丙基–β–d–1–硫代半乳糖苷（isopropyl β-D thiogalactopyranoside, IPTG）此一藥物，誘導重組gIGF-1之蛋白質產生。經純化之重組gIGF-1蛋白質，日後可於CMC細胞株中進行gIGF-1功能性探討。 綜合上述與依據細胞免疫螢光染色之結果得知選殖gIGF-1 cDNA於所構築之載體可成功表現於CMC細胞株中，而重組gIGF-1蛋白質是否會分泌至細胞外仍需進一步確認。
關鍵字
乳腺上皮細胞並列摘要
Type 1 insulin-like growth factor (IGF-1), is a pluripotent growth factor and has been implicated in all phases of cell cycle dynamics, including promoting cell proliferation, differentiation and inhibiting cell death. IGF-1 applies its actions primarily through the type 1 insulin-like growth factor receptor (IGF-1R), which possesses a heterotetrameric structure with internal tyrosine kinase activity and structural homology to the insulin receptor. The bioactivity of IGF-1 is controlled by a complex system which consists of six high affinity binding proteins (IGFBP-1 to -6) and other IGFBP-interacting molecules. In mouse, IGF-1 expressed in the mammary gland nonepithelial portion in early puberty, and expressed in the epithelial cells of the terminal end buds as well as the stromal compartment during the pubertal ductal growth, and also will express in the alveolar and ductal epithelium during late pregnancy age. The spatially and temporally restricted expression of IGF-1 in the epithelium proposed that it may have the important function in the development of mouse mammary gland, but in one of the economic animals, goat, is not clear yet. A goat IGF-1 (gIGF-1) cDNA (465 base pairs) was cloned from ear tissue. The similarity of amino acid sequences between species is from 70% to 90%. The gIGF-1 cDNA was subcloned into a mammalian expression vector and derived by a human cytomegalovirus (CMV) promoter. The gIGF-1 act as a transient expression in an immortalized caprine mammary epithelial cell line (CMC). The localization of gIGF-1 in CMC was observed by immunocytochemistry approach. The expression pattern inferred to drive endoplasmic reticulum-golgi (ER-golgi) pathway that may go through the auto-paracrine by secreting gIGF-1 out of the CMC cell. In order to overcome the question of unpredictable amount of transient expression of gIGF-1 in CMC, the pET/E. coli expression system will apply to express the mature, signal peptide fused, and gas7 gene fused recombinant gIGF-1 proteins. With all the the constructions mentioned above, the recombinant gIGF-1 protein will be induced expression by the isopropyl-β-D-1-thiogalactopyranoside (IPTG) induction. The scaled-up recombinant gIGF-1 protein purified from the pET system will be used to investigate the function of gIGF-1 in CMC in the near future. To sum up, the immuno-staining result indicated that the constructions mentioned above could express in CMC, and according to the pattern which expressed in the cell, it is inferred that the recombinant gIGF-1 in cells transport follows the endoplasmic reticulum-golgi (ER-golgi) pathway. The confirmation whether the recombinant gIGF-1 can secrete out of cells needs further evidence.