以大腸桿菌表現人類胎盤促乳激素之研究

Abstract

胎盤素於近年來廣為使用於化妝保養品中，而非法的醫療性用途也時有所聞。胎盤素是否真如同廣告宣傳中所描述，具有抗老回春的效果？至今仍缺乏科學化的根據。胎盤素中包含了多種激素成分，各種成分對人體有其獨特的作用，也有些成分其作用至今尚未明瞭。當這些成分混合於胎盤素中而使用於人體時，會因補不足而有益健康，還是會過剩而有害人體？唯有進行實驗才能得知。 hPL是胎盤素的成分之一，而且也僅能由胎盤製造，它是一種single polypeptide hormone由191個氨基酸組成，在Cys53-Cys164與Cys181-Cys188這兩個位置有雙硫鍵形成。其氨基酸組成序列與生長激素（Growth Hormone）有高達85 %的相似。截至目前的研究報告指出hPL主要作用於母體，藉由拮抗胰島素的作用，來改變醣類以及脂肪的代謝平衡，以確保對胎兒的營養供應；此外也可能藉由刺激泌乳細胞的增生，間接達成促乳的效果。藉由動物以及細胞培養模式，科學家發現hPL也能直接作用於胚胎組織，促進組織的生長發育。 本實驗的目的在於建立hPL的原核細胞表現系統，並尋找出可行的純化方法，以提供有效而穩定的hPL來源，供應未來研究hPL對成人組織細胞影響的需求。 本實驗是將hPL cDNA以PCR方式放大後轉殖到pET expression system，並由E. coli BL21 ( DE3 )進行表現。大量表現的結果造成hPL以inclusion body的形式堆積在細菌體內，必須另行萃取並且溶解進行再折疊，才能進一步進行純化。純化後的hPL以colony forming assay-觀察能否刺激K562細胞形成群落-檢測是否具有活性，結果成功的證明了純化所得的hPL仍具有活性。

Recently, placental hormones were applied in cosmetic products and some illegal medical usage. Placental hormones are composed of numerous hormones, however, there is a lack of scientific evidence to support their anti-aging effect as described in commercial advertisements. Therefore, it is important to understand by performing scientific research whether and how these hormones are useful or harmful to human body. Human placental lactogen ( hPL ) is a member of placental hormones. It is a single-chain polypeptide hormone of 22,000 daltons, which can only be synthesized and secreted by the placenta. hPL is composed of 191 amino acids with two intramolecular disulfide bridges at Cys53-Cys164 and Cys181-Cys188. hPL contains no carbohydrate residues. The amino acid sequence of hPL is 85% similar to human growth hormone ( hGH ). In vitro and animal model studies, reveal that hPL acts on fetal tissue as a direct-acting growth-promoting hormone. Substantial evidence suggests hPL influences fetal growth in an indirect manner by altering maternal metabolism such as inducing glucose tolerance, lipolysis, and proteolysis in mother. The major role of hPL in human pregnancy is believed to function as an insulin antagonist. The role of hPL in human mammary gland is to stimulate cell proliferation rather than directly stimulate milk secretion. This research object is to establish a bacterial expression system and a purification procedure to produce suitable amounts of hPL for further structural and biological studies. To produce hPL in E. coli, we employed polymerase chain reaction to amplify hPL cDNA, then the gene was cloned into a pET expression vector. Recombinant hPL synthesized in E. coli BL21( DE3 ) was accumulated in inclusion bodies in an unfolded form. The inclusion bodies were purified from cultures of E. coli, solubilized in 8M urea, and folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7. Refolded recombinant hPL was then purified by FPLC system. In order to show that the purified recombinant hPL was biologically active, we performed the in vitro growth-promoting activity study of recombinant hPL using K562 cells. By colony forming assay, the result suggested that we successfully obtained biologically active recombinant hPL.