Kiss1對黃體細胞增生與類固醇生成之調控

The Regulations of Kiss1 on Cell Proliferation and Steroidogenesis in Luteal Cell

張家昇(Chia-Sheng Chang)

指導教授 ： 邱智賢 ； 鍾德憲

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02758

摘要

KISS1與KISS1受體（KISS1 receptor, KISS1R）是現今已知調控性成熟與動情周期的重要分子機制之一，其機制為促使激性腺素釋放激素神經細胞（gonadotropin-releasing hormone neuron, GnRH neuron）生成與分泌GnRH，進而刺激下游傳遞路徑調控生殖生理。而KISS1與KISS1R在生殖生理調控系統中，亦發現在黃體也有表現，但其功能角色尚未瞭解。
本試驗首先選殖小鼠的Kiss1基因，並將其表現在pET21b載體（vector）上，經 由異丙基-β-D-硫代半乳糖苷（isopropyl β-d-1-thiogalactopyranoside, IPTG）誘導ECOSTM 21勝任細胞（competent cell）表現系統生成大量KISS1，再使用高選擇性親和二價鎳離子金屬螯合親和層析管柱（nickel-nitrilotriacetic acid affinity column, Ni-NTA affinity column）純化，最後產物KISS1約佔總蛋白質80%，純化倍率為十倍，產量為0.7 mg/ml。此產物可提供未來抗體製備，或再經過活性測定與純化，而作為藥物試驗使用。
此外，利用母山羊黃體組織，觀察正常母山羊黃體組織的組織切片，並使用山羊黃體細胞株進行體外試驗（In vitro），以了解KISS1對黃體細胞功能之影響。我們在山羊黃體組織上發現KISS1與KISS1R不論基因還是蛋白質皆有表現，且表現的細胞為具有分泌孕酮（progesterone, P4）功能的黃體細胞。體外試驗則使用KISS1的功能片段Kp-10刺激temperature sensitive caprine luteal cell D （tsCLC-D）山羊黃體細胞株。試驗結果發現在Kp-10 10 μM環境下tsCLC-D山羊黃體細胞株P4分泌量會被抑制，但細胞增生率上升，其分子層次結果顯示Kp-10會使tsCLC-D山羊黃體細胞株類固醇生成相關蛋白質及酵素的信使核糖核酸（messenger ribonucleic acid, mRNA）與蛋白質生成被抑制，而細胞增生相關蛋白質的mRNA與蛋白質生成則增加，推測KISS1調控黃體細胞是經由調節黃體細胞轉錄（transcription）與轉譯（translation）之功能，促使其細胞增生能力增強並使類固醇生成能力減弱。此外，Kp-10刺激tsCLC-D山羊黃體細胞株後，細胞質內Ca2+會增加，此結果顯示，當KISS1與KISS1R結合時，會引起細胞外或內質網（endoplasmic reticulum，ER）內的Ca2+運向細胞質，但黃體細胞內與Ca2+變化之相關蛋白質的mRNA並不受Kp-10調節。綜上所述，Kiss1對黃體細胞的調節，是使黃體細胞增生能力上升，而類固醇生成能力下降。

關鍵字

黃體細胞 ； KISS1 ； KISS1受體 ； 孕酮 ； 細胞增生

並列摘要

KISS1 and its receptor (KISS1R) are essential gatekeepers of reproduction system. Previous studies showed that KISS1 controls reproductive functions mainly through regulating gonadotropin-releasing hormone (GnRH) secretion from hypothalamus. However, its expression in corpus luteum with uncertain functions was also discovered recently.
At first, we cloned mouse Kiss1 cDNA (396 base pairs) from hypothalamic tissue into pET21b prokaryotic expression vector between restriction enzyme digest sites Nde I and Xho I. Then we overexpressed the vector in ECOS21TM competent cells by isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. After the nickel-nitrilotriacetic acid (Ni-NTA) chromatography purification, about 10-fold purified KISS1 recombinant protein (0.7 mg/ml) was obtained. This KISS1 product can be used in antibody production and biomedical research in the future.
We also investigated KISS1 functions in corpus luteum. First we confirmed the presence of both KISS1 and KISS1R in corpus luteum of various estrual stages and caprine luteal cell line (tsCLC-D) by RT-PCR, Western blotting, and immunostaining approaches. Next, we demonstrated the inhibitory effect of Kp-10 (the essential fragment for complete KISS1 function) on progesterone (P4) secretion in tsCLC-D cells by ELISA system. Further, by XTT assay we found Kp-10 does not cause cytotoxicity, but increase cell viability, which indicated that the inhibitory effect may not be caused by decreased cell numbers. Instead, by real-time PCR analysis, we demonstrated the inhibitory effect of Kp-10 was caused by down-regulating expression of mRNA encoding steroidogenic genes (Star, Cyp11a1 and Hsd3b). Furthermore, we analyzed the expression of proliferative markers (Pcna and Mki67) using Western blot method, and found the two cell proliferation markers were significantly increased by Kp-10 treatment. In the last part, we monitor the cytoplasmic calcium level changes in tsCLC-D cells by fluorescent calcium probe before and after Kp-10 treatment. Kp-10 (10 μM) can evoke intracellular calcium level in tsCLC-D cells. But further experiment found out the mRNA level of calcium signal transduct pathway related gene remained unchanged.
In summary, we demonstrated that the locally produced KISS1 might play regulatory roles in cell proliferation and P4 secretion in corpus luteum.

並列關鍵字

corpus luteum ； KISS1 ； KISS1R ； progesterone ； proliferation

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