Caveolin-1在大鼠腦垂腺瘤細胞株的功能性研究

泌乳素瘤（prolactinoma）為一常見的腦垂體腺瘤，主要症狀為腦垂體分泌過量泌乳素（prolactin，PRL）。在治療上常使用的藥物為bromocriptine，一種多巴胺D2接受體（dopamine D2 receptor）的促進劑，在研究報告中指出bromocriptine可以引起大鼠腦垂腺瘤細胞株（GH3）的計畫性凋亡，但其機制尚未明瞭。泌乳素可以調控乳腺發育，而乳腺是哺乳動物一生中少數能經歷數次細胞增殖及退化的組織，若其發育的調控機制不正常，會導致上皮細胞增生及癌化。以雜合扣除法（subtractive hybridization）自正常乳腺上皮細胞扣除癌化乳腺上皮細胞，可以發現caveolin-1基因。Caveolin-1在乳腺細胞中可以抑制經由泌乳素所引起的乳腺發育及泌乳，但在相同會受到泌乳素刺激的腦神經細胞所扮演的角色則尚不清楚。本試驗利用大鼠腦垂腺瘤細胞株GH3進行caveolin-1在腦中的弁酮膍s。以RT-PCR技術檢測GH3及小鼠大腦組織caveolin-1 mRNA，發現二者皆有表現caveolin-1，而前者caveolin-1 mRNA表現量較後者少。自小鼠大腦組織選殖caveolin-1基因序列，並將caveolin-1及EGFP序列分別架構在含有CMV promoter載體上，再將其轉型到GH3細胞株。在轉型48小時後，GH3細胞表現外源性caveolin-1，其細胞呈現萎縮形態且其細胞核呈現計畫性死亡的核體分解現象；但表現EGFP的細胞仍維持正常形態。以治療泌乳素瘤使用的藥物bromocriptine處理GH3細胞，在處理24小時後，GH3細胞會進行計畫性死亡，且細胞內caveolin-1的mRNA表現量增加。綜合以上研究證明大量表現caveolin-1可導致GH3細胞計畫性凋亡，並推測caveolin-1的弁鉬P腦垂體腺瘤生成及抑制其生長有關。

關鍵字

caveolin-1 ；細胞凋亡

並列摘要

Mammary gland can proliferate and regress many times in the life time of female mammals. The abnormal regulation of its development may induce irregular proliferation and tumorigenesis of mammary epithelial cells. Prolactin （PRL） is a major regulator for mammary gland development and lactation and is secreted by anterior pituitary gland. Prolactinoma is a common disease in human, its symptoms are accompanied with abnormal proliferation of anterior pituitary gland and hyperprolactinemia. Bromocriptine, a dopamine D2 receptor agonist, is commonly used to cure prolactinoma. Bromocriptine can induce cell apoptosis and decrease PRL secretion and shrink prolactinoma. GH3 cell line, from rat pituitary tumor, which can synthesize and secrete both prolactin and grow hormone, is a useful model system for the study of hormonal dysfunctions. However, its molecular mechanism in regulating GH3 cell apoptosis is still not clear. Caveolin-1, a 23 kDa membrane anchored protein, correlates with lipid transport, membrane traffic, signal transduction, is also been hypothesized as a negative regulator of many signal transduction. Caveolin-1 was cloned by RNA substraction technology and it expressed abundantly in normal mammary epithelial cells compared to tumorous mammary epithelial cells, suggesting that it is a tumor suppressing gene. Here we focus on the role of caveolin-1 in neuro-endocrine system. According to the RT-PCR results, we found that caveolin-1 was expressed both in GH3 and mouse brain tissue. The expression level in mouse brain was elevated at gestation day 18. We successfully cloned caveolin-1 gene from adult mouse brain tissue which was then fused to myc-tag in N-terminal of caveolin-1. Enhanced green fluorescent protein （EGFP） was constructed in the same strategy and was used as a control. These recombinant DNAs were subcloned into a vector driven by cytomegalovious promoter and then transfected separately into GH3 cells. The morphology of GH3 cells appeared to shrink following expression of caveolin-1, while the cells expressing EGFP maintained the normal cell morphology even at 48 hours after transfection. Further more, hoechst stained caveolin-1 expressing GH3 cells showed chromatolysis and the character of cell and cell apoptosis was confirmed by TUNEL assay. To obtain this data, we hypothesize that caveolin may play a role in bromocriptine induced GH3 apoptosis. To explore this, bromocriptine was used to treat GH3 cells for 24 hours, total RNA from the cells were extracted and expression level of caveolin-1 mRNA estimated by semi-quantitative PCR method. The data shows that expression of endogenous caveolin-1 mRNA was elevated following bromocriptine treatment. These results indicate that caveolin-1 possibly play a pivotal role in bromocriptine induce apoptosis in GH3 cells.

並列關鍵字

apoptosis ； caveolin-1

參考文獻

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Caveolin-1在大鼠腦垂腺瘤細胞株的功能性研究

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