胰島素對血漿激肽釋放酶基因表現之影響

The effects of insulin on plasma kallikrein gene expression

洪瑞駿(Ruei-Jyun Hong)

指導教授 ： 譚棉心

高雄醫學大學/生命科學院/生物醫學暨環境生物學研究所/碩士(2010年)

https://doi.org/10.6832/KMU.2010.00020

摘要

血漿激肽釋放酶(plasma kallikrein)為絲氨酸分解酶(serine protease)，由肝臟細胞hKLKB1基因經轉錄及轉譯產生，並分泌至血液循環全身。前人研究發現，血漿激態釋放酶大量表現於高血壓及第一型糖尿病患者血液中。在無法分泌胰島素的第一型糖尿病模式鼠血液中，亦發現異常高量的血漿激肽釋放酶，對其進行腹腔胰島素注射後，血液中血漿激肽釋放酶含量大幅下降，這一現象暗示胰島素似乎可抑制血漿激肽釋放酶表現。為釐清胰島素在肝臟細胞表現血漿激肽釋放酶的影響，HepG2肝臟細胞株以胰島素刺激，並利用反轉錄聚合酵素鏈鎖反應及西方點墨法分析血漿激肽釋放酶是否受胰島素影響。在胰島素刺激24小時，肝臟細胞所表現之血漿激肽釋放酶訊息核糖核酸受到顯著抑制。為研究參與其中的訊息傳遞路徑，利用抑制劑PD98059與LY294002抑制胰島素主要的兩條訊息路徑：MAPK及PI3K/Akt pathway。受胰島素抑制之血漿激肽釋放酶在PI3K/Akt路徑抑制劑處理後表現量回升，因而推測胰島素藉由PI3K/Akt訊息傳遞路徑調控肝臟細胞血漿激肽釋放酶的表現。受胰島素調控的血漿激肽釋放酶啟動子區域則利用重組質體探討，經由報導基因(secreted alkaline phosphatase)的分析，指出胰島素的調控區域可能介於啟動子上-1259至-1159區段。截至目前為止無相關研究探討體內如何血漿激肽釋放酶之表現，透過本篇論文研究胰島素調控血漿激肽釋放酶表現之機制，可增進我們瞭解胰島素在肝臟中不僅止於協調能量代謝平衡，透過調控血漿激肽釋放酶，胰島素在肝臟亦扮演調控血壓的要角。

關鍵字

胰島素 ； 血漿激肽釋放酶第一型糖尿病

並列摘要

Plasma kallikrein, a serine protease, is encoded by KLKB1 gene and is secreted by hepatocytes. It can regulate blood pressure through the proteolytic activation of bradykinin and angiotensin II. In previous studies, the plasma kallikrein was significantly increased in hypertension patients and type I diabetic patients. After insulin injection in streptozotocin-induced diabetes rats, the plasma kallikrein was decreased dramatically. It assumed KLKB1 gene expression might be down-regulated by insulin. To clarify the effect of insulin on KLKB1 mRNA expression, we treated human hepatoma cell line HepG2 with insulin, and the effect of insulin on KLKB1 gene expression was detected by RT-PCR and Western blotting. With insulin treatment, KLKB1 gene expression was repressed significantly after 24 hours. It indicated insulin could inhibit KLKB1 gene expression in HepG2 cells. To understand which insulin pathway involved in KLKB1 gene expression, the inhibitors for PI3K/Akt and MAPK pathway were used. With inhibition of PI3K/Akt pathway, the insulin-repressed KLKB1 mRNA level was recovered. These data suggested insulin repressed KLKB1 gene expression through PI3K/Akt pathway. To investigate the potential regulated elements on KLKB1 promoter, a series recombinant plasmids were constructed, the reporter assay indicated the insulin-regulated element may lie in between -1259 to -1159 of the KLKB1 promoter. With the research of insulin regulation on plasma kallirein expression, it may contribute to our growing understanding of the role of insulin in liver and the regulatory mechanisms of insulin on KLKB1 gene expression.

並列關鍵字

insulin ； plasma kallikrein ； type I diabetes

參考文獻

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