高糖對人類臍帶靜脈內皮細胞之生物效應與訊息傳遞途徑之探討

Study on the biological responses and signal transduction pathways of high glucose in human umbilical vein endothelial cells (HUVECs)

陳燕輝(Yen-Hui Chen)

指導教授 ： 莊麗月

高雄醫學大學/醫學院/醫學研究所/博士(2007年)

https://doi.org/10.6832/KMU.2007.00125

摘要

高糖對於糖尿病血管病變的影響十分深遠，對血管病變而言，血管內皮細胞的損傷是最初的病理特徵。為了進一步研究致病機轉，本實驗將人類臍帶血管內皮細胞培養於高糖(28.5 mM)之培養基中，由胸腺嘧啶標誌反應( 3[H]thymidine incorporation )及計算細胞數目，得知高糖會呈時間效應抑制細胞分裂( cellular mitogenesis)，進而使細胞停滯在G0/G1期。其次，高糖誘發細胞膠原蛋白合成與TGF-??1 mRNA與 轉錄表現的增加。同時，N-acetylcysteine能逆轉高糖抑制細胞生長和促進膠原蛋白合成的效應。由於首次發現高糖會促進p15 INK4b和p15.5 INK4b蛋白質的增加，所以，我們針對p15 INK4b和p15.5 INK4b做研究分析，逆轉錄聚合脢鏈反應(reverse transcription polymerase chain reaction, RT-PCR)實驗的結果證實，人類臍帶靜脈血管內皮細胞的p15 mRNA會呈時間效應增加，同時高糖處理也呈劑量與時間效應的增加p15 INK4b和p15.5 INK4b蛋白質。接著我們實驗中送入p15 antisense也能回復高糖對人類臍帶靜脈血管內皮細胞生長抑制的效應，顯示具有保護血管功能的作用。另外，針對高糖促進p15 INK4b和p15.5 INK4b蛋白質的訊息傳遞途徑作研究，我們發現高糖刺激12小時，使得ERK1/2活性增加到最高。進一步使用PD98059 (MEK抑制劑)培養一天後，能有效的回復高糖所造成細胞滯留在G0/G1期與p15 INK4b和p15.5 INK4b蛋白質增加。另外，使用TGF-?狺予M性抗體和TGF-???n接受器抑制劑 (SB431542)培養一天後，無法有效的回復高糖所造成p15 INK4b和p15.5 INK4b蛋白質增加。但是，N-acetylcysteine (NAC)和calphostin C可以有效被逆轉高糖所增加p15 INK4b和p15.5 INK4b蛋白質和細胞外訊息調控激酶(Extracellular signal-regulated kinase, ERK1/2)活性。綜合上述結果，高糖可以藉由增加p15 INK4b和p15.5 INK4b的mRNA和蛋白質的表現而抑制細胞分裂，並且透過PKC/ROS/ERK1/2訊息傳遞的路徑。同時，高糖也透過PKC/ROS/ERK1/2訊息傳遞的路徑誘發p21 WAF1/CIP1、p53蛋白質增加和 p53(ser15)的磷酸化以及抑制cyclin B1蛋白質。除了p21 WAF1/CIP1和cyclin B1之外，我們也首次發現高糖透過p53路徑誘發p15 INK4b和p15.5 INK4b蛋白質增加。總之，高糖對人類臍帶靜脈血管內皮細胞的生物效應會經由PKC/ROS/ERK1/2訊息傳遞的路徑。

關鍵字

內皮細胞 ； 細胞生長 ； 細胞週期 ； 細胞外訊息調控激酶氧化壓力 ； 周期數依賴型蛋白激酶抑制劑

並列摘要

Hyperglycemia is a major cause of diabetic vascular disease. High glucose is associated with reduce [3H] thymidine incorporation into DNA and cell proliferation in human umbilical vein endothelial cell (HUVECs). Although high glucose may inhibit cell growth and several serine-threonine kinase, the exact mechanism by which it promotes cell cycle arrest is still unclear. In this study, we demonstrated that high glucose at a range of concentrations (18.5-38.5 mM) dose and time-dependently inhibited [3H] thymidine incorporation and decreased cell number in culture HUVECs. Flow cytometry analyses demonstrated that hyperglycemia arrested the cell at the G0/G1 phase of the cell cycle. High glucose also induced collagen and TGF-?? mRNA. N-acetylcysteine (an antioxidant, NAC) attenuated high glucose-inhibited cell proliferation and high glucose-induced collagen. Both mRNA and protein of the CDK inhibitor p15 INK4b and p15.5INK4b in HUVECs were time-dependently induced by high glucose (28.5 mM). In addition, pretreatment of HUVECs with 0.5 ?嵱 p15INK4b antisense oligonucleotide reversed the hyperglycemia-induced inhibition of [3H] thymidine incorporation and up-regulation of p15 INK4b and p15.5 INK4b protein into HUVECs for 24 h. The phosphorylation of ERK1/2 was increased by high glucose exposure 12 h and gradually reduced after long exposure in HUVECs. Pretreatment of 15 ?嵱 PD98095, an inhibitor of MEK which is the upstream kinase of ERK1/2, reverse high glucose-induced G0/G1 phase of cell cycle arrest in HUVECs. PD98095 also blocked induction of p15INK4b and p15.5INK4b by high glucose for 24 h in HUVECs. These results indicate that high glucose-induced HUVECs growth inhibition are associated with p15INK4b and p15.5INK4b and stimulation of ERK pathway can result in an induction of CDK inhibitor p15INK4b and p15.5INK4b and cell cycle arrest. High glucose increased transforming growth factor-?? (TGF-??) gene transcriptional activity and mRNA expression. However, neither SB431542 (type I TGF-?? receptor blocker) nor TGF-??1 antibody affected high glucose-induced p15INK4b and p15.5INK4b protein expression. NAC and calphostin C could decrease high glucose-induced ERK1/2 activity. Thus, high glucose induced p15INK4b and p15.5INK4b via PKC/ROS/ERK1/2 pathway. Similary, high glucose induced p21 WAF1, p53 and p53(ser15) and inhibited cyclinB1 via PKC/ROS/ERK1/2 pathway. High glucoe induced p15INK4b and p15.5INK4b via p53 pathway. Taken together, these result suggest that high glucose-induced growth arrest is dependent on p15 INK4b、 p15.5 INK4b、 p21 WAF1/CIP1、 p53 and cyclin B1 via PKC/ROS/ERK1/2 pathway.

並列關鍵字

Endothelial cells ； Proliferation ； Cell cycle ； ERK ； Oxidative stress ； CDKIs

參考文獻

參考文獻

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