- 學位論文
第二型糖尿病致病機轉中金屬依賴型基質分解酶與第四型介白素之間的交互作用
Interaction between matrix metalloproteinases and interleukin-4 in type 2 diabetes mellitus
摘要
糖尿病是常見的內分泌疾病，但是其詳細的致病機轉仍然不清楚。有許多文獻報導指出，個體免疫作用和發炎反應對於第二型糖尿病之發病與後續併發症之衍生過程扮演重要的角色。本實驗室先前的研究結果顯示，與健康個體做比較，較多的第二型糖尿病患者帶有轉錄活性較高之第四型介白素(interleukin-4, IL-4)啟動子基因型，而且週邊血液單核細胞分泌IL-4的能力也比健康個體之單核細胞高。很多研究指出糖尿病患者腎病變病灶處matrix metalloproteinase 2 (MMP2)與matrix metalloproteinase 9 (MMP9)活性較高，臨床研究證實高血糖對間質細胞和血管平滑肌細胞的影響包括使基底膜增厚以及細胞外基質的堆積，這些都是造成糖尿病腎病變的關鍵因素；在糖尿病患者視網膜病變病過程中，視網膜細胞MMP9的表現量及活性也較正常人高。但上述大部份的研究皆主要針對病灶處探討高血糖對MMP的影響，然而，高血糖對全身性MMPs的表現量以及活化的影響目前尚未知。因此本論文利用3T3-L1細胞為實驗模式，檢測在高或低濃度葡萄糖環境下，IL-4對MMPs表現量的影響以探討發炎反應與MMPs系統之間的交互作用，進一步推論IL-4參與第二型糖尿病的致病機轉。結果顯示在高濃度葡萄糖環境下，與未做任何處理的細胞組相比較，在IL-4前處理30分鐘後，再以低濃度胰島素處理18小時，3T3-L1纖維母細胞MMP2的mRNA表現量上升；但若IL-4和/或胰島素的處理之下，MMP9的mRNA表現量上升。我們利用gelatin-zymography分析MMP2和MMP9的蛋白質活性，發現在高濃度葡萄糖環境下，與未做任何處理的細胞組相比較，IL-4處理30分鐘、高濃度胰島素處理30分鐘或是IL-4前處理30分鐘再以高濃度胰島素處理30分鐘，3T3-L1纖維母細胞MMP2的蛋白質活性有下降的趨勢，但若以低濃度胰島素處理18小時或是IL-4前處理30分鐘後再以低濃度胰島素處理18小時，MMP2的蛋白質活性卻有上升的趨勢；另一方面，發現在高濃度葡萄糖環境下，與未做任何處理的細胞組相比較，IL-4處理30分鐘或是IL-4前處理30分鐘後再以高濃度胰島素處理30分鐘，3T3-L1纖維母細胞MMP9的蛋白質活性有下降的趨勢，但若以低濃度胰島素處理18小時、高濃度胰島素處理30分鐘或是IL-4前處理30分中後再以低濃度胰島素處理18小時，MMP9的蛋白質活性有上升的趨勢。另外在低濃度葡萄糖環境下，IL-4前處理30分鐘後，與換置低濃度葡萄糖環境下並未做任何處理的細胞組相比較，不論是換置高或低濃度的葡萄糖培養液中再以低濃度胰島素/高濃度胰島素處理之下，3T3-L1纖維母細胞MMP2的蛋白質活性有上升的趨勢；更發現在低濃度葡萄糖環境下，IL-4前處理30分鐘後，與換置低濃度葡萄糖環境下並未做任何處理的細胞組相比較，如果換置低濃度葡萄糖培養液下並以低濃度胰島素處理下，3T3-L1纖維母細胞MMP9的蛋白質活性有下降的趨勢，但若換置在高或低濃度葡萄糖培養液下並以高濃度胰島素處理下，MMP9的蛋白質活性卻有上升的趨勢。我們利用分化技術將3T3-L1纖維母細胞成功分化成為3T3-L1脂肪細胞，發現在高濃度葡萄糖環境下，與未做任何處理的細胞組相比較，IL-4處理30分鐘、高濃度胰島素處理30分鐘、低濃度胰島素處理18小時或是IL-4前處理再以高/低濃度胰島素處理下，3T3-L1脂肪細胞MMP2的mRNA表現量有下降的趨勢；MMP9的mRNA表現量卻有上升的趨勢。因為糖尿病患者通常有肥胖的現象，脂肪組織會分泌許多脂肪細胞激素，這些激素在調控能量衡定中扮演著重要的角色。整合先前研究結果，我們推論在糖尿病患者中，透過週邊血液高濃度IL-4調控體內纖維母細胞以及脂肪細胞MMPs的表現量以及活性，影響細胞正常功能而導致一些糖尿病併發症的產生，以證明IL-4參與第二型糖尿病的致病機轉。
關鍵字
基底膜 ； 細胞外基質 ； 介白質素-4 ； 金屬依賴型基質水解酵素 ； 金屬依賴型基質水解酵素2 ； 金屬依賴型基質水解酵素9 ； 第二型糖尿病 ； 轉型生長因子b ； 腫瘤壞死因子a ； 金屬依賴型基質水解酵素之組織抑制劑並列摘要
Diabetes mellitus (DM) is a common endocrine disease with unknown etiology. Immune response and inflammation are suggested to play important roles in the development and complications of type 2 diabetes mellitus (T2DM). Our previous studies revealed that individuals carrying genotypes of high interleukin-4 (IL-4) secreting ability and higher IL-4 levels were susceptible to diabetic development. Several studies documented that MMP2 and MMP9 were down-regulated in diabetic nephropathy. However, whether and to what degree hyperglycemia affects the expression and activity of the MMPs induction/activation system in patients with diabetes remains unknown. Therefore, we designed to examine the putative cross talk between inflammation and MMP system by investigating the putative regulation and effect of IL-4 to MMP expression by using 3T3-L1 cells to further elucidate the roles of MMP and IL-4 in the pathogenesis of T2DM. Our data showed that MMP2 mRNA expression was increased in 3T3-L1 fibroblasts by pre-treatment of IL-4 for 30 min and then chronic insulin for 18 hrs under high glucose condition in contrast of control without any treatment. The MMP9 mRNA expression was increased by treatment of IL-4 and/or insulin under high glucose condition in contrast of control without any treatment. We detect MMP2 and MMP9 activity by using gelatin-zymography analysis. We find that the MMP2 activity was decreased by treatment of IL-4 for 30 min or acute insulin for 30 min or pre-treatment of IL-4 for 30 min and then acute insulin for 30 min under high glucose condition in contrast of control without any treatment. But, the MMP2 activity was increased by treatment of chronic insulin for 18 hrs or pre-treatment of IL-4 for 30 min and then treatment of chronic insulin for 18 hrs under high glucose condition in contrast of control without any treatment. Otherwise, the MMP9 activity was decreased by treatment of IL-4 for 30 min or pre-treatment of IL-4 for 30 min and then acute insulin for 30 min under high glucose condition in contrast of control without any treatment. The MMP9 activity was increased fibroblasts by treatment of chronic insulin for 18 hrs or acute insulin for 30 min or pre-treatment of IL-4 for 30 min and then chronic insulin for 18 hrs under high glucose condition in contrast of control without any treatment. Besides, we find that MMP2 activity was increased fibroblasts by pre-treatment of IL-4 for 30 min under low glucose condition and then insulin in high or low glucose condition in contrast of control with only pre-treatment of IL-4 for 30 min under low glucose condition. The MMP9 activity was decreased by pre-treatment of IL-4 for 30 min under low glucose condition and then chronic insulin for 18 hrs in contrast of control with only pre-treatment of IL-4 for 30 min. But, the MMP9 activity was increased by pre-treatment of IL-4 for 30 min and then acute insulin for 30 min under high or low glucose condition in contrast of control with only pre-treatment of IL-4 for 30 min in low glucose condition. Using well-differentiation technique, 3T3-L1 fibroblasts were induced to 3T3-L1 adipocytes. We find that the MMP2 mRNA expression was decreased and the MMP9 mRNA expression was increased in 3T3-L1 adipocytes by treatment of IL-4 for 30 min or acute insulin for 30 min or chronic insulin for 18 hrs or pre-treatment of IL-4 for 30 min and then insulin under high glucose condition in contrast f control without any treatment. This common form of diabetes is often associated with obesity. Adipose tissues will secrete many adipocytokines and play a role in energy balance. Thus, we can evidence the putative mechanism of IL-4 regulation to MMPs system in type 2 diabetes. Above all, we can suggest that IL-4 may be involved in some complications of diabetes by regulating MMPs system.