臺灣博碩士論文加值系統

Diabetes mellitus (DM) is characterized by impaired insulin signaling , elevated plasma glucose, and a predisposition toward chronic complications involving several tissues. Among the chronic complication of DM, the dermatological complications are among the least thoroughly studied. Impaired wound healing is one of the major dermatological complications in diabetic patients. Normal wound healing can be divided into four phases: (1) hemostasis and inflammation phase; (2) re-epithelialization phase; (3) contraction and synthetic phase; (4) remodeling phase. In the re-epithelialization process during cutaneous wound healing, the migration and proliferation of keratinocytes (KCs) are crucial steps. Although numerous studies discussing the poor wound healing process in diabetics have been reported, the effects of glucose on KC migration and proliferation are scarcely studied. Our current studies aim to explore the effects of high glucose on cultured human KC migration and proliferation. Our results showed that the proliferation of KCs was significantly decreased by high glucose treatment. In addition, using in vitro wound scratch assay and Transwell migration assay, we demonstrated that the motility of KCs was significantly reduced under hyperglycemic condition. According to the aforementioned phenomena, we further investigated the possible molecular mechanisms involved. The results of RT-PCR revealed that high glucose treatment downregulated the expressions of MMP-1, MMP-2 and MMP-9 in KCs. Furthermore, the expression of TIMP-1 was increased after high glucose treatment. Focal adhesion kinase (p125FAK) plays a pivotal role in cell migration. In our study, expressions of p125FAK and phosphorylated p125FAK (pp125FAK) in KCs were decreased under hyperglycemic condition. In this study, we also demonstrated that high glucose inhibited the expression of integrin α2β1、α3β1 on KCs.
In conclusion, our results indicated that the decrease in the migration and proliferation of KCs after high glucose treatment might be related to the inhibition of re-epithelialization during wound healing. Our results may provide in vitro evidences for poor wound healing in diabetic patients.

目錄…………………………………………………………………… 1
中文摘要……………………………………………………………… 3
英文摘要……………………………………………………………… 5
前言…………………………………………………………………… 7
材料與實驗方法……………………………………………………….14
人類表皮角質細胞之培養………………………………………. 14
細胞外基質蛋白 (Extracellular matrix,ECM) 塗覆…………… 14
以不同濃度D-glucose 作用人類培養角質細胞……………….15
MTS 法檢測細胞增生能力……………………………… 15
細胞增生能力試驗 (BrdU-ELISA)………………………………16
刮傷實驗 (Wound scratch assay)……………………………….. 17
細胞移動能力試驗 (Transwell migration assay)…………….. 17
以 RT-PCR 分析 mRNA之表現………………………………. 19
西方墨點法 (Western blotting analysis)………………………20
Cellular Enzyme-Linked Immunosorbant Assay……………… 22
統計分析方法…………………………………………………… 23
結果…………………………………………………………………… 25
高濃度 D-glucose 對 KCs 增生之研究……………………… 25
高濃度 D-glucose 對 KCs 移動之研究……………………….27
利用刮傷試驗觀察 KCs 移動的影響……………………. 27
利用細胞移動能力試驗觀察 KCs 移動的影響…… 28
高濃度 D-glucose 對 KCs MMP-1、MMP-2、MMP-9、
TIMP-1與TIMP-2 mRNA表現的影響…………………… 29
高濃度 D-glucose 對 KCs p125FAK 和pp125FAK 表現的影
響………………………………………………………… 31
高濃度 D-glucose 對 KCs integrins 表現之調控…… 33
討論…………………………………………………………………… 39
未來研究方向………………………………………………………… 44
圖表…………………………………………………………………… 45
參考文獻……………………………………………………………… 78

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