臺灣博碩士論文加值系統

背景：在糖尿病腎病變造成的纖維化中，高血糖是一個重要的危險因子。先前的研究發現，IL-7具有抗肺纖維化的潛力。然而，IL-7在腎纖維化的過程扮演的角色未知。因此，我們想探討IL-7在腎近端小管上皮細胞高糖的環境下所扮演的角色。方法：細胞培養在高糖最終濃度為27.5 mM兩天，在最後24小時加入不同濃度的IL-7 (10、 50、100與 200 ng/ml)。利用ELISA來偵測細胞外分泌的纖維蛋白及乙型生長轉型因子濃度。再使用Western blot來觀察EMT的標記【包括α-smooth muscle actin ( α-SMA) 及 E-cadherin】，訊息傳遞，及EMT的啟動子( Snail, Slug )之表現。以及使用免疫螢光染色來觀察原位蛋白的表現（譬如Fibronectin, α-SMA, E-cadherin, Snail, Slug）。結果：我們發現IL-7抑制高糖誘導減少的細胞生長及抑制高糖誘導的細胞纖維化。並且IL-7能夠阻斷高糖誘導所升高的纖維蛋白，第一型乙型轉型生長因子，第一型乙型轉型生長因子接受器以及磷酸化Smad2/3。相反的，IL-7可以逆轉高糖所誘導減少的Smad7。除此之外，IL-7能夠抑制高糖所誘導增加的α-SMA, Snail及Slug，並且能逆轉高糖誘導減少的E-cadherin。結論：IL-7抑制高糖誘導的腎近端小管纖維化可能通過調節 Smads和EMT的途徑。

Background: Hyperglycemia is the most important risk factor in the progression of renal fibrosis in diabetic kidney. Based on previous studies, interleukin-7 (IL-7) may possess antifibrotic activities in pulmonary fibrosis model. However, the role of IL-7 in the pathogenesis of renal tubulointerstitial fibrosis remains unclear. Thus, we hereby elucidate the effects of IL-7 in cultured renal proximal tubular epithelial cells (designated as HK-2) treated under hyperglycemic media. Material and Methods: Cells were cultured in high glucose (HG, 27.5mM) for 2 days. Different concentration of IL-7 (10、 50、100 or 200ng/ml) was added in the last 24 hours of culture. ELISA was used to evaluate the secreted protein such as fibronectin and TGF-β1. Western blot was used to examine the EMT marker (including α-smooth muscle actin (α-SMA) and E-cadherin), signal transducer (including Smad Smad2/3 and Smad7) and EMT initiator (e.q Snail, Slug). Immunofluorescence staining was used to assay the in situ expression of proteins (e.q. α-SMA, E-cadherin and Snail). Results: We found that IL-7 significantly attenuated HG-inhibited cellular growth and HG-induced fibrosis. HG-induced up-regulation of fibronectin, TGF-β, TGF-β RII and p-smad2/3 was markedly inhibited by IL-7. On the contrary, HG-induced down-regulation of smad7 was significantly reversed by IL-7 instead. Moreover, IL-7 markedly inhibited HG-induced increase in α-smooth muscle actin and snail. Whereas HG-induced decrease in E-cadherin expression was reversed as well. Conclusion, IL-7 has the potential to inhibit high glucose-induced renal tubular fibrosis possibly by modulating Smads and EMT pathway.

口試委員會審定書…………………………………………………i
授權書…………………………………………………………...….ii
誌謝………………………………………………………………....iii
中文摘要……………………………………………...……………iv
英文摘要………………………………………………………...…v
縮寫表…………………………………………………………....…vi
第一章 緒論 1
一、糖尿病腎病變之盛行率 2
二、糖尿病腎臟纖維化之致病與機轉 3
三、腎臟纖維化與EMT過程之相關 6
四、高糖與糖尿病腎病變之關係 8
五、乙型轉型生長因子在糖尿病腎病變中扮演之角色………10
六、纖維化之訊息傳導路徑 ...13
七、白介素-7簡介 18
第二章 研究目的 22
第三章 材料與方法 24
一、試劑 25
二、方法 25
1. 細胞培養 26
( 1 ) 繼代細胞 26
( 2 ) 活化細胞 26
( 3 ) 細胞保存 27
2. 細胞數目 27
3. 酵素連結免疫吸附分析 ( ELISA ) 27
4. 免疫螢光分析 ( Immunofluorescence ) 28
5. 細胞蛋白質總量測定 28
6. 西方墨點分析 29
7. 統計分析 30
第四章 結果 31
一、IL-7對27.5 mM的D-glucose誘導纖維化情形下腎近端小管細胞生長的影響 ….. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...…32
二、IL-7對27.5 mM的D-glucose誘導纖維化情形下腎近端小管纖維蛋白的影響……………………………………………………32
三、IL-7減少27.5 mM的D-glucose誘導腎近端小管細胞外分泌TGF-β1.. .….….….….….….….….….….….….…..….….….…33
四、IL-7對27.5 mM的D-glucose誘導纖維化情形下腎近端小管細胞TGF-β1受器的影響……………………………………………34
五、IL-7對27.5 mM的D-glucose誘導下腎近端小管細胞Smad2/3, pSmad2/3, Smad4 以及 Smad7的影響………………...………34
六、IL-7抑制27.5 mM的D-glucose誘導下腎近端小管細胞胞內上皮型態的影響……………………………………………...…….35
七、IL-7對27.5 mM的D-glucose誘導下腎近端小管細胞於免疫螢光觀察上皮型態的影響……………………………………..…..36
八、IL-7抑制27.5 mM的D-glucose誘導下腎近端小管細胞纖維化的調控機制…………………………………………………....37
第五章 討論 …….38
第六章 結論 42
第七章 圖表 44
第八章 參考文獻 53

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