臺灣博碩士論文加值系統

本論文以人類角質細胞株 (HaCaT cells) 作為研究材料，探討紫外光 (UV) 對 HaCaT 細胞分化的影響及其機制。文獻指出角質細胞在高鈣離子濃度或低溫下會易於分化。HaCaT 細胞株在原始選殖之時，培養在高溫(38℃)及低鈣 (0.03 mM ) 下，但50代以後 (本實驗所用) 已喪失對溫度及鈣之靈敏度，的確 HaCaT 細胞在基礎態，培養在有鈣或無鈣的情況下，對其生長與分化並無影響。HaCaT 細胞經由 UVB (10mJ/cm2) 照射24小時後，會使細胞生長減緩，早期分化指標蛋白 K1 及 K10 之表現及 ERK 與 AKT 之磷酸化均會增加。移除細胞外鈣離子對 UVB 引起的 K1及 K10 之表現增加及 ERK 與 AKT 磷酸化增加，均會抑制，但對基礎態的細胞則不影響，顯示 UVB 能促進細胞外鈣內流，再使細胞 ERK 及 AKT磷酸化及 K1 及 K10 之表現增加。此外 UVB 亦使得 JNK 磷酸化增加，移除細胞外鈣離子不改變 JNK 磷酸化上升的作用。EGF 能反轉UVB 引起的 K1 及 K10 表現增加及 ERK 磷酸化上升，對 AKT 及 JNK 的磷酸化增加作用則不影響，顯示ERK 磷酸化增加與 K1 及 K10 的表現相關。加入 TrpV 4 通道抑制劑 (RN1734)，對 UVB 之作用不影響。此些結果顯示 UVB 促使細胞外鈣內流，進而造成胞內鈣上升，再活化 ERK，促使細胞分化，表現 K1 及 K10。EGF 能抑制ERK 的磷酸化而抑制此作用。

HaCaT cells were used in this study to investigate the effect of ultraviolet (UV) on the differentiation of keratinocytes and the underlying mechanisms. It has been suggested that keratinocytes undergo terminal differentiation under high Ca2+ concentration and low temperature. During the isolation and selection of HaCaT cell line, the cells were grown in medium containing 0.03 mM Ca2+ at 38oC. However, after 50 passages used in this study, this cell lost its temperature and Ca2+ sensitivity. Indeed, under basal state, the growth and differentiation are indistinguishable in the presence or absence of extracellular Ca2+. Twenty four hr after UVB (10 mJ/cm2) irradiation, the growth of HaCaT cells decreased and the expression of K1 and K10 and the phosphorylation of ERK and Akt increased compared with control cells. Removal of extracellular Ca2+ blocked UVB induced increases of K1 and K10 expression and ERK and Akt phosphorylation, while it has no effect on those in control cells suggesting that UVB induced Ca2+ influx which in turn activated phosphorylation of ERK and Akt and the expression of K1 and K10. In addition, JNK phosphorylation is also induced by UVB which is insensitive to the removal of extracellular Ca2+. Co-treatment of EGF with UVB reversed the increases of K1 and K10 expression and ERK phosphorylation, while it has no effect on Akt and JNK phosphorylation suggesting the correlation between ERK phosphorylation and K1 and K10 expression. Co-treatment of TrpV4 channel blocker RN1734 with UVB had no effect on the action of UVB. Taken together, our results indicate that UVB promotes the differentiation of keratinocytes. This effect depends on Ca2+ influx which in turn activates phosphorylation of ERK and expression of K1 and K10. EGF reverses the phosphorylation of ERK and blocks the differentiation.

目錄
目錄.................................................I
圖文目錄............................................II
縮寫表.............................................III
中文摘要.............................................V
英文摘要...........................................VII
第一章 緒論.........................................1
第一節、角質細胞的生理功能及分化過程.................1
第二節、處理角質細胞的藥物...........................2
第二章 實驗材料與方法................................5
第一節、實驗材料.....................................5
第二節、實驗方法.....................................9
第三章 實驗結果.....................................16
第一節 細胞型態之觀察...............................17
第二節 Baicalein、UVB 與 EGF 對細胞的死亡的影響 ....19
第三節 細胞型態改變之機制討論.......................20
第四章 討論.........................................25
第五章 結論.........................................27

圖文目錄
圖一、Baicalein 與 UV 對 HaCaT 細胞型態的影響.......28
圖二、不同處理下用 Hematoxylin and eosin 染色.......29
圖三、不同條件處理下細胞形態的影響..................30
圖四、Baicalein、UV 與 EGF 對 HaCaT 死亡的影響......31
圖五、不同處理下對各種 kinases 之磷酸化的影..……….32
圖六　、　不同處理與抑制劑處理對 K1 與 K10 的影響...33
圖七、不同處理與抑制 TRPV 4 對 K1 與 K10 的影響 ....34
圖八、處理藥物對 Kinases 磷酸化的影響...............35
圖九、不同的處理下 baicalein 刺激下胞內鈣的變化.....36
圖十、HaCaT 處理各種條件下對 ROS 的影響.............37

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