中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2013年
12期
877-880
,共4页
李聪颖%沈征宇%汪蓓青%徐慧%刘俊岭%王克敏%陈向东
李聰穎%瀋徵宇%汪蓓青%徐慧%劉俊嶺%王剋敏%陳嚮東
리총영%침정우%왕배청%서혜%류준령%왕극민%진향동
基因,ADAR1%Wnt11%黑素细胞%角蛋白细胞
基因,ADAR1%Wnt11%黑素細胞%角蛋白細胞
기인,ADAR1%Wnt11%흑소세포%각단백세포
Genes,ADAR1%Wnt11%Melanocytes%Keratinocytes
目的 探讨遗传性对称性色素异常症致病基因ADAR1对Wnt11表达和酪氨酸酶活性的影响.方法 将HaCaT细胞等细胞数量分为对照组、实验1组、实验2组、实验3组.用3种针对ADAR1基因的shRNA质粒分别沉默3个实验组中HaCaT细胞的ADAR1基因,用Western印迹法检测4个组HaCaT细胞相关蛋白的表达水平.建立HaCaT细胞与黑素瘤细胞A375共培养模型,对实验组(HaCaT细胞中的ADAR1和Wnt 11蛋白低表达)与对照组(HaCaT细胞中的ADAR1和Wnt11蛋白正常表达)的细胞形态和酪氨酸酶活性进行比较.结果 蛋白印迹法发现,ADAR1基因沉默的HaCaT细胞中的Wnt 11蛋白条带灰度值明显低于正常HaCaT细胞中的Wnt 11蛋白条带灰度值,证实HaCaT细胞中的ADAR1基因沉默后,Wnt11蛋白的表达水平明显下降.在共培养模型中,实验组HaCaT细胞与A375细胞连接处的树突状突起明显少于对照组,实验组A375细胞的酪氨酸酶活性与对照组相比显著降低,减去空白组A值后,实验组A值为0.0168±0.0069,对照组为0.0490±0.0132,P＜ 0.01.结论 HaCaT细胞中ADAR1基因沉默可降低Wnt 11的表达,并影响A375细胞酪氨酸酶活性.
目的 探討遺傳性對稱性色素異常癥緻病基因ADAR1對Wnt11錶達和酪氨痠酶活性的影響.方法 將HaCaT細胞等細胞數量分為對照組、實驗1組、實驗2組、實驗3組.用3種針對ADAR1基因的shRNA質粒分彆沉默3箇實驗組中HaCaT細胞的ADAR1基因,用Western印跡法檢測4箇組HaCaT細胞相關蛋白的錶達水平.建立HaCaT細胞與黑素瘤細胞A375共培養模型,對實驗組(HaCaT細胞中的ADAR1和Wnt 11蛋白低錶達)與對照組(HaCaT細胞中的ADAR1和Wnt11蛋白正常錶達)的細胞形態和酪氨痠酶活性進行比較.結果 蛋白印跡法髮現,ADAR1基因沉默的HaCaT細胞中的Wnt 11蛋白條帶灰度值明顯低于正常HaCaT細胞中的Wnt 11蛋白條帶灰度值,證實HaCaT細胞中的ADAR1基因沉默後,Wnt11蛋白的錶達水平明顯下降.在共培養模型中,實驗組HaCaT細胞與A375細胞連接處的樹突狀突起明顯少于對照組,實驗組A375細胞的酪氨痠酶活性與對照組相比顯著降低,減去空白組A值後,實驗組A值為0.0168±0.0069,對照組為0.0490±0.0132,P＜ 0.01.結論 HaCaT細胞中ADAR1基因沉默可降低Wnt 11的錶達,併影響A375細胞酪氨痠酶活性.
목적 탐토유전성대칭성색소이상증치병기인ADAR1대Wnt11표체화락안산매활성적영향.방법 장HaCaT세포등세포수량분위대조조、실험1조、실험2조、실험3조.용3충침대ADAR1기인적shRNA질립분별침묵3개실험조중HaCaT세포적ADAR1기인,용Western인적법검측4개조HaCaT세포상관단백적표체수평.건립HaCaT세포여흑소류세포A375공배양모형,대실험조(HaCaT세포중적ADAR1화Wnt 11단백저표체)여대조조(HaCaT세포중적ADAR1화Wnt11단백정상표체)적세포형태화락안산매활성진행비교.결과 단백인적법발현,ADAR1기인침묵적HaCaT세포중적Wnt 11단백조대회도치명현저우정상HaCaT세포중적Wnt 11단백조대회도치,증실HaCaT세포중적ADAR1기인침묵후,Wnt11단백적표체수평명현하강.재공배양모형중,실험조HaCaT세포여A375세포련접처적수돌상돌기명현소우대조조,실험조A375세포적락안산매활성여대조조상비현저강저,감거공백조A치후,실험조A치위0.0168±0.0069,대조조위0.0490±0.0132,P＜ 0.01.결론 HaCaT세포중ADAR1기인침묵가강저Wnt 11적표체,병영향A375세포락안산매활성.
Objective To estimate the influence of ADAR1 gene,which is considered to be responsible for the pathogenesis of dyschromatosis symmetrica hereditaria,on Wnt1 1 expression and tyrosinase activity.Methods Some cultured HaCaT cells were equally divided into four groups:control group remaining untreated,three experimental groups transfected with three different ADAR1-specific shRNAs respectively.Then,Western blot was performed to quantify the expression of Wnt11 protein in HaCaT cells so as to select the most potent shRNA.Some human A375 melanoma cells were cocultured with untransfected HaCaT cells (normally expressing ADAR1 and Wnt1 1 proteins) or HaCaT cells transfected with the selected specific shRNA (lowly expressing ADAR1 and Wnt11 proteins).Thereafter,cell appearance was observed using inverted microscopy at 24,48 and 72 hours,and tyrosinase activity was estimated at 48 hours.Results As Western blot showed,the expression of Wnt 11 protein was significantly lower in the three ADAR1-silenced experimental groups than in the control group.The number of dendritic protrusions at the junction sites between HaCaT cells and A375 cells was significantly decreased,together with a significant reduction in tyrosinase activity (absorbance value:0.0168 ± 0.0069 vs.0.0490 ± 0.0132,P ＜0.01),in A375 cells cocultured with transfected HaCaT cells compared with those cocultured with normal control HaCaT cells.Conclusion ADAR1 gene silencing in HaCaT cells can attenuate the expression of Wnt11 protein,and affect tyrosinase activity in A375 cells.
