中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2014年
4期
233-236
,共4页
金慧%陈旭%徐松%曹蕾%黄丹%鞠梅%陈崑%李新宇%周之海
金慧%陳旭%徐鬆%曹蕾%黃丹%鞠梅%陳崑%李新宇%週之海
금혜%진욱%서송%조뢰%황단%국매%진곤%리신우%주지해
紫外线%角蛋白细胞%原癌基因蛋白质类%Beclin-1%Atg12%Atg3
紫外線%角蛋白細胞%原癌基因蛋白質類%Beclin-1%Atg12%Atg3
자외선%각단백세포%원암기인단백질류%Beclin-1%Atg12%Atg3
Ultraviolet rays%Keratinocytes%Proto-oncogene proteins%Beclin-1%Atg12%Atg3
目的 探讨不同剂量中波紫外线(UVB)照射培养的HaCaT细胞后不同时间点对p62、Beclin-1、Atg12和Atg3蛋白表达水平的调控效应.方法 使用4.5、10和50 J/cm2 UVB照射HaCaT细胞,照射后加入新鲜培养基继续培养4h和12 h,同时设相同处理但不照射UVB的细胞为对照细胞.另设观察组,在照射后立即(包括对照细胞),使用含蛋白酶抑制剂E64D(10 μg/L)和胃酶抑素(10 μg/L)的培养基孵育4h和12h,以阻断对p62的降解.使用Western印迹法测定HaCaT细胞p62、Beclin-1、Atg12和Atg3蛋白的表达水平.结果 50 mJ/cm2 UVB照射HaCaT细胞后4h,p62表达水平(p62与内参的比值为0.473±0.022)较对照细胞(0.246±0.038)升高(t=15.27,P＜0.05);阻断溶酶体后,细胞新生p62的水平(0.445±0.035)与阻断溶酶体的对照(0.244±0.016)相比,仍为上调(t=7.62,P＜ 0.05).在4.5 mJ/cm2 UVB照射细胞后12h,与对照(0.254±0.035)相比,HaCaT细胞p62表达上调(0.497±0.047,t=22.89,P＜0.05);阻断溶酶体后,与阻断溶酶体的对照(0.257±0.025)相比,p62表达水平仍然上调(0.548±0.051,t=17.42,P＜0.05).4.5 mJ/cm2和50 mJ/cm2UVB照射后4h和12h,对照细胞和照射细胞间的Beclin-1、Atg12和Atg3的表达差异均无统计学意义(P＞0.05),阻止溶酶体对自噬体内容物的降解也未能发现Beclin-1、Atg12和Atg3的表达差异(P＞0.05).结论 p62表达在不同剂量UVB照射和照射后不同时间存在差异调控,并且这种调控效应与自噬体形成可能没有生物学联系.
目的 探討不同劑量中波紫外線(UVB)照射培養的HaCaT細胞後不同時間點對p62、Beclin-1、Atg12和Atg3蛋白錶達水平的調控效應.方法 使用4.5、10和50 J/cm2 UVB照射HaCaT細胞,照射後加入新鮮培養基繼續培養4h和12 h,同時設相同處理但不照射UVB的細胞為對照細胞.另設觀察組,在照射後立即(包括對照細胞),使用含蛋白酶抑製劑E64D(10 μg/L)和胃酶抑素(10 μg/L)的培養基孵育4h和12h,以阻斷對p62的降解.使用Western印跡法測定HaCaT細胞p62、Beclin-1、Atg12和Atg3蛋白的錶達水平.結果 50 mJ/cm2 UVB照射HaCaT細胞後4h,p62錶達水平(p62與內參的比值為0.473±0.022)較對照細胞(0.246±0.038)升高(t=15.27,P＜0.05);阻斷溶酶體後,細胞新生p62的水平(0.445±0.035)與阻斷溶酶體的對照(0.244±0.016)相比,仍為上調(t=7.62,P＜ 0.05).在4.5 mJ/cm2 UVB照射細胞後12h,與對照(0.254±0.035)相比,HaCaT細胞p62錶達上調(0.497±0.047,t=22.89,P＜0.05);阻斷溶酶體後,與阻斷溶酶體的對照(0.257±0.025)相比,p62錶達水平仍然上調(0.548±0.051,t=17.42,P＜0.05).4.5 mJ/cm2和50 mJ/cm2UVB照射後4h和12h,對照細胞和照射細胞間的Beclin-1、Atg12和Atg3的錶達差異均無統計學意義(P＞0.05),阻止溶酶體對自噬體內容物的降解也未能髮現Beclin-1、Atg12和Atg3的錶達差異(P＞0.05).結論 p62錶達在不同劑量UVB照射和照射後不同時間存在差異調控,併且這種調控效應與自噬體形成可能沒有生物學聯繫.
목적 탐토불동제량중파자외선(UVB)조사배양적HaCaT세포후불동시간점대p62、Beclin-1、Atg12화Atg3단백표체수평적조공효응.방법 사용4.5、10화50 J/cm2 UVB조사HaCaT세포,조사후가입신선배양기계속배양4h화12 h,동시설상동처리단불조사UVB적세포위대조세포.령설관찰조,재조사후립즉(포괄대조세포),사용함단백매억제제E64D(10 μg/L)화위매억소(10 μg/L)적배양기부육4h화12h,이조단대p62적강해.사용Western인적법측정HaCaT세포p62、Beclin-1、Atg12화Atg3단백적표체수평.결과 50 mJ/cm2 UVB조사HaCaT세포후4h,p62표체수평(p62여내삼적비치위0.473±0.022)교대조세포(0.246±0.038)승고(t=15.27,P＜0.05);조단용매체후,세포신생p62적수평(0.445±0.035)여조단용매체적대조(0.244±0.016)상비,잉위상조(t=7.62,P＜ 0.05).재4.5 mJ/cm2 UVB조사세포후12h,여대조(0.254±0.035)상비,HaCaT세포p62표체상조(0.497±0.047,t=22.89,P＜0.05);조단용매체후,여조단용매체적대조(0.257±0.025)상비,p62표체수평잉연상조(0.548±0.051,t=17.42,P＜0.05).4.5 mJ/cm2화50 mJ/cm2UVB조사후4h화12h,대조세포화조사세포간적Beclin-1、Atg12화Atg3적표체차이균무통계학의의(P＞0.05),조지용매체대자서체내용물적강해야미능발현Beclin-1、Atg12화Atg3적표체차이(P＞0.05).결론 p62표체재불동제량UVB조사화조사후불동시간존재차이조공,병차저충조공효응여자서체형성가능몰유생물학련계.
Objective To evaluate the regulatory effects of different doses of ultraviolet B (UVB) radiation on the expressions of p62,Beclin-1,Atg12 and Atg3 proteins in the human keratinocyte cell line HaCaT.Methods Cultured HaCaT cells were divided into several groups to be irradiated with three doses of UVB (4.5,10 and 50 mJ/cm2) immediately followed by additional culture with or without the lysosomal protease inhibitors E64D of 10 μg/L and pepstatin of 10 μg/L,both of which were used to block p62 degradation,for 4 and 12 hours respectively.Those HaCaT cells receiving the same treatment but no irradiation served as the control.Subsequently,Western blot was performed to determine the expression levels of p62,Beclin-1,Atgl2 and Atg3 proteins in these cells.The protein expression level was represented as the ratio of the product of absorbance value and area of the electrophoretic bands of target proteins to that of β-tubulin.Paired t test was performed to compare the expression levels of these proteins among different groups.Results After irradiation with UVB of 50 mJ/cm2,the expression level of p62 was significantly upregulated at 4 hours in unblocked HaCaT cells compared with unblocked unirradiated cells (0.473 ± 0.022 vs.0.246 ± 0.038,t =15.27,P ＜ 0.05),and in blocked HaCaT cells compared with blocked unirradiated cells (0.445 ± 0.035 vs.0.244 ± 0.016,t =7.62,P ＜ 0.05).Increased p62 expression was also observed in unblocked and blocked HaCaT cells at 12 hours after irradiation with UVB of 4.5 mJ/cm2 compared with the corresponding unirradiated cells (unblocked cells:0.497 ± 0.047 vs.0.254 ± 0.035,t =22.89,P ＜ 0.05; blocked cells:0.548 ± 0.051 vs.0.257 ± 0.025,t =17.42,P ＜ 0.05).No significant differences were noted in the protein expression levels of Beclin-1,Atgl2 or Atg3 between unblocked or blocked HaCaT cells and corresponding unirradiated cells at 4 or 12 hours after irradiation with UVB at 4.5 or 50 mJ/cm2 (all P ＞ 0.05).Conclusions The expression level of p62 is different at different time points after different doses of UVB radiation,and there seems no biological association between UVB radiation and autophagosome formation.
