CAJ | 학술논문

[目的]探讨不同剂量中波紫外线(UVB)照射人皮肤成纤维细胞诱导的自噬对细胞凋亡活性的影响.[方法]人皮肤成纤维细胞来自于23岁健康男性环切术后包皮的原代培养,连续传代培养后取第3～ 10代的细胞进行实验.通过单丹酰戊二胺(MDC)染色和免疫荧光标记微管相关蛋白1轻链3(LC3)的方法,确定不同剂量3-甲基腺嘌呤(3-MA)对自噬的抑制作用.UVB照射后立即加入0.5 mmol/L 3-MA孵育细胞4h作为自噬的抑制方法.Hoechst和碘化丙啶(PI)染色结合Annexin V-异硫氰酸荧光索(FITC)和PI标记细胞后流式细胞仪检测作为细胞凋亡的定性和定量方法.[结果]0.5 mmol/L 3-MA能明显抑制人皮肤成纤维细胞自噬活性(饥饿诱导组自噬细胞阳性百分数为63.037％±5.876％,3-MA孵育后下降至34.425％±5.183％).空白对照组、30、50、100mJ/cm2UVB照射组标记LC3绿色荧光信号逐渐增强,照射后立即对上述4组加入3-MA孵育4h则LC3蛋白表达差异不明显.0.5 mmol/L 3-MA对细胞活性影响最小.50 mJ/cm2 UVB照射下加入3-MA孵育较未加3-MA孵育细胞强Hoechst和强PI双染细胞增多；100 mJ/cm2 UVB照射后加3-MA孵育较未加3-MA孵育细胞强Hoechst和强PI双染细胞减少.Annexin V -FITC和PI标记细胞后流式细胞仪分析显示,在50 mJ/cm2 UVB照射下,抑制自噬的细胞中晚期凋亡水平[( 10.933±0.839)％]较未抑制细胞[(7.267±0.473)％]上升,两者之间差异具有统计学意义(t=5.20,P＜ 0.05)；而在100mJ/cm2UVB照射下,抑制自噬的细胞中晚期凋亡水平[(7.100±0.781)％]较未抑制细胞[( 10.133±0.681)％]下降,两者之间差异具有统计学意义(t=6.29,P＜ 0.05).[结论]50 mJ/cm2 UVB照射诱导人皮肤成纤维细胞自噬通过抑制凋亡对细胞起到保护作用,而在100 mJ/cm2 UVB照射下发生的较高水平自噬可能诱导了自噬性细胞死亡.
[목적]탐토불동제량중파자외선(UVB)조사인피부성섬유세포유도적자서대세포조망활성적영향.[방법]인피부성섬유세포래자우23세건강남성배절술후포피적원대배양,련속전대배양후취제3～ 10대적세포진행실험.통과단단선무이알(MDC)염색화면역형광표기미관상관단백1경련3(LC3)적방법,학정불동제량3-갑기선표령(3-MA)대자서적억제작용.UVB조사후립즉가입0.5 mmol/L 3-MA부육세포4h작위자서적억제방법.Hoechst화전화병정(PI)염색결합Annexin V-이류청산형광색(FITC)화PI표기세포후류식세포의검측작위세포조망적정성화정량방법.[결과]0.5 mmol/L 3-MA능명현억제인피부성섬유세포자서활성(기아유도조자서세포양성백분수위63.037％±5.876％,3-MA부육후하강지34.425％±5.183％).공백대조조、30、50、100mJ/cm2UVB조사조표기LC3록색형광신호축점증강,조사후립즉대상술4조가입3-MA부육4h칙LC3단백표체차이불명현.0.5 mmol/L 3-MA대세포활성영향최소.50 mJ/cm2 UVB조사하가입3-MA부육교미가3-MA부육세포강Hoechst화강PI쌍염세포증다；100 mJ/cm2 UVB조사후가3-MA부육교미가3-MA부육세포강Hoechst화강PI쌍염세포감소.Annexin V -FITC화PI표기세포후류식세포의분석현시,재50 mJ/cm2 UVB조사하,억제자서적세포중만기조망수평[( 10.933±0.839)％]교미억제세포[(7.267±0.473)％]상승,량자지간차이구유통계학의의(t=5.20,P＜ 0.05)；이재100mJ/cm2UVB조사하,억제자서적세포중만기조망수평[(7.100±0.781)％]교미억제세포[( 10.133±0.681)％]하강,량자지간차이구유통계학의의(t=6.29,P＜ 0.05).[결론]50 mJ/cm2 UVB조사유도인피부성섬유세포자서통과억제조망대세포기도보호작용,이재100 mJ/cm2 UVB조사하발생적교고수평자서가능유도료자서성세포사망.
[Objective] To observe the effects of autophagy induced by different doses of ultraviolet B (UVB) irradiation on the apeptosis in human skin fibroblasts.[Methods] Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male,and subjected to a primary culture.After 3 to 10 passages,the cells were collected and applied in the following experiment.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations (0,0.5,2.0,5.0and 10.0 mmol/L) of 3-methyladenine (3-MA).To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/Lfollowing UVB irradiation,monodansylcadaverine (MDC) staining was carried out,and immunofluorescence was used to detect the expression of microtubule-associated protein 1 light chain (LC3).Some fibroblasts were classified into 8 groups to remain untreated,be irradiated with UVB of 30,50 and 100 mJ/cm2 alone,treated with 3-MA of 0.5 mmol/L alone,or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30,50 and 100 mJ/cm2,respectively,then,cell apoptosis was qualitatively detected by Hoechst and propidium iodide (PI)staining,and quantitatively detected by flow cytometry with annexin V and PI.[Results] The percentage of autophagic cells was (63.037 ± 5.876) ％ in fibroblasts treated with starvation condition,significantly decreased to (34.425 ± 5.183) ％ in fibroblasts treated with 3-MA of 0.5 mmol/L.The expression of LC3 showed a gradually increasing trend from untreated fibroblasts,to fibroblasts irradiated with UVB of 30,50 and 100 mJ/cm2,while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation.Compared with the other concentrations,the 3-MA of 0.5 mmol/L showed the least influence on the viability of fibroblasts.The addition of 3-MA of 0.5 mmol/L increased the percentage of cells both positive for Hoechst and PI staining in fibroblasts irradiated with UVB of 50 mJ/cm2,but decreased that in fibroblasts irradiated with UVB of 100 mJ/cm2.Similarly,the percentage of middle and late apoptotic cells was significantly higher in fibroblasts irradiated with UVB of 50 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 50 mJ/cm2alone ((10.933 ± 0.839) ％ vs.(7.267 ± 0.473) ％,t =5.20,P＜ 0.05),but lower in fibroblasts irradiated with UVB of 100 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 100 mJ/cm2alone ( (7.100 ± 0.781 ) ％ vs.( 1 0.133 ± 0.681 ) ％,t =6.29,P ＜ 0.05 ).[Conclusion]s The irradiation with UVB of 50 mJ/cm2 may protect fibroblasts by inducing autophagy and suppressing apoptosis,while the high level of autophagy induced by UVB of 100 mJ/cm2 may lead to autophagic cell death in fibroblasts.