尖锐湿疣角质形成细胞中陷窝蛋白1的表达与细胞增殖和凋亡的相关性  

Expression of caveolin.1 in keratinocytes and its correlation with cellular proliferation and apoptosis in condyloma acuminatum skin lesions

作　　者：熊浩[1] 黎倩[1] 刘清秀[1] 陈平姣[1] 陈名华[1] 张静[1] 高琰[1] 曾抗[1] 

机构地区：[1]南方医科大学南方医院皮肤科,广州510515

出　　处：《中华皮肤科杂志》2016年第5期318-323,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81171627）

摘　　要：目的探讨陷窝蛋白1在尖锐湿疣角质形成细胞中的表达及其与角质形成细胞增殖和凋亡的相关性。方法免疫组化EnVision法和TUNEL技术检测40例尖锐湿疣皮损组织和10例健康人包皮组织中角质形成细胞陷窝蛋白1、增殖细胞核抗原的表达和细胞凋亡，计算陷窝蛋白1平均吸光度、增殖指数和凋亡指数，比较两组之间的差异，并分析尖锐湿疣组中陷窝蛋白1表达与增殖指数、凋亡指数的相关性。针对陷窝蛋白1基因设计3条siRNA，用脂质体转染法将siRNA导入HaCaT细胞，用荧光定量PCR方法筛选出干扰效果最佳的siRNA，转染HaCaT细胞为实验组，转染非特异序列的细胞为阴性对照组，未转染的细胞为空白对照组。Western印迹和免疫荧光检测转染48h后陷窝蛋白1的表达水平；MTS法分别测定干扰陷窝蛋白1基因24、48、72h后HaCaT细胞增殖的变化；流式细胞仪（FCM）检测干扰陷窝蛋白1基因48h后HaCaT细胞的凋亡情况。结果尖锐湿疣角质形成细胞中陷窝蛋白1的表达（0．042±0．021）显著低于正常包皮组织（0．181±0．075），差异有统计学意义（t=5．843，P〈0．001）。尖锐湿疣皮损中角质形成细胞的增殖指数（65．63％±19．86％）和凋亡指数（24．12％±10．86％）均显著高于正常包皮组织（23．51％±4．00％，3．13％±1．12％），两组差异有统计学意义（t=12．440、11．970，均P〈0．001）。尖锐湿疣组陷窝蛋白1表达与增殖指数呈显著负相关（r=一0．798，P〈0．05），与凋亡指数呈显著正相关（r=0．825，P〈0．05）。荧光定量PCR显示siRNA一2在浓度25nmol／L下抑制效果最佳。Western印迹和免疫荧光显示，转染siRNA48h后，实验组陷窝蛋白1的表达显著低于空白对照组和阴性对照组（P〈0．05）。MTS结果显示，实验组细胞24、48和72h时的A值分别为0．563±0．013、0．628±0．006和0．811±0．018，高于空白对�Objective To investigate the expression of eaveolin-1 in keratinocytes of condylomata acuminatum （CA） skin lesions, and to explore its correlation with keratinoeyte proliferation and apoptosis. Methods Tissue specimens were obtained from lesions of 40 patients with CA （CA group） and normal skin of 10 healthy human controls （control group ）, then fixed with formalin and embedded with paraffin. An immunohistoehemieal analysis was done using the EnVision system to measure the expressions of caveolin-1 （expressed as absorbance value） and proliferating cell nuclear antigen （PCNA） in keratinocytes, and terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） assay to evaluate apoptosis of keratinocytes in these tissue specimens. The proliferative index and apoptotic index of cells were calculated. Independent samples t test was conducted to compare these parameters between the two groups, and Pearson correlation analysis to assess the relationship of caveolin-1 expression with proliferative index and apoptotic index. Three small interfering RNAs （siRNAs） were designed targeting the caveolin-1 gene, and transfected into HaCaT cells separately by using liposomes. Then, fluorescence-based quantitative PCR （qPCR） was performed to detect the expression of caveolin-1, so as to find the most efficient siRNA. Cultured HaCaT cells were divided into 3 groups：experimental group transfected with caveolin-1 siRNA, negative control group transfected with nonspecific siRNA, and blank control group receiving no treatment. Western blot analysis and immunofluorescenee assay were carried out to quantify the protein expression of caveolin-1 in HaCaT cells at 48 hours after transfection, MTS assay was performed to evaluate proliferative activity of HaCaT cells at 24, 48 and 72 hours, and flow cytometry （FCM） to detect cell apoptosis at 48 hours. Results Compared with the control group, the CA group showed significantly decreased caveolin-1 expression （0.042 ± 0.021 vs. 0.181 ± 0.075, t

关 键 词：尖锐湿疣 窖蛋白1 角蛋白细胞 细胞凋亡 细胞增殖 

分 类 号：R752.53[医药卫生—皮肤病学与性病学]