中耳胆脂瘤中DAPI核染色、FOXO3和Bim的表达和意义  

Expression and Significance of DAPI Staining, FOXO3 and Bim in Middle Ear Cholesteatoma

作　　者：刘东亮[1] 马秀岚[1] Liu Dongliang;Ma Xiulan(Department of Otolaryngology Head and Neck Surgery,Shengjing Hospital of China Medical University,Shenyang,110004,China)

机构地区：[1]中国医科大学附属盛京医院耳鼻咽喉科,沈阳110004

出　　处：《听力学及言语疾病杂志》2021年第4期393-397,共5页Journal of Audiology and Speech Pathology

摘　　要：目的检测中耳胆脂瘤中人类叉头框O3(forkhead box O3,FOXO3)、唯BH3域蛋白(Bcl-2 interacting mediator of cell death,Bim)的表达和相关性,以及Bim和4',6-二脒基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)染色的相关性,探讨FOXO3和Bim在中耳胆脂瘤中的作用。方法应用免疫组化染色(immunohistochemistry)检测30例中耳胆脂瘤标本及15例正常皮肤中FOXO3、Bim的表达和定位;通过免疫荧光技术(immunofluorescence,IF)检测这些标本中Bim和DAPI的染色情况;应用Western blot检测以上标本FOXO3、Bim及内参的表达情况;分析Bim和DAPI核染色的相关性、Bim和FOXO3表达的相关性。结果①免疫组化染色显示,FOXO3在胆脂瘤和正常皮肤的细胞核及胞质均有着色,在胆脂瘤细胞核中FOXO3的阳性样本率(36.7%,11/30)明显低于正常皮肤(86.7%,13/15)(χ^(2)=10.04,P<0.05);胆脂瘤细胞胞质中FOXO3的阳性率(30.0%,10/30)也显明低于正常皮肤(80.0%,12/15)(χ^(2)=8.72,P<0.05)。Bim主要定位于胆脂瘤和正常皮肤的细胞质,其在胆脂瘤中的阳性率(53.3%,16/30)明显低于正常皮肤组织(93.3%,14/15)(χ^(2)=7.20,P<0.05)。②免疫荧光染色显示DAPI和Bim在胆脂瘤的荧光值(分别为25.37±3.67和35.16±4.21)均弱于正常皮肤(分别为91.45±10.14和82.51±7.34),差异有统计学意义(t=1.93,P<0.05;t=1.71,P<0.05)。③Western blot检测显示胆脂瘤中FOXO3和Bim的表达均明显低于正常皮肤,差异具有统计学意义(t=2.01,P<0.05;t=1.99,P<0.05)。④在中耳胆脂瘤标本中,FOXO3和Bim呈正相关(r=0.362,P<0.05);Bim和DAPI呈显著正相关(r=0.501,P<0.01)。结论FOXO3/Bim信号通路在中耳胆脂瘤的形成过程中发挥了作用,且该作用与抗凋亡有关。Objective To study the expression and correlation of fork frame O3(forkhead box O3,FOXO3),only BH3 domain proteins(Bcl-2 interacting mediator of cell death,Bim)in human middle ear cholesteatoma,as well as the expression and correlation of Bim and 4',6-diamidino-2-phenylindole(DAPI)stain,and investigate their roles in the formation of cholesteatoma in the middle ear.Methods Immunohistochemistry was applied to detect the expression and localization of FOXO3 and Bim in 30 cases of middle ear cholesteatoma specimens and 15 cases of normal skin from July to December 2019 in shengjing hospital affiliated to China medical university.Staining of Bim and DAPI in these specimens was detected by immunofluorescence(IF).The expressions of FOXO3,Bim and GAPDH were detected by Western blot.The correlation between FOXO3 and DAPI staining and the correlation between Bim and FOXO3 expression were further analyzed by SPSS 22.0 statistical software.Results①Immunohistochemical experiments showed that FOXO3 was stained in the nuclei and cytoplasm of cholesteatoma and normal skin.The rate of positive samples(36.7%)of nucleus in cholesteatoma significantly lower than that of normal skin(86.7%)with statistically significant difference(χ^(2)=10.04,P<0.05).Cytoplasm FOXO3 also exhibits a lower rate(30.0%)than normal skin(80.0%)with statistically significant difference(χ^(2)=8.72,P<0.05).The Bim mainly locate in the cytoplasm of cholesteatoma and normal skin,the positive rate in the cholesteatoma(53.3%),significantly lower than the normal skin(93.3%)with statistically significant difference(χ^(2)=7.20,P<0.05).②Immunofluorescence experiments showed that the fluorescence value of DAPI in cholesteatoma was weaker than that in normal skin,and the difference was statistically significant(t=1.93,P<0.05).The fluorescence value of Bim in cholesteatoma was also weaker than that of normal skin,and the difference was statistically significant(t=1.71,P<0.05).③Western blot analysis showed that the total expression of FOXO3 in cholesteatoma was

关 键 词：中耳胆脂瘤 人类叉头框O3 唯BH3域蛋白 4' 6-二脒基-2-苯基吲哚 凋亡 

分 类 号：R764.21[医药卫生—耳鼻咽喉科]