中耳胆脂瘤中P-Akt和Foxo3的表达及其和凋亡的关系

Expression of P-Akt and Foxo3 in Middle Ear Cholesteatoma and Association with Apoptosis

目的检测人类叉头框O3(forkhead box O3,Foxo3)蛋白、磷酸化蛋白激酶B(phosphorylated protein kinase B,P-Akt)在中耳胆脂瘤的表达和DAPI核染色情况。方法应用免疫荧光技术(immunofluorescence,IF)检测中国医科大学附属盛京医院2019年8月-11月期间30例中耳胆脂瘤标本(分为原发性胆脂瘤组和继发性胆脂瘤组)及15例正常皮肤标本中Foxo3和DAPI核染色情况;应用Western blot免疫印迹法检测以上标本中P-Akt(ser473)、Foxo3及内参GAPDH的表达情况;应用SPSS 22.0统计软件进行统计学分析。结果①免疫荧光实验显示,Foxo3定位于胆脂瘤和正常皮肤的细胞核和细胞质,DAPI染色于细胞核;Foxo3在中耳胆脂瘤的荧光值明显弱于正常皮肤,差异具有统计学意义(t=2.78,P=0.007,P<0.01);DAPI在中耳胆脂瘤的荧光值弱于正常皮肤,差异具有统计学意义(t=2.08,P=0.03,P<0.05);Foxo3和DAPI在原发性胆脂瘤和继发性胆脂瘤的染色均无统计学差异。②Western blot免疫印迹法检测显示:胆脂瘤中P-Akt(ser473)的表达量明显高于正常皮肤中的表达量,两者差异具有统计学意义(t=2.05,P=0.02,P<0.05);Foxo3的表达量明显低于正常皮肤,差异具有统计学意义(t=1.94,P=0.04,P<0.05);两者在原发性胆脂瘤和继发性胆脂瘤的表达均无统计学差异。③在中耳胆脂瘤标本中,Foxo3和DAPI呈显著正相关(r=0.451,P=0.001,P<0.01);P-Akt(ser473)和Foxo3的表达呈显著负相关(r=-0.466,P=0.0007,P<0.01)。结论中耳胆脂瘤的DAPI和Foxo3荧光染色均弱于正常皮肤组织,且两者呈正相关;胆脂瘤中P-Akt表达明确高于正常皮肤组织,而Foxo3在胆脂瘤中的表达明确低于正常皮肤,且两者呈负相关。这提示PI3K/Akt/Foxo3通路通过抑制凋亡的方式参与中耳胆脂瘤的发病过程。

Objective To report expression of forkhead box O3(Foxo3)protein and phosphorylated protein kinase B(P-Akt)protein in middle ear cholesteatoma and their correlation for their roles in the formation of cholesteatoma.Methods Immunofluorescence(IF)was used to detect Foxo3 expression and DAPI nuclear staining in samples of primary and secondary middle ear cholesteatoma(n=30),as well as samples of normal skin(n=15).Western blot was used to detect expression of P-Akt(ser473),Foxo3 and GAPDH.Their correlations were further analyzed by the SPSS 22.0 software.Results 1)immunofluorescence showed Foxo3 expression in nucleus and cytoplasm and DAPI staining in the nucleus.Foxo3 expression in cholesteatoma was significantly lower than that in normal(t=2.78,P=0.007).DAPI staining in cholesteatoma was also lower than that in normal skin(t=2.08,P=0.03).There was no significant difference in Foxo3 and DAPI expression between primary and secondary cholesteatoma.2)Western blot showed significantly higher levels of P-Akt(ser473)expression in cholesteatoma than in normal skin(t=2.05,P=0.02)and significantly lower levels of Foxo3 expression in cholesteatoma than in normal skin(t=1.94,P=0.04)with no significant difference between primary and secondary cholesteatoma.3)Foxo3 and DAPI expression was positively correlated in cholesteatoma(r=0.451,P=0.001),while P-Akt(ser473)and Foxo3 expression was negatively correlated(r=-0.466,P=0.0007).Conclusion Expression of both DAPI and Foxo3 in cholesteatoma was weaker than in normal skin,and positively correlated.P-Akt expression in cholesteatoma was significantly higher than in normal skin,while Foxo3 protein expression in cholesteatoma was significantly lower than in normal skin,and negatively correlated with P-Akt expression.These findings suggest that the PI3K/Akt/Foxo3 signaling pathway is involved in the pathogenesis of middle ear cholesteatoma by inhibiting apoptosis.