烟曲霉菌诱导大鼠呼吸道上皮细胞IKKα调控maspin蛋白表达的初步研究  

Preliminary study on Aspergillus fumigates-induced IKKa controlled maspin protein expression inrespiratory epithelial cells of rats

作　　者：张芳[1,2] 安云芳[1] 赵长青[1] 

机构地区：[1]山西医科大学第二医院耳鼻咽喉科,030001 [2]山西医科大学口腔医学系

出　　处：《中华耳鼻咽喉头颈外科杂志》2013年第1期48-53,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金，山西省归国留学基金

摘　　要：目的研究烟曲霉诱导大鼠呼吸道上皮细胞时是否存在IkB激酶-α（IKBkinase-α，IKKα）调控maspin蛋白表达下调。方法体外培养大鼠呼吸道上皮细胞（ratbronchialepithelialcells，REC），制备灭活的烟曲霉菌菌丝（Aspergillusfumigatushyphae，AFH）悬液诱导REC细胞，以磷酸盐缓冲液（PBS）为对照，采用逆转录聚合酶链反应（RT-PCR）技术检测REC中maspin基因的表达，并通过免疫细胞化学技术观察maspin蛋白及IKKα在REC的表达情况。进一步制备浓度依次增高的AFHl—3悬液诱导REC细胞，WesternBlot技术检测REC的maspin和IKKα蛋白表达。采用SPSS13．0软件进行方差分析和两变量的相关性分析。结果RT—PCR检测显示AFH组与对照组的maspin蛋白的mRNA相对表达量分别为0．128±0．059和2．972±0．353，差异具有统计学意义（t=15．883P〈0．05）。免疫细胞化学结果显示，maspin蛋白在两组中均表达，其中AFH组呈弱阳性，而对照组呈中度阳性，综合计分两组差异有统计学意义（t=3．721P〈0．05）；IKKα显色在AFH组中度阳性，在对照组弱阳性，综合计分差异有统计学意义（t=6．825P〈0．05）。WesternBlot结果maspin与B—actin的灰度比值对照组为0．912±0．023，AFHl—3组分别为0．607±0．030、0．476±0．019、0．416±0．017，组间差异有统计学意义（F=281．91，P〈0．05），且任意两组比较差异均有统计学意义；IKKα与β-actin灰度比值对照组为0．624±0．012，AFHl—3组分别为0．739±0．020、0．778±0．010、0．927±0．017，组间差异有统计学意义（F=200．91，P〈0．05），且任意两组比较差异均有统计学意义。两变量相关性分析结果显示，maspin蛋白表达与IKKα蛋白表达呈负相关关系（r=-0．911，P〈0．05）。结论AFH诱导大鼠REC中IKKα表达上调并伴有maspin蛋白表达下调。Objective To study if the expression of maspin protein in respiratory epithelial cells was downregulated through IKB kinase-α （IKKα）-controlled mechanism in an Aspergillus fumigates-induced model in rat. Methods Inactivated Aspergillus fumigatus hyphae （AFH） suspension was used to induce respiratory epithelial cells （ REC ） cultured in vitro in rat, with PBS buffer as control. By RT-PCR, the mRNA expression of maspin was quantified, and by immunocytochemistry, the expression of maspin and IKKα in REC was observed. Furthermore AFH （from level 1 to level 3 ） suspension was prepared to induce REC. Then Western blot hybridization technique was used to detect the expression of maspin and IKKα protein. All data were processed by analysis of variance and two-variable correlation analysis. Results By RT-PCR, a statistically significant （t = 2. 463, P 〈 0. 05 ） expression of maspin mRNA was found （0. 128 ±0. 059 in AFH group, 2. 972 ±0. 353 in control group）. By Immunocytoehemistry, the difference of maspin protein color in different groups was shown statistically significant in integrated scoring （t = 3. 721, P 〈 O. 05, weak positive in AFH group, moderately positive in control group）. While in IKKc~ colorstudy, the difference between the two groups was also statistically significant （ t = 6. 825, P 〈 0. 05 ） in integrated scoring, with a moderate positive in AFH group and weak positive in control group. By Western Blot hybridization, grayscale ratio of maspin and [3-actin was 0. 912 ± 0. 023 in control group, 0. 607 ± O. 030, 0. 476 ± O. 019, O. 416 ± 0. 017 in AFH 1 - 3 groups, respectively, with a statistically significant difference within the four groups （ F = 281.91, P 〈 0. 05 ） ; While grayscale ratio of IKKot and [3-actin was 0. 624 ± 0. 012 in control group, 0. 739 ± O. 020, O. 778 ± 0. 010, 0. 927 ± 0. 017, respectively, in AFH 1-3 groups; with a statistically significant difference within the four groups （F = 200. 9l, P 〈 0. 05）. Moreover, the dif

关 键 词：丝氨酸蛋白酶抑制剂 舍平类 I-KB激酶 曲霉菌 烟 鼻窦炎 真菌病 

分 类 号：R765[医药卫生—耳鼻咽喉科]