中华结核和呼吸杂志
中華結覈和呼吸雜誌
중화결핵화호흡잡지
Chinese Journal of Tuberculosis and Respiratory Diseases
2009年
1期
27-32
,共6页
黏蛋白类%白细胞弹性蛋白酶%受体%表皮生长因子%茶黄素
黏蛋白類%白細胞彈性蛋白酶%受體%錶皮生長因子%茶黃素
점단백류%백세포탄성단백매%수체%표피생장인자%다황소
Mucins%Leukocyte elastase%Receptor,epidermal growth factor%Theaflavins
目的 探讨茶黄素对炎症反应时气道上皮细胞黏液分泌的影响.方法 通过人中性粒细胞弹性蛋白酶(HNE)刺激人肺腺癌细胞A549,构建炎症反应时气道黏液高分泌模型,以茶黄素和表皮生长因子受体(EGFR)阻断剂表皮生长因子受体阻断剂(AG1478)进行干预,观察黏蛋白5AC(MUCSAC)、EGFR、磷酸化EGFR(P-EGFR)、磷酸化细胞外信号调节激酶1/2(P-ERK1/2)、兔抗磷酸化p38(P-p38)及磷酸化JNK丝裂原活化蛋白激酶(P-JNK)的表达.用四甲基偶氮唑盐法测定细胞活性,再将A549细胞分为对照组、HNE处理组、茶黄素组、AG1478组和茶黄素+AG1478组.用逆转录PCR方法检测各组MUC5AC mRNA、EGFR mRNA的变化;Western blot法检测EGFR、P-EGFR、P-ERK1/2、P-p38和P-JNK蛋白的表达;酶联免疫吸附测定法观察MUCSAC蛋白表达的变化,并用细胞免疫激光共聚焦显微镜观察作用前后黏蛋白的分布.两样本均数间比较采用t检验,多样本均数间比较采用单因素方差分析.结果 HNE处理组MUC5AC的mRNA和蛋白的积分吸光度值分别为0.99±0.03和(169±6)μg/mg,EGFR mRNA和蛋白表达的积分吸光度值分别为0.98±0.02和(0.89±0.03)μg/mg,均较对照组[0.53±0.02、(105±4)μg/mg和0.61±0.11、0.21±0.05]明显升高;P-EGFR、P-ERK1/2的蛋白表达也较对照组显著增加,而P-p38的表达则有较低幅度的增强,P-JNK无明显变化.给予茶黄素及AG1478预处理后,与HNE刺激组相比,EGFR、P-EGFR、P-ERK1/2、P-p38均明显下调,P-JNK无相应改变;而茶黄素+AG1478组MUCSAC mRNA和MUC5AC的下调较单独用茶黄素或AG1478处理更为明显,其积分吸光度值分别为0.20±0.02和(125±3)μg/mg,差异均有统计学意义(t值分别为3.02和1405.94,均P<0.05).结论 茶黄素可通过下调EGFR水平、减少EGFR的活化、部分阻遏EGFR信号转导途径及细胞外信号调节激酶来实现对下游途径的影响,从而发挥茶黄素抑制炎性气道黏液高分泌形成的作用.
目的 探討茶黃素對炎癥反應時氣道上皮細胞黏液分泌的影響.方法 通過人中性粒細胞彈性蛋白酶(HNE)刺激人肺腺癌細胞A549,構建炎癥反應時氣道黏液高分泌模型,以茶黃素和錶皮生長因子受體(EGFR)阻斷劑錶皮生長因子受體阻斷劑(AG1478)進行榦預,觀察黏蛋白5AC(MUCSAC)、EGFR、燐痠化EGFR(P-EGFR)、燐痠化細胞外信號調節激酶1/2(P-ERK1/2)、兔抗燐痠化p38(P-p38)及燐痠化JNK絲裂原活化蛋白激酶(P-JNK)的錶達.用四甲基偶氮唑鹽法測定細胞活性,再將A549細胞分為對照組、HNE處理組、茶黃素組、AG1478組和茶黃素+AG1478組.用逆轉錄PCR方法檢測各組MUC5AC mRNA、EGFR mRNA的變化;Western blot法檢測EGFR、P-EGFR、P-ERK1/2、P-p38和P-JNK蛋白的錶達;酶聯免疫吸附測定法觀察MUCSAC蛋白錶達的變化,併用細胞免疫激光共聚焦顯微鏡觀察作用前後黏蛋白的分佈.兩樣本均數間比較採用t檢驗,多樣本均數間比較採用單因素方差分析.結果 HNE處理組MUC5AC的mRNA和蛋白的積分吸光度值分彆為0.99±0.03和(169±6)μg/mg,EGFR mRNA和蛋白錶達的積分吸光度值分彆為0.98±0.02和(0.89±0.03)μg/mg,均較對照組[0.53±0.02、(105±4)μg/mg和0.61±0.11、0.21±0.05]明顯升高;P-EGFR、P-ERK1/2的蛋白錶達也較對照組顯著增加,而P-p38的錶達則有較低幅度的增彊,P-JNK無明顯變化.給予茶黃素及AG1478預處理後,與HNE刺激組相比,EGFR、P-EGFR、P-ERK1/2、P-p38均明顯下調,P-JNK無相應改變;而茶黃素+AG1478組MUCSAC mRNA和MUC5AC的下調較單獨用茶黃素或AG1478處理更為明顯,其積分吸光度值分彆為0.20±0.02和(125±3)μg/mg,差異均有統計學意義(t值分彆為3.02和1405.94,均P<0.05).結論 茶黃素可通過下調EGFR水平、減少EGFR的活化、部分阻遏EGFR信號轉導途徑及細胞外信號調節激酶來實現對下遊途徑的影響,從而髮揮茶黃素抑製炎性氣道黏液高分泌形成的作用.
목적 탐토다황소대염증반응시기도상피세포점액분비적영향.방법 통과인중성립세포탄성단백매(HNE)자격인폐선암세포A549,구건염증반응시기도점액고분비모형,이다황소화표피생장인자수체(EGFR)조단제표피생장인자수체조단제(AG1478)진행간예,관찰점단백5AC(MUCSAC)、EGFR、린산화EGFR(P-EGFR)、린산화세포외신호조절격매1/2(P-ERK1/2)、토항린산화p38(P-p38)급린산화JNK사렬원활화단백격매(P-JNK)적표체.용사갑기우담서염법측정세포활성,재장A549세포분위대조조、HNE처리조、다황소조、AG1478조화다황소+AG1478조.용역전록PCR방법검측각조MUC5AC mRNA、EGFR mRNA적변화;Western blot법검측EGFR、P-EGFR、P-ERK1/2、P-p38화P-JNK단백적표체;매련면역흡부측정법관찰MUCSAC단백표체적변화,병용세포면역격광공취초현미경관찰작용전후점단백적분포.량양본균수간비교채용t검험,다양본균수간비교채용단인소방차분석.결과 HNE처리조MUC5AC적mRNA화단백적적분흡광도치분별위0.99±0.03화(169±6)μg/mg,EGFR mRNA화단백표체적적분흡광도치분별위0.98±0.02화(0.89±0.03)μg/mg,균교대조조[0.53±0.02、(105±4)μg/mg화0.61±0.11、0.21±0.05]명현승고;P-EGFR、P-ERK1/2적단백표체야교대조조현저증가,이P-p38적표체칙유교저폭도적증강,P-JNK무명현변화.급여다황소급AG1478예처리후,여HNE자격조상비,EGFR、P-EGFR、P-ERK1/2、P-p38균명현하조,P-JNK무상응개변;이다황소+AG1478조MUCSAC mRNA화MUC5AC적하조교단독용다황소혹AG1478처리경위명현,기적분흡광도치분별위0.20±0.02화(125±3)μg/mg,차이균유통계학의의(t치분별위3.02화1405.94,균P<0.05).결론 다황소가통과하조EGFR수평、감소EGFR적활화、부분조알EGFR신호전도도경급세포외신호조절격매래실현대하유도경적영향,종이발휘다황소억제염성기도점액고분비형성적작용.
Objective To investigate the effects of theaflavins (TFs) on airway mucous hypersecretion and on the signal transduction pathway of epidermal growth factor receptor (EGFR). Methods The cell model of mucous hypersecretion was made by human lung A549 cell stimulated by human ueutrophil elastae (HNE), and treated with TFs and AG1478, a blocking agent of EGFR. The expression of mucin (MUC) 5AC, EGFR, P-EGFR, phosphorylation extracellular signal regulated kinase 1/2 (P-ERK1/2), P-p38, phosphorylation c-Jun N-terminal kinase (P-JNK) were detected. The cell activity after TFs treatment was assessed by methyl thiazolyl tetrazolium method. The cells were divided into 5 groups : a negative control group, an HNE treatment group, a TFs pre-treatment group, an AG1478 pre-treatment group and a TFs +AG1478 group. The changes of MUC5AC mRNA and EGFR mRNA were examined by the use of reverse transcriptase-polymerase chain reaction (RT-PCR). The protein expression changes of EGFR, P-EGFR, P-ERK1/2, P-p38 and P-JNK were measured by Western blot. The protein expression changes of MUC5AC were detected by enzyme-linked immunosorbent assay, while the protein morphological changes of MUC5AC were observed by immunofluorescence and confocal laser technology. The data were analyzed with SPSS 12.0 software. Differences between groups were assessed for significance by t test. Results The expression levels of MUC5AC mRNA and its protein in the HNE group were (0.99±0.03 )and(169±6)μg/mg, and those of EGFR were (0.98±0.02) and (0.89±0.03), both of them increased significantly as compared to those in the control group [(0.53±0.02), (105±4)μg/mg and (0.61±0.11), (0.21+0.05)]. The protein expressions of P-EGFR, P-ERK1/2, P-p38 were increased significantly as compared with the control group. But the increase of P-p38 was not significant as compared to P-ERK1/2. The protein expression of P-JNK did not change markedly. After the cells were pre-treated with TFs and AG1478 respectively, the above measurements were decreased significantly as compared with the HNE group. The decrease was more significant in the TFs + AG1478 group (t=3.02, P<0.05). Conclusions Theaflavins decreased the level of EGFR and inhibited the activation of EGFR, and attenuated airway mucous hypersecretiou via the ERK pathway.
