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
2010年
11期
801-805
,共5页
余红梅%李琪%Juliy M.Perelman%Victor P.Kolosov%周向东
餘紅梅%李琪%Juliy M.Perelman%Victor P.Kolosov%週嚮東
여홍매%리기%Juliy M.Perelman%Victor P.Kolosov%주향동
弹性蛋白酶抑制因子%黏蛋白类%吸烟%转录因子AP-1%NF-κB
彈性蛋白酶抑製因子%黏蛋白類%吸煙%轉錄因子AP-1%NF-κB
탄성단백매억제인자%점단백류%흡연%전록인자AP-1%NF-κB
Elafin%Mucins%Smoking%Transcription factor AP-1%NF-kappa B
目的 探讨内生多肽Elafin调节气道黏液高分泌的分子机制.方法 构建人 Elafin 重组质粒,原代培养正常人支气管上皮细胞 HBE16,分为对照组、香烟抽提物(CSE)刺激组、CSE 刺激+转染重组质粒、CSE 刺激+空质粒组、单纯转染重组质粒以及空质粒组6组.四甲基偶氮唑盐法检测各组细胞活力,Western blot法检测p-JNK、p-ERK、p-P38和IκBα蛋白含量,荧光素酶报告基因系统测定激活蛋白-1(AP-1)及核转录因子-κB(NF-κB)活性,RT-PCR检测各组黏蛋白(MUC)5AC mRNA 表达水平,ELISA 法分析各组细胞 MUC5AC 蛋白的相对含量.结果 CSE刺激组的p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC和MUC5AC mRNA 含量分别为(0.55±0.03)μg/mg、(0.64±0.06)μg/mg、(0.60±0.07)μg/mg、(0.27±0.03)μg/mg、7.49±0.31、4.42±0.22、(0.71±0.04)mg/L和0.81±0.04,与对照组的(0.26±0.02)μg/mg、(0.30±0.05)μg/mg、(0.19±0.04)μg/mg(0.61±0.04)μg/mg、2.54±0.22、2.37±0.16、(0.23±0.02)mg/L和0.32±0.03比较差异有统计学意义(均P＜0.05).转染重组Elafin后再给予CSE刺激,p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC和MUC5AC mRNA含量分别为(0.38±0.04)μg/mg、(0.31±0.04)μg/mg、(0.14±0.03)μg/mg、(0.54±0.03)μg/mg、2.60±0.19、2.55±0.21、(0.28±0.03)mg/L、0.35±0.05,与CSE组相比差异有统计学意义(均P＜0.05);p-P38蛋白含量在CSE刺激及转染Elafin前后无明显变化.结论 内生多肽Elafin可降低JNK和ERK磷酸化水平并抑制IκBα蛋白降解,从而降低转录激活蛋白-1和核因子-κB的活性,下调黏蛋白5AC的高表达,而p38MAPK在其中的作用并不明显.
目的 探討內生多肽Elafin調節氣道黏液高分泌的分子機製.方法 構建人 Elafin 重組質粒,原代培養正常人支氣管上皮細胞 HBE16,分為對照組、香煙抽提物(CSE)刺激組、CSE 刺激+轉染重組質粒、CSE 刺激+空質粒組、單純轉染重組質粒以及空質粒組6組.四甲基偶氮唑鹽法檢測各組細胞活力,Western blot法檢測p-JNK、p-ERK、p-P38和IκBα蛋白含量,熒光素酶報告基因繫統測定激活蛋白-1(AP-1)及覈轉錄因子-κB(NF-κB)活性,RT-PCR檢測各組黏蛋白(MUC)5AC mRNA 錶達水平,ELISA 法分析各組細胞 MUC5AC 蛋白的相對含量.結果 CSE刺激組的p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC和MUC5AC mRNA 含量分彆為(0.55±0.03)μg/mg、(0.64±0.06)μg/mg、(0.60±0.07)μg/mg、(0.27±0.03)μg/mg、7.49±0.31、4.42±0.22、(0.71±0.04)mg/L和0.81±0.04,與對照組的(0.26±0.02)μg/mg、(0.30±0.05)μg/mg、(0.19±0.04)μg/mg(0.61±0.04)μg/mg、2.54±0.22、2.37±0.16、(0.23±0.02)mg/L和0.32±0.03比較差異有統計學意義(均P＜0.05).轉染重組Elafin後再給予CSE刺激,p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC和MUC5AC mRNA含量分彆為(0.38±0.04)μg/mg、(0.31±0.04)μg/mg、(0.14±0.03)μg/mg、(0.54±0.03)μg/mg、2.60±0.19、2.55±0.21、(0.28±0.03)mg/L、0.35±0.05,與CSE組相比差異有統計學意義(均P＜0.05);p-P38蛋白含量在CSE刺激及轉染Elafin前後無明顯變化.結論 內生多肽Elafin可降低JNK和ERK燐痠化水平併抑製IκBα蛋白降解,從而降低轉錄激活蛋白-1和覈因子-κB的活性,下調黏蛋白5AC的高錶達,而p38MAPK在其中的作用併不明顯.
목적 탐토내생다태Elafin조절기도점액고분비적분자궤제.방법 구건인 Elafin 중조질립,원대배양정상인지기관상피세포 HBE16,분위대조조、향연추제물(CSE)자격조、CSE 자격+전염중조질립、CSE 자격+공질립조、단순전염중조질립이급공질립조6조.사갑기우담서염법검측각조세포활력,Western blot법검측p-JNK、p-ERK、p-P38화IκBα단백함량,형광소매보고기인계통측정격활단백-1(AP-1)급핵전록인자-κB(NF-κB)활성,RT-PCR검측각조점단백(MUC)5AC mRNA 표체수평,ELISA 법분석각조세포 MUC5AC 단백적상대함량.결과 CSE자격조적p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC화MUC5AC mRNA 함량분별위(0.55±0.03)μg/mg、(0.64±0.06)μg/mg、(0.60±0.07)μg/mg、(0.27±0.03)μg/mg、7.49±0.31、4.42±0.22、(0.71±0.04)mg/L화0.81±0.04,여대조조적(0.26±0.02)μg/mg、(0.30±0.05)μg/mg、(0.19±0.04)μg/mg(0.61±0.04)μg/mg、2.54±0.22、2.37±0.16、(0.23±0.02)mg/L화0.32±0.03비교차이유통계학의의(균P＜0.05).전염중조Elafin후재급여CSE자격,p-JNK、p-ERK、p-c-Jun、IκBα、AP-1、NF-κB、MUC5AC화MUC5AC mRNA함량분별위(0.38±0.04)μg/mg、(0.31±0.04)μg/mg、(0.14±0.03)μg/mg、(0.54±0.03)μg/mg、2.60±0.19、2.55±0.21、(0.28±0.03)mg/L、0.35±0.05,여CSE조상비차이유통계학의의(균P＜0.05);p-P38단백함량재CSE자격급전염Elafin전후무명현변화.결론 내생다태Elafin가강저JNK화ERK린산화수평병억제IκBα단백강해,종이강저전록격활단백-1화핵인자-κB적활성,하조점단백5AC적고표체,이p38MAPK재기중적작용병불명현.
Objective To explore the effects of endogeny polypeptide elafin on mucin (MUC)5AC overexpression. Methods Eukaryotic expression vector pEGFP-N1-Elafin was constructed. HBE16 cells were cultured and divided into 6 groups:a control group, a cigarette smoke extract (CSE) stimulated group,a CSE and Elafin transfected group, a CSE and pEGFP-N1 transfected group, a single elafin transfected group, and a single pEGFP-N1 transfected group. After 24 h, the protein levels of phosphorylation Jun Nterminal kinase (p-JNK), phosphorylation extracellular signal-regulated kinase (p-ERK), p-P38 and inhibitor of NF-κB (IκB) α were detected by Western blot. The transcription activities of activiator protein-1(AP-1) and nuclear factorκB (NF-κB) were detected by luciferase reporter gene detection system. The levels of MUC5AC protein and mRNA were detected by ELISA and RT-PCR. Results In CSE group, there was a significant increase of MUC5AC protein (0. 71 ±0. 04) mg/L and mRNA expression (0. 81 ±0. 04),with elevation of p-JNK production ( 0. 55 ± 0. 03 ) μg/mg, p-ERK production (0. 64 ± 0. 06) μg/mg, p-c-Jun production (0. 60 ±0. 07) μg/mg, AP-1 activity (7. 49 ±0. 31 ) and NF-κB activity (4. 42±0. 22), all significantly higher than those in the control group ( t = 4. 50-14. 28, P ＜ 0. 01 ), and IκBα protein production was (0. 27 ± 0. 03 ) mg/L, significantly lower than that in the control group ( t = 6. 82, P =0. 008). Transfected recombinant elafin reduced MUC5AC protein (0. 71 ± 0. 04) mg/L and mRNA level (0. 81 ± 0. 04 ), decreased p-JNK (0. 38 ± 0. 04 ) μg/mg and p-ERK (0. 31 ± 0. 04 ) μg/mg production,inhibited AP-1 activity (2.60 ± 0. 19) and NF-κB activity (2. 55 ± 0. 21 ), but increased IκBα protein (0. 54 ± 0. 03 ) μg/mg, compared with single CSE-stimulated group ( all P ＜ 0. 05 ). p-P38 showed no significant change after CSE stimulation or transfection of elafin. Conclusion Endogeny polypeptide elafin may down-regulate MUC5AC overexpression, and this is through the inhibition of AP-1 and NF-κB activation via effects on mitogen-activated protein kinases and IκB pathway by Elafin.
