CAJ | 학술논문

目的探讨降钙素基因相关肽(CGRP)对高氧暴露下肺泡Ⅱ型上皮细胞(AEC Ⅱ)的促增殖作用及其蛋白激酶Cα/核转录因子-κgB(PKCa/NF-κB)信号途径的调控机制.方法将原代分离培养的孕21 d胎鼠AEC Ⅱ分为空气组、高氧组、高氧CGRP组、高氧CGRP拮抗剂组.空气组和高氧组分别在21%O2或85%O2中暴露24 h;高氧CGRP组在高氧处理前加入CGRP,高氧CGRP拮抗剂组同时加入CGRP和CGRP受体拮抗剂CGRP8-37.采用四甲基偶氮唑盐(MTT)比色法和流式细胞术测定细胞增殖能力和不同细胞周期细胞比例;蛋白质免疫印迹法(Western blotting)检测胞膜和胞质PKCα的表达;激光共聚焦检测NF-κB p65的细胞核表达.结果高氧组G0/G1期细胞比例明显高于空气组[(80.652±6.253)%比(45.825±2.899)%],而细胞增殖率[(68.752±5.766)%比(100.000±6.682)%]及S期、G2/M期细胞比例[分别为(14.198±4.785)%比(27.470±2.775)%,(5.148±1.688)%比(26.708±1.863)%]均低于空气组(均P<0.01).CGRP干预可提高高氧暴露AEC Ⅱ的增殖能力[(94.813±6.102)%],使S期和G2/M期细胞增多((30.547±9.861)%,(17.668±9.509)%,均P<0.01].高氧组胞膜与胞质PKCα比值显著低于空气组(0.63±0.10比1.00±0.09),而NF-κB p65的荧光强度高于空气组(22.98±2.20比14.54±2.35);高氧CGRP组胞膜与胞质PKCα比值(1.41±0.23)及核内NF-κB p65荧光强度(35.38±3.37)均高于高氧组(0.63±0.10,22.98±2.20)及高氧CGRP拮抗剂组(O.74±0.10,24.88±1.81,均P<0.01).结论 CGRP可促进高氧暴露下AEC Ⅱ的生长增殖;PKCα参与了CGRP对细胞作用的信号传递,而NF-κB是PKCα的下游分子之一,仅部分执行PKCα的效应.
목적 탐토강개소기인상관태(CGRP)대고양폭로하폐포Ⅱ형상피세포(AEC Ⅱ)적촉증식작용급기단백격매Cα/핵전록인자-κgB(PKCa/NF-κB)신호도경적조공궤제.방법 장원대분리배양적잉21 d태서AEC Ⅱ분위공기조、고양조、고양CGRP조、고양CGRP길항제조.공기조화고양조분별재21%O2혹85%O2중폭로24 h;고양CGRP조재고양처리전가입CGRP,고양CGRP길항제조동시가입CGRP화CGRP수체길항제CGRP8-37.채용사갑기우담서염(MTT)비색법화류식세포술측정세포증식능력화불동세포주기세포비례;단백질면역인적법(Western blotting)검측포막화포질PKCα적표체;격광공취초검측NF-κB p65적세포핵표체.결과 고양조G0/G1기세포비례명현고우공기조[(80.652±6.253)%비(45.825±2.899)%],이세포증식솔[(68.752±5.766)%비(100.000±6.682)%]급S기、G2/M기세포비례[분별위(14.198±4.785)%비(27.470±2.775)%,(5.148±1.688)%비(26.708±1.863)%]균저우공기조(균P<0.01).CGRP간예가제고고양폭로AEC Ⅱ적증식능력[(94.813±6.102)%],사S기화G2/M기세포증다((30.547±9.861)%,(17.668±9.509)%,균P<0.01].고양조포막여포질PKCα비치현저저우공기조(0.63±0.10비1.00±0.09),이NF-κB p65적형광강도고우공기조(22.98±2.20비14.54±2.35);고양CGRP조포막여포질PKCα비치(1.41±0.23)급핵내NF-κB p65형광강도(35.38±3.37)균고우고양조(0.63±0.10,22.98±2.20)급고양CGRP길항제조(O.74±0.10,24.88±1.81,균P<0.01).결론 CGRP가촉진고양폭로하AEC Ⅱ적생장증식;PKCα삼여료CGRP대세포작용적신호전체,이NF-κB시PKCα적하유분자지일,부부분집행PKCα적효응.
Objective To explore the effects of caleitonin gene-related peptide (CGRP) on type Ⅱ alveolar epithelial cell (AEC Ⅱ ) exposed to hyperoxia, and to determine whether the mechanism is mediated by protein kinase Cα/nuclear factor-κB (PKCa/NF-κB) signal pathway. Methods AEC Ⅱ were isolated from the lung of 21 days fetal rat and cultured for 15 hours to coalesce. Then AEC Ⅱ were randomly assigned into four groups: air, hyperoxia, O2/CGRP, and O2/CGRP8-37 (a receptor antagonist against CGRP).AEC Ⅱ were exposed to FiO2 21% (air) or 85% (hyperoxia) for 24 hours respectively. In O2/CGRP and O2/CGRP8-37 groups CGRP or both CGRP and CGRPS-37 were added into cultural fluid before placing the plate into 85% oxygen. Cell proliferation ability was determined by methl thiazolyl tetrazolium (MTT)assay and cell cycles by flow cytometry. Western blotting was employed to detect the fraction of PKCα inmembrane and cytosol, and translocation of NF-κB was observed under laser confocal microscopy. Results AEC Ⅱ in hyperoxia group showed a decreased viability of AEC Ⅱ [(68. 752±5. 766)%vs. (100. 000±6. 682)%] and had an enhanced percentage of G0/G1 phase [(80. 652±6. 253)% vs. (45. 825±2. 899)%]with a corresponding decline in percentage of S phase [(14. 198±4. 785)% vs. (27. 470± 2. 775)%] and G2/M phases [(5. 148± 1. 688)% vs. (26. 708±1. 863)%] compared with AEC Ⅱ in air (all P<0. 01).Addition with CGRP before hyperoxia exposure promoted AEC Ⅱ proliferation [(94. 813±6. 102)%] and enhanced the cell proportions in S and G2/M phases [(30. 547±9. 861)% and (17. 668±9. 509)%, all P＜0. 01]. The ratio of membrane to cytoplasm fraction of PKCα declined (0. 63±0. 10 vs. 1.00±0. 09) and the fluorescence of NF-κB in nucleus enhanced (22.98±2.20 vs. 14.54±2.35) in hyperoxia compared with that in air, while both the ratio of PKCα and intensity of NF-κB were increased in O2/CGRP group (1.41±0. 23,35.38±3. 37) compared with those in hyperoxia (0. 63±0. 10, 22. 98±2. 20) and O2/CGRP8-37 groups(0. 74±0. 10, 24. 88±1.81, all P<0. 01). Conclusion CGRP could promote prolferation of AEC Ⅱ when exposed to high oxygen tension. PKCα participates in the signal transduction process and NF-κB is a downstream molecular of PKCα, executing in part the function of PKCα signal.