摘要
目的探讨Notch信号通路在血小板源生长因子AA（PDGF-AA）诱导血管平滑肌细胞（VSMC）增殖和迁移中的作用及机制。方法脱臼处死1～2月龄雄性SD大鼠,无菌取腹主动脉,剥弃外膜及去除内皮后,贴块法培养VSMC,α-SM-actin染色鉴定。实验分为四组：对照组、γ-secretase抑制剂组（DAPT组）、PDGF-AA组和PDGFAA＋γ-secretase抑制剂组（PDGF-AA＋DAPT组）。实时荧光定量PCR（Real-time q PCR）检测Notch信号分子mRNA表达;Cell Titer96 Aqueous One Solution试剂盒检测细胞增殖活性;采用细胞划痕实验检测VSMC的迁移能力。结果腹主动脉VSMC表达Notch信号通路Jagged1、Jagged2、Notch1～3等几种主要的配体及受体;PDGF-AA刺激24h和48 h后分别导致Jagged2和Jagged1 mRNA表达显著增强（P〈0.01,n=4）;与0 h相比,PDGF-AA刺激72 h后,Notch1、Notch3 mRNA表达显著增高（P〈0.01,n=4）,而刺激24 h后Notch2 mRNA表达显著降低。PDGF-AA显著促进HES1（P〈0.05,n=4）、HEY2（P〈0.05,n=4）和转录因子Rbp-jКmRNA（P〈0.05,n=4）表达,DAPT抑制PDGF-AA介导的HES1、HEY2 mRNA表达增强（P〈0.01,n=4）,但是进一步促进PDGF-AA诱导的Rbp-jКmRNA表达（P〈0.05,n=4）;PDGF-AA刺激促进VSMC增殖（P〈0.01,n=5）和减少划痕余留面积（P〈0.01,n=4）,两种效应均可被DAPT显著阻抑（P〈0.01,n=4）。结论 PDGF-AA调节了VSMC Notch信号分子表达,PDGF-AA部分通过激活Notch信号通路调节VSMC的增殖和迁移。
Aim To explore the role of Notch signaling pathway in platelet derived growth factor-AA（ PDGFAA） induced vascular smooth muscle cell（ VSMC） proliferation and migration. Methods 1-2 months old Sprague Dawley（ SD） male rats were used in present study. Rat abdominal aorta was gained by aseptic operation,after removing the outer membrane of vascular and the endothelium,smooth muscle cells（ SMC） was acquired by block pasting method and identified by immunofluorescence assay for α-SM-actin staining. The experiment was divided into four groups： normal group（ Control）,γ-secretase inhibitor group（ DAPT）,PDGF-AA group（ PDGF-AA）,PDGF-AA combined γ-secretase group（ PDGF-AA ＋ DAPT）. The expression of Jagged1,Jagged2,Notch1-4,HES1,HEY1 and HEY2 mRNA was detected by real-time quantitative polymerase chain reaction（ real-time qP CR）,cell proliferation activity was detected by CellT iter96 Aqueous One Solution kit,and the migration of SMC was detected by cell scratching assay. Results Notch signals including Jagged1,Jagged 2,and Notch1-3 were expressed in cultured SMC. PDGF-AA induced increased Jagged 2 mRNA expression 24 h post-stimulation（ P〈0. 01,n = 4） and increased Jagged1 mRNA expression 48 h poststimulation（ P〈0. 01,n = 4）; Notch1 and 3 mRNA expression also significantly increased 72 h after PDGF-AA stimulation compared with 0 h group（ P〈0. 01,n = 4）,whereas Notch2 mRNA were obviously decreased by PDGF-AA 48 h post-stimulation（ P〈0. 01,n = 4）. PDGF-AA also significantly promoted the HES1（ P〈0. 05,n = 4）,HEY2（ P〈0.05,n = 4） and transcription factor Rbp-j kappa（ P〈0. 05,n = 4） mRNA expression in SMC,DAPT markedly inhibited PDGF-AA induced HES1,HEY2 mRNA expression（ P〈0. 01,n = 4）,but DAPT further increased PDGF-AA induced Rbp-j kappa mRNA expression（ P〈0. 05,n = 4）; PDGF-AA stimulation obviously promoted SMC proliferation（ P〈0.01,n = 5） and reduced remaining scratches area（ P〈0. 01,n = 4）,whereas the effects were significantly attenuated by DAPT（ P〈0. 01,n = 4）. Conclusions PDGF-AA can directly affect the expressions of Notch signaling molecules in SMC,Notch signaling pathway is partly in responsible for PDGF-AA-induced SMC proliferation and migration.
作者
王金祥
周芳
朱光旭
刘菊芬
朱向情
康华莉
庞荣清
黄岚
潘兴华
WANG Jin-Xiang ZHOU Fang ZHU Guang-Xu LIU Ju-Fen ZHU Xiang-Qing KANG Hua-Li PANG Rong- Qing HUANG Lan and PAN Xing-Hua(Cell Biological Therapy Center of Kunming General Hospital, Chengdu Military Region of PLA ＆ Cell Biological Medicine Integrated Engineering Laboratory of State and Region of Yunnan province, Kunming, Yunnan 650032, China Clinical College of Medicine, PLA Kunming General Hospital, Kunming Medical University, Kunming, Yunnan 650032, China Cardiovascular Institute of Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China)
出处
《中国动脉硬化杂志》
CAS
北大核心
2016年第9期882-888,共7页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金(81170316)资助
