摘要
目的观察MEK、ERK及p-ERK在体外培养血管瘤内皮细胞中的表达，探讨MEK／ERK信号通路在小儿血管瘤的发生、发展及消退过程中的作用。方法用组织块结合酶消化法原代培养血管瘤内皮细胞，待细胞处于对数生长期，换无血清培养基培养48h，使细胞同步化，然后加入0．1ng／L雌二醇，50μmol／L普萘洛尔作为干预组，加入完全内皮细胞培养基作为对照组继续孵育24h。采用Western blot法检测血管瘤内皮细胞MEK、ERK及p-ERK的表达水平，同期用流式细胞计数仪检测血管瘤内皮细胞细胞周期及凋亡的变化，并分析MEK、ERK及p-ERK的表达水平与血管瘤内皮细胞周期及凋亡的关系。结果雌二醇干预后，MEK（0．14±0．01）、ERK1／2（1．36±0．11／0．52±0．10）和p-ERK1／2（0．12±0．03／0．85±0．10）与对照组MEK（0．08±0．00）、ERK1／2（1．16±0．06／0．29±0．04）和p-ERK1／2（0．06±0．02／0．42±0．08）比较，差异均有统计学意义（t=10．01，2．86／3．45，3．57／5．95，P〈0．05）；G0／G1期比例为（90．52±1．28）％，较对照组（94．02±0．85）％明显降低，差异有统计学意义（t=-3．98，P〈0．05）；细胞总凋亡率为（0．70±0.10）％，较对照组（1．77±0．21）％明显降低，差异有统计学意义（t=-8．00，P〈0．05）。普萘洛尔干预后，MEK（0．06±0．01）、ERK1／2（0．87±0．05／0．14±0．02）和p-ERK1／2（0．02±0．01／0．11±0．05）与对照组各项比较，差异均有统计学意义（t=-2．88，-6．79／-6．20，-3．96／-5．74，P〈0．05）；G0／G1期比率为（96．42±1．16）％，较对照组明显升高，差异有统计学意义（t=-2．89，P〈0．05）；细胞总凋亡率为（2．87±0．57）％，对照组明显升高，差异有统计学意义（t=3．14，P〈0．05）。结论MEK／ERK信号通路可能参与调控体外培养血管瘤内皮细胞的细胞周期和凋亡。雌激素、普萘洛尔可能通过干预MEK／ERK信号通路来调控体外培养血管瘤内皮细胞的增殖与凋亡。
Objective To observe the expression and regulation of MEK, ERK and p-ERK in hemangioma derived endothelial cells （HemECs） and examine the effects of MEK/ERK signal pathway in different phases of infantile hemangioma. Methods HemECs were isolated from freshly resected hemangioma specimens. During logarithmic phase, culture medium was changed to serum-free and cell synchronization started within 48 hours. HemECs were treated with 50 μm/L propranolol, 0. 1 ng/L estradiol for treatment group and eulture medium with serum for control group after 24 hours. Then the protein levels of MEK, ERK and p-ERK, cell cycle distribution and apoptosis were observed. Finally the relationship between apoptosis, cell cycle and protein levels of MEK, ERK and p-ERK was analyzed. Results After estradiol treatment for 24 hours, the protein levels of MEK （0. 14±0. 01）,ERK1/2 （1.36 ± 0. 11/0. 52 ± 0. 10）, p-ERK1/2（0, 12 ± 0. 03/0. 85 ± 0. 10） were higher than those of control group MEK（0. 08 ± 0. 00）, ERK1/2 （1. 16 ± 0. 06/0. 29 ± 0. 04） and p-ERK1/2 （0. 06 ± 0. 02/0. 42 ± 0. 08）. And there were significant differences （t = 10. 01,2. 86/3. 45, 3. 57/5.95, P〈0. 05）. The total ratio of cells in Go/Gt phase （90. 52 ± 1.28） % was lower than that of control group （94. 02 ± 0. 85） %. And there were significant differences （t = - 3. 98, P〈0. 05）. The total ratio of apoptosis cells （0. 70 ± 0. 10）% declined compared to control group （1.77 ± 0. 21）%. And the difference was statistically significant （t = - 8. 00, P〈0. 05）. After with a 24-hour treatment of propranolol, the protein levels of MEK （0. 06 ± 0. 01）, ERK1/2（0. 87 ± 0. 05/0. 14 ± 0. 01） and p-ERK1/2 （0. 02 ± 0. 01/0. 11 ± 0. 05） were lower than those of control group. And there was significant difference（t = - 2. 88, - 6. 79/- 6. 20, - 3.96/- 5.74, P〈0. 05）. Cell cycle were arrested in G0/G1 phase. Compared to control group, propranolol group （96. 42 ± 1. 16）% was higher than control group. And there was significant difference（t = - 2. 89,P±0. 05）. The total ratio of apoptotic cells （2. 87 ± 0. 57） % increased versus control group. And the difference was statistically significant （t = 3. 14,P〈0. 05）. Conelus｜ons MEK/ERK pathway plays an important role in G0/G1 phase arrest and apoptosis of hemangioma vascular endothelial cells in vitro. Estradiol and propranolol may regulate the proliferation and apoptosis of HemECs through MEK/ERK signaling pathway.
出处
《中华小儿外科杂志》
CSCD
北大核心
2014年第12期890-894,共5页
Chinese Journal of Pediatric Surgery
基金
国家自然科学基金（81160330,81460476）
贵州省优秀科技教育人才省长资金[黔省专合字（2010）67号]
关键词
血管瘤
内皮
血管
基因表达调控
Hemangioma
Endothelium, vascular
Gene expression regulation
