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1.450042 郑州,解放军第988医院心内科
2.400037 重庆,陆军军医大学(第三军医大学)第二附属医院心内科
[黄岚,E-mail:huanglan260@126.comHUANG Lan, E-mail:huanglan260@126.com
收稿:2018-09-17,
修回:2018-12-20,
纸质出版:2019-03-15
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王连友, 杨杰, 黄岚. 钙库操纵性钙内流参与调节血管内皮细胞增殖与迁移[J]. 第三军医大学学报, 2019,41(5):424-429.
Lianyou WANG, Jie YANG, Lan HUANG. Store-operated calcium entry is involved in proliferation and migration of vascular endothelial cells[J]. Journal of Third Military Medical University, 2019, 41(5): 424-429.
王连友, 杨杰, 黄岚. 钙库操纵性钙内流参与调节血管内皮细胞增殖与迁移[J]. 第三军医大学学报, 2019,41(5):424-429. DOI: 10.16016/j.1000-5404.201809115.
Lianyou WANG, Jie YANG, Lan HUANG. Store-operated calcium entry is involved in proliferation and migration of vascular endothelial cells[J]. Journal of Third Military Medical University, 2019, 41(5): 424-429. DOI: 10.16016/j.1000-5404.201809115.
目的
2
利用体外培养的人脐静脉血管内皮细胞,初步探讨钙库操纵性钙内流(store-operated calcium entry,SOCE)对血管内皮细胞(endothelial cell,EC)功能的影响。
方法
2
培养于激光共聚焦培养皿的EC随机分为对照组和不同剂量(10、25、50、75、100 μmol/L)SOCE抑制剂(2-APB)干预组。干预组细胞以2-APB干预5 min后,检测各组细胞的SOCE强度(
n
=10)。培养于6孔板的细胞,根据实验目的,随机分为对照组和不同剂量(50、75、100 μmol/L)的2-APB干预组。2-APB干预24 h后,以CCK-8检测各组EC的增殖功能(
n
=9)、以Transwell培养法检测各组EC的迁移功能(
n
=10)。2-APB干预2 h后,以Griess Reagent方法检测各组EC的NO产量(
n
=6)以及用Western blot方法检测各组EC的eNOS变化(
n
=3)。
结果
2
较低剂量的2-APB(10、25 μmol/L)对SOCE有抑制趋势,但无统计学意义(
P
>
0.05),较高剂量的2-APB(50、75、100 μmol/L)能够显著抑制SOCE(
P
<
0.05)。采用具有显著抑制作用浓度的2-APB(50、75、100 μmol/L)抑制SOCE后,能够显著抑制EC的增殖能力(与对照组比较,EC的增殖分别下降33.2%、46.3%和61.2%,
P
<
0.05)、迁移功能(与对照组比较,分别下降33.5%、54.3%和64.9%,
P
<
0.05)、NO产量(与对照组比较,分别下降40.5%、61.9%和73.1%,
P
<
0.05)以及eNOS的合成(与对照组比较,75和100 μmol/L组分别下降45.1%和62.4%,
P
<
0.05)。
结论
2
SOCE参与调节EC的增殖和迁移功能,抑制SOCE能引起EC功能异常,此作用可能是通过抑制eNOS的合成实现。
Objective
2
To investigate the effects of store-operated calcium entry (SOCE) on the bio-functions of vascular endothelial cells (ECs) based on human umbilical vein endothelial cells (HUVECs).
Methods
2
The cultured HUVECs were divided into control group and 2-APB (SOCE inhibitor
10
25
50
75 and 100 μmol/L) intervention groups. The SOCE was mea
sured by the fluorescence intensity of calcium probe in 5 min after 2-APB treatment (
n
=10). The proliferation (
n
=9) and migration (
n
=10) capacities of HUVECs after different doses of 2-APB treatment (50
75 and 100 μmol/L) for 24 h were measured by CCK-8 assay and Transwell assay. In addition
at 2 h after 2-APB treatment
Griess reagent kit was employed to detect the production of NO (
n
=6)
and Western blotting was used to measure the expression of eNOS (
n
=3) in different groups.
Results
2
Low doses of 2-APB (10 and 20 μmol/L) showed an inhibitory effect on SOCE
but the inhibition had no statistical significance (
P
<
0.05)
while high doses of 2-APB (50
75 and 100 μmol/L) inhibited SOCE significantly (
P
>
0.05). Treatment of 2-APB (50
75 and 100 μmol/L) also suppressed the proliferation (decreased by 33.2%
46.3% and 61.2%
respectively when compared with control cells
all
P
<
0.05)
the migration (decreased by 33.5%
54.3% and 64.9%
respectively when compared with control cells
all
P
<
0.05)
NO production (decreased by 40.5%
61.9% and 73.1%
respectively when compared with control cells
all
P
<
0.05) as well as the synthesis of eNOS (decreased by 45.1% and 62.4% in the 75 and 100 μmol/L treated cells
respectively when compared with control cells
both
P
<
0.05) in ECs.
Conclusion
2
SOCE is involved in the proliferation and migration of ECs
and inhibition of SOCE can induce ECs dysfunction
which may act through suppressing eNOS synthesis.
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