ANGPTL4对LPS诱导的人肺微血管内皮细胞损伤的保护作用

doi:10.3877/cma.j.issn.1674-6902.2017.02.016

目的

探讨ANGPTL4对脂多糖(LPS)诱导的人肺微血管内皮细胞(HPMVECs)损伤的保护作用。

方法

以体外培养的HPMVECs作为实验对象，将细胞随机分为四组：正常对照组(C组)、LPS诱导组(L组)、ANGPTL4表达组(A组)和ANGPTL4表达＋ LPS诱导组(A＋L组)。各处理组细胞分别培养12、24、48、72 h后采用CCK-8法观察细胞增殖；细胞孵育12 h后，采用荧光定量PCR和Wester-blot检测ANGPTL4表达水平，ELISA法检测细胞培养液中TNF-α、IL-1β和MCP-1的蛋白含量。

结果

与C组相比，L组和A组细胞的ANGPTL4表达明显增高(P<0.01)；与L组相比，A＋L组细胞的ANGPTL4表达明显增高(P<0.01)，LPS刺激可以抑制细胞增殖，而经过pcDNA3.1-ANGPTL4重组质粒转染后这种抑制作用又被削弱了；高表达ANGPTL4抑制炎症因子TNF-α、IL-1β和MCP-1的生成。

结论

ANGPTL4可以减弱LPS抑制HPMVECs细胞增殖的作用，降低细胞中TNF-α，IL-1β和MCP-1等炎症因子的生成，这些效应可能是ANGPTL4保护肺微血管内皮细胞的作用机制之一。

关键词: ANGPTL4, 急性肺损伤, 人肺微血管内皮细胞

Objective

To investigate the protective effect of ANGPTL4 to human lung microvascular endothelial cell injury.

Method

Human lung microvascular endothelial cells cultured in vitro were randomly divided into four groups: normal control group (group C), LPS induced group (group L), ANGPTL4 expression (group A) and ANGPTL4+ LPS induced group (group A+ L). CCK8 method was used to observe cell proliferation; fluorescence quantitative PCR and Western blot were applied to test ANGPTL4 expression level, Inflammatory factor TNF-α, IL-1β and MCP-1in cell culture fluid were also detected by ELISA method.

Results

Compared with group C, the expression of ANGPTL4 in group L and group A was significantly higher(P<0.01); Compared with group L, The expression of ANGPTL4 in A+ L group was significantly higher (P<0.01), LPS stimulation can inhibit cell proliferation, and after pcDNA3.1- ANGPTL4 recombinant plasmid transfection, this inhibition was weakened; high ANGPTL4 expression can also inhibit the generation of inflammatory factor TNF-α, IL-1β and MCP-1.

Conclusions

ANGPTL4 can inhibit LPS induced HPMVECs cell proliferation decreased, and reduce the generation of Inflammatory factor TNF-α, IL-1β and MCP-1, which may be one of the mechanism of the protective effect of ANGPTL4 to human lung microvascular endothelial cells.

Key words: ANGPTL4, Acute lung injury, Human lung microvascular endothelial cell

表1 相关引物序列

基因	引物	引物序列
ANGPTL4	ANGPTL4_F	5′-ACAACTCAAGGCTCAGAA-3′
?	ANGPTL4_R	5′-CTCATGGTCTAGGTGCTT-3′
ALB	ALB_F	5′-TGAAGGTCGGAGTCAACGGATTTG-3′
?	ALB_R	5′-ATGTGGGCCATGAGGTCCACCAC-3′
FOXP2	FOXP2_F	5′-TCCATCAAAGAAACATGACTAAATGC-3′
?	FOXP2_R	5′-GTCAACTCCCTGAGGATAGGAACAT-3′

表1 相关引物序列

基因	引物	引物序列
ANGPTL4	ANGPTL4_F	5′-ACAACTCAAGGCTCAGAA-3′
?	ANGPTL4_R	5′-CTCATGGTCTAGGTGCTT-3′
ALB	ALB_F	5′-TGAAGGTCGGAGTCAACGGATTTG-3′
?	ALB_R	5′-ATGTGGGCCATGAGGTCCACCAC-3′
FOXP2	FOXP2_F	5′-TCCATCAAAGAAACATGACTAAATGC-3′
?	FOXP2_R	5′-GTCAACTCCCTGAGGATAGGAACAT-3′

图1 LPS刺激和pcDNA3.1-ANGPTL4重组质粒转染后细胞中ANGPTL4的表达；注：A：RT-PCR检测各组细胞ANGPTL4基因的表达；B：Western blot检测各组细胞ANGPTL4蛋白的表达(^*P<0.05)

图2 LPS刺激和pcDNA3.1-ANGPTL4重组质粒转染后对细胞增殖和炎症因子的影响；注：A：CCK8检测各组细胞的增殖；B：ELISA检测各组细胞培养液中炎症因子TNF-α、IL-1β和MCP-1的含量(^*P<0.05)

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Protective effect of ANGPTL4 on human lung microvascular endothelial cell injury

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Protective effect of ANGPTL4 on human lung microvascular endothelial cell injury