载脂蛋白M在非小细胞肺癌A549细胞增殖、迁移和侵袭中的作用

doi:10.3969/j.issn.1673-8640.2020.09.019

摘要/Abstract

摘要：

目的探讨载脂蛋白M（apo M）对非小细胞肺癌（NSCLC）A549细胞系增殖、迁移、侵袭的影响及与基质金属蛋白酶（MMP）-10的关系。方法以转染apo M慢病毒的A549细胞作为实验组（apo M-OE组）,转染空慢病毒的A549细胞作为阴性对照组。分别采用CCK-8法、划痕实验和Transwell侵袭实验检测各组细胞的增殖、迁移和侵袭能力。采用实时荧光定量聚合酶链反应（qRT-PCR）和免疫印迹法检测各组细胞apo M及MMP-10的表达水平。结果与阴性对照组相比,apo M-OE组apo M表达明显上调（P<0.01）,表达量约为阴性对照组的28倍。apo M-OE组细胞增殖倍数及侵袭率明显高于阴性对照组（P<0.001）。与阴性对照组比较,apo M-OE组空白视野显著减少,闭合速率显著加快,即细胞迁移率更大（P<0.001）。apo M-OE组中MMP-10 mRNA及apo M、MMP-10蛋白表达量显著高于阴性对照组（P<0.05）。结论过表达apo M可增强A549细胞的增殖、迁移和侵袭能力,同时使细胞高表达MMP-10。

关键词: 载脂蛋白M, 基质金属蛋白酶-10, 非小细胞肺癌

Abstract:

Objective To investigate the influence of apolipoprotein M（apo M） on the proliferation,migration and invasion of non-small cell lung cancer（NSCLC） A549 cell line and its relationship with matrix metalloproteinase（MMP）-10. Methods A549 cells being transfected with apo M lentivirus were used as apo M-OE group,and those being transfected with A549 empty lentivirus were used as negative control group. CCK-8 method,scratch test and Transwell invasion test were used to determine the proliferation,migration and invasion abilities. The expression levels of apo M and MMP-10 were determined by real-time fluorescence quantitative polymerase chain reaction（qRT-PCR） and immunoblotting. Results Compared with negative control group,the expression level of apo M in apo M-OE group was up-regulated（P<0.01）,and the expression level was about 28 times higher than that of negative control group. The proliferation and invasion rates of apo M-OE group were higher than those of negative control group（P<0.001）. Compared with negative control group,the blank field of vision in apo M-OE group was reduced,the closing rate was accelerated,and the migration rate was greater（P<0.001）. The expression levels of MMP-10 mRNA,apo M protein and MMP-10 protein in apo M-OE group were higher than those in negative control group（P<0.05）. Conclusions Over-expressing apo M can promote the proliferation,migration and invasion of A549 cells,and can make the cells highly expressing MMP-10.

Key words: Apolipoprotein M, Matrix metalloproteinase-10, Non-small cell lung cancer

中图分类号:

R446.1

王子谨, 张晓膺. 载脂蛋白M在非小细胞肺癌A549细胞增殖、迁移和侵袭中的作用[J]. 检验医学, 2020, 35(9): 937-942.

WANG Zijin, ZHANG Xiaoying. Role of apolipoprotein M in the proliferation,migration and invasion of non-small cell lung cancer A549 cells[J]. Laboratory Medicine, 2020, 35(9): 937-942.

图/表 6

表1 apo M基因、MMP-10基因及内参基因的引物及探针序列

名称	序列（5´~3´）	长度/bp
apo M正义引物	TGCCCCGGAAATGGATCTA	60
apo M反义引物	CAGGGCGGCCTICAGTT	54
apo M探针	FAM-CACCTGACTGAAGGGAGCACAGATCTCA-TAMRA	87
MMP-10正义引物	TCGCCCAGTTCCGCCTT	54
MMP-10反义引物	GCACCAGGGGTTCCTCAGTAG	66
MMP-10探针	TGGCATTCAGTCTCTCTACGGACCTC	81
GAPDH正义引物	GGAAGGTGAAGGTCGGAGTC	63
GAPDH反义引物	CGTTCTCAGCCTTGACGGT	60
GAPDH探针	FAM-TTTGGTCGTATTGGGCGCCTG-TAMRA	66

图1 A549细胞的转染情况及apo M mRNA的表达水平注：（a）荧光下与白光下A549细胞的转染情况（×200）;（b）apo M-OE组及阴性对照组apo M mRNA的表达水平

图2 过表达apo M对A549细胞增殖的影响注： ● apo M-OE组; ■ 阴性对照组;与阴性对照组比较,*P<0.001

图3 过表达apo M对A549细胞迁移、侵袭能力的影响注：（a）划痕实验中apo M-OE组和阴性对照组空白视野变化率; ● apo M-OE组; ■ 阴性对照组;与阴性对照组比较,*P<0.001;（b）apo M-OE组和阴性对照组0和96 h的细胞迁移情况（×40）;（c）Transwell小室实验中apo M-OE组与阴性对照组每视野下的侵袭细胞数;与阴性对照组比较,*P<0.001;（d）apo M-OE组细胞的相对迁移能力（以阴性对照组侵袭细胞数为对照计算）;（e）apo M-OE组48 h的细胞侵袭情况（×400）;（f）阴性对照组48 h的细胞侵袭情况（×400）

图4 apo M-OE组和阴性对照组apo M的表达注：（a）apo M蛋白表达的免疫印迹图,1、3为apo M-OE组,2、4为阴性对照组;（b）apo M-OE组及阴性对照组apo M蛋白的相对表达量

图5 apo M-OE组和阴性对照组MMP-10的表达注：（a）apo M-OE组及阴性对照组MMP-10 mRNA的相对表达量;（b）MMP-10蛋白表达的免疫印迹图,1、3为apo M-OE组,2、4为阴性对照组;（c）apo M-OE组及阴性对照组MMP-10蛋白的相对表达量

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