摘要
目的探讨趋化因子配体2(CCL2)对白血病细胞的趋化作用及高表达后降低白血病细胞迁移侵袭的能力。方法以白血病细胞K562为研究对象,在Transwell小室的下室加入重组CCL2蛋白进行趋化,检测细胞迁移和侵袭能力,Western blot检测重组CCL2蛋白作用后的K562细胞中基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(MMP-9)的表达水平。K562细胞转染CCL2过表达载体,RT-PCR和Western blot检测转染效果,ELISA法检测转染后细胞培养液上清中CCL2含量。Transwell小室检测过表达CCL2的细胞迁移和侵袭能力,ELISA法检测侵袭实验Transwell小室上室和下室中CCL2含量。结果 Transwell小室下室中加入CCL2蛋白后K562细胞的迁移和侵袭穿膜指数分别为:2. 35±0. 26、2. 58±0. 18,均明显高于不加CCL2蛋白的K562细胞迁移和侵袭穿膜指数1. 00±0. 09、1. 01±0. 09。重组CCL2蛋白作用后K562细胞中MMP-2表达水平分别由0. 26±0. 12升高至0. 99±0. 08,MMP-9水平由0. 39±0. 06升高至1. 18±0. 13。K562细胞转染CCL2过表达载体后能够成功过表达细胞中CCL2水平,提高细胞分泌CCL2水平。过表达CCL2后的K562细胞穿膜侵袭指数从0. 99±0. 11降低至0. 58±0. 16,侵袭实验小室上室和下室中CCL2含量依次为:(900. 58±69. 63) ng/L、(98. 84±58. 64) ng/L。过表达CCL2后迁移穿膜指数无明显变化(1. 02±0. 14 vs. 0. 99±0. 08)。结论 CCL2能够定向趋化白血病细胞迁移和侵袭,而高表达CCL2后白血病细胞由于自分泌功能导致侵袭能力降低,作用机制可能与MMP-2和MMP-9水平有关。
Objective The study of CCL2 on the chemotactic action of leukemia cells and the ability to reduce the invasion of leukemia cells after high expression were discussed.Methods Leukemia cell K562 was used as the object of study,recombinant CCL2 protein was added to the lower chamber of the Transwell chamber for chemotaxis,cell migration and invasion were examined,Western blot was used to detect the expression levels of MMP-2 and MMP-9 in K562 cells after recombinant CCL2 protein.K562 cells were transfected with CCL2 overexpression vector,transfection effects were detected by RT-PCR and Western blot,the content of CCL2 in the supernatant of culture medium after transfection was detected by ELISA.the cell migration and invasion ability of CCL2 expressing Transwell was detected,ELISA assay was used to detect the CCL2 content in the Transwell chamber and lower chamber.Results The migration and invasion of CCL2 protein in the lower chamber of Transwell chamber were 2.35±0.26,2.58±0.18 respectively,the migration and invasion of protein were significantly higher than those without CCL2,and the transmembrane index was 1.00±0.09,1.01±0.09.After recombinant CCL2 protein,the expression levels of MMP-2 in K562 cells increased from 0.26±0.12 to 0.99±0.08 respectively,the level of MMP-9 increased from 0.39±0.06 to 1.18±0.13.After transfection of CCL2 overexpression vector,K562 cells can successfully express CCL2 levels in cells,increase cell secretion of CCL2 levels.After overexpression of CCL2,the transmembrane attack index of K562 cells decreased from 0.99±0.11 to 0.58±0.16,the contents of CCL2 in the upper and lower chambers of the invasive experiment were(900.58±69.63)ng/L,(98.84±58.64)ng/L.After overexpression of CCL2,there was no significant change in the migration through membrane index(1.02±0.14 vs.0.99±0.08).Conclusion CCL2 can direct chemotaxis,migration and invasion of leukemic cells.However,after high expression of CCL2,leukemic cells decrease invasiveness because of autocrine function,the mechanism of action may be related to the levels of MMP-2 and MMP-9.
作者
黄欣
秦云
黄江涛
HUANG Xin;QIN Yun;HUANG Jiang-tao(The Bood Internal Medicine,Xiaogan Center Hospital,Xiaogan Hubei 432000,China)
出处
《临床和实验医学杂志》
2019年第2期152-156,共5页
Journal of Clinical and Experimental Medicine
关键词
白血病
趋化因子配体2
迁移侵袭
Leukemia
Chemokine C-C motif ligand 2
Migration
Invasion
