锌离子对人脐静脉内皮细胞的影响

Influence of zinc ion on human umbilical vein endothelial cells

目的探究氯化锌(Zn Cl2)对人脐静脉内皮细胞(HUVECs)的影响。方法体外培养HUVECs,氯化锌浓度6~34μM分别处理细胞,MTS检测生长活性,筛选促细胞增殖的最佳浓度作为实验组浓度。实验组和对照组(未处理)流式细胞术检测HUVECs凋亡情况;鬼笔环肽-异硫氰酸荧光素(FITC)染色检测HUVECs骨架重构;Transwell小室检测HUVECs迁移和侵袭;Real-time PCR检测细胞迁移相关蛋白基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)m RNA相对表达水平;明胶酶谱法检测MMP-2、MMP-9的活性;ELISA法检测HUVECs上清液MMP-2、MMP-9水平。结果与对照组相比,浓度范围在10~22μM内Zn Cl2处理的细胞,其相对增殖率升高,差异具有统计学意义(P均<0.05)。选取促进HUVECs增殖的最佳浓度14μM作为实验组浓度。实验组的HUVECs凋亡率为(12.02±0.84)%,高于对照组的(4.05±0.52)%,差异有统计学意义(P<0.01)。实验组HUVECs细胞迁移个数为(112.20±10.83)个,高于对照组的(94.80±9.50)个,差异有统计学意义(P<0.05)。实验组HUVECs细胞侵袭个数高于对照组,[(36.20±3.22)个vs.(32.80±1.07)个],差异有统计学意义(P<0.05)。实验组HUVECs细胞迁移相关蛋白MMP-2和MMP-9的m RNA相对表达水平高于对照组,差异有统计学意义(P均<0.05)。实验组细胞迁移相关蛋白MMP-2的光密度值显著高于对照组,差异有统计学意义(P<0.05);实验组细胞MMP-9的光密度值高于对照组,差异有统计学意义(P<0.05)。实验组细胞上清液中MMP-2和MMP-9的吸光度值高于对照组,差异有统计学意义(P均<0.05)。结论 14μM氯化锌处理HUVECs,使其处于增殖和凋亡平衡的生长活跃状态,并且促进HUVECs骨架重塑和细胞迁移。

Objective To study the influence of zinc chloride （ZnCl2） on human umbilical vein endothelial cells （HUVECs）. Methods HUVECs were in vitro cultured and treated with ZnCl2 respectively in doses from 6 μM to 34 μM. The growth activity was detected by using MTS test for screening the best dose of ZnCl2 to promote HUVECs proliferation as test group dose. The apoptosis of HUVECs was detected by using flow cytometry in test group and control group （without ZnCl2 treating）. The cytoskeleton remodeling of HUVECs was detected after FITC-Phalloidine staining. The migration and invasiveness of HUVECs were detected by using Transwell chamber test. The relative expressions of matrix metalloproteinase-2 （MMP-2） mRNA and matrix metalloproteinase-9 （MMP-9） mRNA were detected by using real-time fluorescence quantitative polymerase chain reaction （RT-PCR）. The activities of MMP-2 and MMP-9 were detected by using gelatin zymography. The levels of MMP-2 and MMP-9 in HUVECs supernatant were detected by using enzyme-linked immunosorbent assay （ELISA）. Results The relative growth rate （RGR） of HUVECs treated with ZnCl2 in doses from 10 μM to 22 increased in test group compared with control group （all P〈0.05）. The best dose of ZnCl2 （14 μM） to promote HUVECs proliferation was chosen astest group dose. The apoptosis rate of HUVECs was （12.02±0.84）% in test group and （4.05±0.52）% in control group （P〈0.01）. The migration numbers of HUVECs were （112.20±10.83） in test group and （94.80±9.50） in control group （P〈0.05）. The invasiveness numbers of HUVECs were higher in test group than those in control group [（36.20±3.22） vs. （32.80±1.07）, P〈0.05]. The expressions of MMP-2 mRNA and MMP-9 mRNA of HUVECs were higher in test group than those in control group （all P〈0.05）. The optical density （OD） value of MMP-2 was significantly higher in test group than that in control group （P〈0.05）. The OD value of MMP-9 was higher in test group than that in control group （P〈0.05）. The absorbance values of MMP-2 and MMP-9 in HUVECs supernatant were higher in test group than those in control group （all P〈0.05）. Conclusion HUVECs, treated with ZnCl2 （14μM）, are in an active statement of growth with the balance of proliferation and apoptosis, and their cytoskeleton remodeling and migration are promoted.