河北医科大学学报

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TLR4MMP2/MMP9信号通路在氧糖剥夺-复糖复氧诱导人脐静脉内皮细胞凋亡中的作用

  

  1. 1.中国人民解放军第二五二医院麻醉科，河北 保定 071000；2.河北省保定市第一医院麻醉科，河北 保定 071000；3.河北省安国市医院麻醉科，河北 安国  071200
  • 出版日期:2019-02-25 发布日期:2019-01-16
  • 作者简介:刘琳琳（1981-），女，河北安国人，中国人民解放军第二五二医院主治医师，医学学士，从事临床麻醉学研究。

Role of TLR4MMP2/MMP9 signaling pathway in apoptosis of human umbilical vein endothelial cells induced by oxygenglucose deprivationreoxygenation#br#

  1. 1.Department of Anesthesiology, the 252nd Hospital of the Chinese People′s Liberation Army， Hebei
    Province, Baoding 071000, China; 2.Department of Anesthesiology, the First Hospitol of
    Baoding, Heibei Province, Baoding 071000, China; 3.Department of Anesthesiology,
    Anguo Hospital, Hebei Province, Anguo 071200, China
  • Online:2019-02-25 Published:2019-01-16

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摘要： ［摘要］〓
〖HTH〗目的〖HTSS〗〖KG*2〗探讨Toll样受体4（Toll like receptor 4，TLR4）-基质金属蛋白酶2（matrix metalloprotein，MMP2）/基质金属蛋白酶9（matrix metalloprotein，MMP9）信号通路在氧糖剥夺-复糖复氧诱导人脐静脉内皮细胞凋亡中的作用。
〖HTH〗方法〖HTSS〗〖KG*2〗离体培养人脐静脉内皮细胞株HUVEC12，以1×106/mL接种于6孔板（每孔2 mL）或1×104/mL 接种于96孔板（每孔200 μL），采用随机数字表法将36孔样本分为4组，对照组（C组）、氧糖剥夺组（O组），TAK242+氧糖剥夺组（TO组），CCT+氧糖剥夺处理组（MO组），每组6孔。O、TO和MO组人脐静脉内皮细胞接受氧糖剥夺6 h后复糖复氧24 h；TO和MO组人脐静脉内皮细胞接受氧糖剥夺前1 h向培养基中分别加入TLR4特异性阻断剂TAK242（10 μmol/mL）和MMP2/MMP9特异性抑制剂CCT（200 μg/mL）；对照组（C组）人脐静脉内皮细胞正常培养。按照各组处理收集细胞，Annexin VFITC/PI双染色-流式细胞法测定细胞凋亡率，MTT法测定细胞生存率，Transwell法检测HUVEC12细胞电阻值及渗透性，酶联免疫吸附测定法检测TNFα和IL1β浓度，Western blot法测定MMP2、MMP9表达。
〖HTH〗结果〖HTSS〗〖KG*2〗与C组比较，O组、TO组和MO组HUVEC12细胞凋亡率和葡聚糖FITC荧光强度升高，存活率和电阻值降低，TNFα、IL1β、MMP2和MMP9表达水平升高（P＜005）；与O组比较，TO组HUVEC12细胞凋亡率和葡聚糖FITC荧光强度降低，存活率和电阻值升高，TNFα、IL1β、MMP2和MMP9表达水平下降（P＜005）；与O组比较，MO组HUVEC12细胞凋亡率和葡聚糖FITC荧光强度降低，存活率和电阻值升高，TNFα和IL1β表达水平下降（P＜005）；与TO组比较，MO组HUVEC12细胞凋亡率和葡聚糖FITC荧光强度升高，存活率和电阻值降低，TNFα、IL1β、MMP2和MMP9表达水平升高。
〖HTH〗结论〖HTSS〗〖KG*2〗氧糖剥夺-复糖复氧诱导HUVEC12细胞凋亡的机制可能与激活TLR4MMP2/MMP9信号通路相关。

关键词: 缺氧, 人脐静脉内皮细胞, 细胞凋亡

Abstract: ［Abstract］ Objective〖HTSS〗〓To explore the effects of Toll like receptor 4(TLR4)matrix metalloprotein2(MMP2)/matrix metalloprotein9(MMP9) signal pathway on apoptosis of human umbilical vein endothelial(HUVEC) induced by oxygenglucose deprivation/resuscitation.
〖HTH〗〖WTHZ〗Methods〖HTSS〗〓In vitro, HUVEC12 cells which was exposed to oxygen and glucose(OGD) for 6 h and 〖JP2〗reperfusion for 24 h were divided into 4 groups: control(C) group, OGD/R(O) group, TAK242+〖JP〗OGD/R(TO) group, CCT+OGD/R(MO) group. Before OGD exposure, the cultures in TO and MO groups were treated with TLR4 inhibitor TAK242(10 μmol/mL) or MMP2/MMP9 inhibitor CCT(200 μg/mL), respectively. The effects of apoptosis and viability were assessed by Annexin VFITC/PI and MTT assays. The resistance and permeability were assessed by Transwell assay. The levels of TNFα and IL1β were assessed by ELISA. The expressions of MMP2 and MMP9 were assessed by western blot measurement.
〖HTH〗〖WTHZ〗Results〖HTSS〗〓Compared with group C, the apoptosis rate and glucanFITC fluorescence intensity of HUVEC12 cells in group O, TO and MO increased, while the survival rate and resistance decreased, and the expression levels of TNFa, IL1β, MMP2 and MMP9 increased(P＜005). Compared with O group, the apoptosis rate and fluorescence intensity of glucanFITC in TO group decreased, the survival rate and resistance increased, and the expression levels of TNFα, IL1β, MMP2 and MMP9 decreased(P＜005). Compared with group O, the apoptosis rate of HUVEC12 cells and the fluorescence intensity of dextranFITC in MO group were decreased, the survival rate and resistance value were increased, and the expression of TNFα and IL1β in water skin was decreased(P＜005). Compared with TO group, the apoptosis rate of HUVEC12 cells and the fluorescence intensity of dextranFITC in MO group increased, the survival rate and resistance value decreased, and the expression of TNFα, IL1β, MMP2 and MMP9 in the water skin increased(P＜005).
〖HTH〗〖WTHZ〗Conclusion〖HTSS〗〓Apoptosis of HUVEC12 cells induced by oxygenglucose deprivationreoxygenation may be related to activation of TLR4MMP2/MMP9 signaling pathway.

Key words: anoxia； human umbilical vein endothelial cells, apoptosis

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