| Objective:Environmental pollution has been implicated in the etiology of cardiovascular disease,and the influence is more and more heavy.Benzo(a)pyrene,as a lipophilic Polycyclic aromatic hydrocarbons(PAHs),has obviously carcinogenic.Recent years,some study has found Benzo(a)pyrene may initiate vessel injury and lead to the accelerated atherosclerosis.Thrombomodulin is widely expressed in endothelial cells,contributing to anti-inflammatory and anticoagulant effects,however the link between benzopyrene and thrombomodulin is rarely reported.KLF2,known as endothelial cells’ molecular switch,adjust the physiological functions of endothelial cells through a variety of ways,and keep the endothelial cell function in the stationary state.Previous research has shown that KLF2 can upregulate the expression of TM.The aim of this study was to evaluate the impact of benzo(a)pyrene on the TM and TMmRNA in HUVEC cells and the role of KLF2 in this change.Methods:The primary human umbilical vein endothelial cells were isolated and cultured,and the 2~4 generation of HUVECs were used in experimen.Experiment group: 0.1%DMSO,four groups of different concentrations(0.1mg/L,1mg/L,2mg/L,5mg/L)of benzopyrene solution.(1)CCK-8 test detected the effects of BaP on the proliferation of HUVECs.Every group was incubated different time of 6h,24 h.Microplate Reader was used for detecting A value;(2)After each group of different concentrations incubated cells for 6h,24 h and 2mg/LBap cultured different time of 0h,1h,6h,12 h and 24 h,IgG FITC antibody incubated cells labeled Anti-Thrombomodulin antibody,then Flow cytometric analysis technique was used for detecting MFI of 10000 cells,(3)and RT-PCR was used for detecting TM mRNA;(4)HUVECs was incubated for 24 h by different concentrations,and for 0h,1h,6h,12 h and 24 h by 2mg/LBaP.RT-PCR was used for detecting LKLF mRNA;(5)Using Control siRNA-A or LKLF siRNA-A transfected HUVEC,then 2mg/L BaP and 0.1%DMSO cultured 24 h,Flow cytometric analysis technique detected the TM MFI of each group and RT-PCR was used for detecting TM mRNA.Results:(1)At low magnification,the cells were spindle shaped,polygonal,and the nucleolus was clearly identified by.VIII factor:more than 95% of the endothelial cells contained brown granules,positive staining,confirmed that the cells were endothelial cells.(2)Compared to control group,different concentration of 6h,24 h BaP groups were observed to reduce A value with the increasing of concentration.But only 24h2mg/L,5mg/L BaP groups were significant difference(P<0.05).(3)Compared to0.01%DMSO group,different concentration of Ba P in 6h and 24 h groups were observed to decrease the TM protein expression with the increasing of concentration.Reaching0.1mg/L in 24 h,the difference was significant(P<0.05).Compared to control group,different time of 2mg/L Ba P groups were observed to decrease the TM protein expression with the increasing of time,in 1h and 24 h the difference was significant(P<0.05).(4)Compared to 0.1%DMSO group,different concentration of BaP groups were observed to decrease the TM mRNA expression with the increasing of concentration,and achieve to 2mg/L Ba P group in 6h or 0.1mg/L in 24 h was significant difference(P<0.05),Compared to 0h,different time of tar groups were observed to decrease the TM mRNA expression with the increasing of time(P<0.05).(5)It was also observed that the KLF2 m RNA expression was decreased.And in 2mg/L BaP group,each time was significant difference(P<0.05).(6)Compared with control siRNA-A group,TM MFI and TMmRNA expression in each groups were decreased,there was significant difference(P < 0.05).Compared with LKLF siRNA group,the reduction of LKLF siRNA+2mg/L group significantly decreased(P<0.05).Conclusion:To some extent,benzopyrene can inhibit the proliferation of HUVECs,the expression of thrombomodulin and the production of KLF2 mRNA,and the change of the expression of TM may be affected by the level of KLF2 mRNA. |
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