The Mechansm Of Estrogrn-mediated Protection Against HIV-1Tat-induced Disruption The Tight Junction Protine

Cultivation of human brain endothelial cells【Objective】 To investigate the cell culture,growth cycle of human brainendothelial cells(hCMEC/D3), the toxicity effect of HIV-1Tat and intracellularreactive oxygen species production. To set up a suitable culture system for nextstudy by observing the biological characteristics of hCMEC/D3and theinhibitory effect of drugs。【Method】 Inverted microscope was applied for hCMEC/D3morphologyobservation, then the growth curve of hCMEC/D3was plotted.MTT was used todetect hCMEC/D3activity after treatment with HIV-1Tat protein. The reactiveoxygen species (ROS) in hCMEC/D3were detected by using2’,7’-dichlorofluorescein (DCFH) via fluorescence spectrophotometer.【Results】Under the microscope, hCMEC/D3was polygon or long spindle.The ratio of cell growth inhibition less than30%after treatment with1ug/ml Tat for24h. The average fluorescence intensity of ROS in the group of HIV-1Tattreatment was10.315±0.648(S), which is higher than the blank control.【Conclusion】 hCMEC/D3are active and stable with continuouslypassaging. HIV Tat protein does not affect the activation of hCMEC/D3,Intracellular ROS increase in the treatment of HIV Tat protein. Inhibition of estradiol on HIV-1Tat-induced Tight Junction ProteinExpression in human brain endothelial cells【Objective】 To investigate the effect of17β-estradiol(E2) on HIV-1Tat-induced altertions of the tight junction protein expression.【Methods】 Western blot was used to detected the expression of tightjunction protein in human brain endothelial cells（hCMEC/D3）treated with E2and HIV-1Tat. hCMEC/D3were pretreated with serum free Endothelial CellBasal Medium-2was used as control(CTL group). The other groups includedE2+Tat group (cells treated with10-8mol/L estradiol (E2) for2h, then co-treatedwith HIV-1Tat for24h), E2group ((cells treated with estradiol for24h) and Tatgroup (cells treated with HIV-1Tat for24h).【Results】 The tight junction protein expression in hCMEC/D3ofClaudin-1,zona occludens-1(ZO-1),zona occludens-2(ZO-2) decreased withtreatment Tat for24h（P<0.05，P<0.01，P<0.01, respectively）. E2inducedupregulation of expression of Claudin-1, ZO-1（P<0.01，P<0.05）and attenuatedHIV-1Tat-induced degrdation of Claudin-1and ZO-2（P<0.05，P<0.01）,however, the expression of ZO-1was unaffected (p>0.05).【Conclusions】 HIV-1Tat-induced the tight junction protein degrdation of Claudin-1, ZO-1, ZO-2in human brain microvascular endothelial cells.Moreover, E2can protecte against HIV-1Tat-induced degradation ofClaudin-1, ZO-2.