Effects Of HIV-1 Tat From A HAD Patient And A Non-HAD Patient On Endothelial Cells Activity And Tight Junction Protein Content

OBJECTIVEHIV-1 Tat is an important regulatory protein encoded by HIV-1 tat,which can participate in the transcription and activation of the virus and promote the replication of the virus in cells.At the same time,Tat can be secreted and released to the outside of cells by T cells,macrophages and other cells infected by HIV-1.It can reach multiple parts of the body by virtue of its transmembrane function.It has been shown that most of HIV-1 patients,especially those who develop AIDS encephalopathy and AIDS encephalitis have different degrees of structural damage and functional decline in BBB.Endothelial cells are the first line of defense for BBB and play an important role in controlling substances entry and exit and maintaining homeostasis of BBB.Tight junction proteins are complex structures interconnected by endothelial cells.Reducing the expression of tight junction proteins can increase endothelial cells permeability.The tight junction protein is mainly composed of transmembrane protein,cytoplasmic adhesion protein and cytoskeletal protein.ZO-1 is a cytoplasmic adhesion protein,which maintains the stability of the junction system.It plays an important role in cell signal transduction,regulation of cell proliferation and differentiation,and permeability of BBB.It is often used as an indicator for observing tight junction barrier function and evaluating permeability function.The effect of HIV-1 Tat on endothelial cells can be divided into two categories:?Direct effect:Tat can damage endothelial cells by damaging the organelles of endothelial cells and up-regulating the expression of pro-apoptotic proteins.It also can reduce the expression of tight junction proteins,which can increase the permeability of endothelial cells.?Indirect effect:Tat can stimulate astrocytes to secrete pro-inflammatory mediators,which are participating in inflammatory reactions,and thereby damage endothelial cells.In recent years,there have been many studies on the mechanism of HIV-1 Tat damaging endothelial cells,but the mechanism is still unclear.Therefore,comprehensive and in-depth studies on the mechanism of Tat damaging to endothelial cells is of great significance for the prevention,treatment and control of HIV related neurocognitive disorders.In this study,in order to research the direct effect of endothellial cells,HIV-1 Tat derived from basal ganglia(BG)in HIV-associated dementia(HAD)patients was expressed and purified in prokaryotic cells,and the activity of HIV-1 Tat protein on endothelial cells was studied.At the same time,the spleen(SPL)and BG-derived Tat of HAD,non-HAD(non-HIV-associated dementia,non-HAD)AIDS patients were expressed in U87 cells.The effect of U87 cell supernatant on the content of tight junction protein ZO-1 in primary mouse cerebral microvascular endothelial cells(MCMECs)was studied,in order to explore the indirect effects of HIV-1 Tat protein on endothelial cells and provide a scientific basis for the pathogenesis of HAD.METHODS1.The effect of HIV-1 Tat on endothelial cells activity(1)Construction of pET-32a-tat prokaryotic expression vectorThe HAD-BG group HIV-1 tat gene was amplified by sequencing the correct pGEX-KG-tat recombinant plasmid as a template.The His tag was added to the primer for purification of HIV-1 Tat.The PCR product and pET-32a vector were digested and ligated to construct pET-32a-tat prokaryotic expression vector,which was transformed into E.coli competent DH5?.It was screened by ampicillin and picked up by single colony and identified by PCR.It was sequenced by company and the sequencing results were compared.(2)Expression,purification and quantification of HIV-1 TatThe pET-32a-tat prokaryotic expression vector was transformed into E.coli competent BL2 1(DE3).The single colonies were picked and inoculated into LB medium containing 1‰ampicillin.When the A of bacterial fluid was between 0.6 and 0.8,IPTG was added into it to inducing expression of Tat.Protein expression was identified by SDS-PAGE and Western Blot.After the cells were disrupted,the supernatant was collected by centrifugation and purified by a nickel affinity chromatography column and a gel filtration prepacked column.Tat was concentrated and quantified by BCA protein.(3)Effect of HIV-1 Tat on HUVECs activityHUVECs were placed into 96-well plates and Tat was added at a final concentration of 100 ng/ml,200 ng/ml,300 ng/ml,400 ng/ml,500 ng/ml,and 1000 ng/ml with 5 parallel holes per concentration.Normal cells were used as negative controls and the medium was used as blank control.After 36h,the cck-8 was added,and the A value at 450 nm was detected by a microplate reader.2.Effect of U87 supernatant with HIV-1 Tat on the content of ZO-1 of primary MCMECs(1)Construction of pEGFP-N1-tat eukaryotic expression vectorThe HAD-SPL,HAD-BG,non-HAD-SPL,and non-HAD-BG HIV-1 tat gene were amplified by using pGEX-KG-tat and pMD19-T-tat recombinant plasmids as templates.The PCR product and pEGFP-N1 vector were digested and ligated to construct pEGFP-N1-tat eukaryotic expression vector,which was transformed into E.coli competent DH5a.It was screened by ampicillin and picked up by single colony and identified by PCR.It was sequenced by company and the sequencing results were compared.(2)Expression of HIV-1 Tat in U87 cellsThe HAD-SPL,HAD-BG,non-HAD-SPL,and non-HAD-BG eukaryotic expression vector pEGFP-N1-tat was constructed successfully and transfected into U87 cells.Normal cells were used as blank control,and pEGFP-Nl vector group was used as negative control.The time-dependent changes in HIV-1 Tat expression were determined by observing green fluorescent protein.Immunohistochemistry demonstrated that HIV-1 Tat was expressed in U87 cells after transfection of eukaryotic expression vector pEGFP-N1-tat.(3)Extraction,culture and identification of primary MCMECs6?8 Kunming mice of 4?6 week old were selected,brain tissue was cut aseptically and digested with papain and DNase I.1.7 times volume of 22%BSA was added and centrifuged.The lower layer was resuspended in endothelial cell medium and inoculated on a plate coated with rattailtendon collagen I.After the cells were covered with a single layer,CD31 and VIII factors of endothelial cells were detected and identified by immunofluorescence.(4)Effect of U87 supernatant with HIV-1 Tat on the content of ZO-1 of primary MCMECsThe U87 supernatants expressing the HIV-1 Tat of HAD-SPL,HAD-BG,non-HAD-SPL,and non-HAD-BG were collected and added into the MCMECs.The effect of U87 supernatant on the content of MCMECs ZO-1 was detected by Western Blot.The normal cells were used as blank control and pEGFP-N1 vector group was used as negative control.The gamma values of the ratio of ZO-1 and ?-actin were analyzed by Image J software.The effects of different supernatants of U87 cells on the protein content of ZO-1 were analyzed by calculating ZO-1/?-actin.3.Statistical analysisThe activity of HUVECs and the content of MCMECs ZO-1 protein were statistically analyzed by SPSS 16.0 software.The data were described by mean± standard deviation(x±s),and analyzed by analysis of variance(a = 0.05).RESULTS1.The effect of HIV-1 Tat on endothelial cells activity(1)Construction of pET-32a-tat prokaryotic expression vectorThe PCR amplification product was subjected to agarose gel electrophoresis,and a band corresponding to the size of the predicted tat first exon gene(216 bp)fragment appeared at 250 bp.A single colony was picked for PCR identification and confirmed to be the HIV-1 tat gene.The sequencing results of the company were correct and proved that the prokaryotic expression vector pET-32a-tat was successfully constructed.(2)Expression,purification and quantification of HIV-1 TatThe results of SDS-PAGE and Western Blot showed that there were obvious protein bands at 15KD,which were consistent with the size of HIV-1 Tat,and a small part was present as multimer.At the same time,the results showed that most of the protein of interest was expressed in the supernatant,and a small part was in inclusion.The supernatant was purified by nickel affinity chromatography column.The results of Western Blot showed that the target protein content in the 500 mmol/L eluate was higher,and the addition of DTT was not significant in reducing the target protein multimer.The 500 mmol/L elution peak was collected,concentrated,and further purified by gel filtration prepacked column Column Superdex 75 Increase 10/300 GL.The AKTA explorer UV absorption chart showed a single absorption peak.According to the peak position of the UV absorption map,the protein was a multimer molecular weight multimer,which had been identified as HIV-1 Tat,and was decomposed into a plurality of monomers mainly under the action of DTT.The concentration was 0.47 mg/ml by BCA assay.(3)Effect of HIV-1 Tat protein on HUVECs activityWith the increase of Tat concentration,the activity of HUVECs decreased gradually.Compared with the negative control group,there was no significant difference in cell viability between 100ng/ml and 200ng/ml(P>0.05),but 300ng/ml,400ng/ml,500ng/ml and 1000ng/ml was statistically significant(P<0.05).There was no significant difference between the four groups(P>0.05).2.Effect of U87 supernatant with HIV-1 Tat on the content of ZO-1 of primary MCMECs(1)Construction of pEGFP-N1-tat eukaryotic expression vectorThe PCR amplification product was subjected to agarose gel electrophoresis,and a band corresponding to the size of the predicted tat first exon gene(216 bp)fragment appeared at 250 bp.A single colony was picked for PCR identification and confirmed to be the HIV-1 tat gene.The sequencing results of the company were correct and proved that the of four groups of HAD-SPL,HAD-BG,non-HAD-SPL,and non-HAD-BG pEGFP-N1-tat eukaryotic expression vectors were successfully constructed.(2)Expression of HIV-1 Tat in U87 cellsAfter transfection of U87 cells with pEGFP-N1-tat eukaryotic expression vectors of four groups of HAD-SPL,HAD-BG,non-HAD-SPL,and non-HAD-BG and pEGFP-N1 vector,green fluorescence appeared in U87 cells at 12h,and the fluorescence intensity increased with time.At 48h,the maximum value was reached.It was weakened at 72h.The blank control showed no green fluorescence.U87 cells transfected for 48h were detected by immunohistochemistry.Brown granules were observed clearly in each experimental group,and no brown granules were observed in the negative control and blank control.In summary,HIV-1 Tat protein can be expressed in U87 cells.(3)Extraction,culture and identification of primary MCMECsThe cells extracted and cultured from mouse brain tissue were identified by immunofluorescence.The results of CD31 and ? factors showed green fluorescence,and blue fluorescence was observed in stained nucleus by DAPI,indicating that the cultured cells were primary MCMECs.(4)Effect of U87 supernatant with HIV-1 Tat on the content of ZO-1 of primary MCMECsAfter incubation of the U87 supernatant expressing the Tat of HAD-SPL,HAD-BG,non-HAD-SPL,and non-HAD-BG,the content of tight junction protein ZO-1 in MCMECs showed different degrees of change.Compared with the negative control group,the content of ZO-1 in HAD-SPL and non-HAD-SPL group was decreased,and the content of ZO-1 in HAD-BG and non-HAD-BG group was increased,but the difference was not statistical significance(P>0.05).CONCLUSIONS1.The HIV-1 Tat with biological activity was efficiently expressed in BL21(DE3)and purified,and the activity of HUVECs was significantly decreased when the concentration reached 300ng/ml.2.The U87 supernatant expressing HAD-SPL,HAD-BG,non-HAD-SPL,non-HAD-BG Tat had no effect on the ZO-1 content of MCMECs.