| OBJECTIVEHuman immunodeficiency virus type I(HIV-1)can invade the central nervous system,causing the most serious damage to the nervous system is AIDS dementia syndrome(ADC).The mechanism of HIV invasion into the central nervous system remains unclear.At present,there are several hypotheses,among which the monocyte/macrophage invasion hypothesis is considered as the main mode of HIV infection in the brain.Astrocytes,which regulate and maintain normal structure and function of the blood-brain barrier,can be infected and become the viral reservior when HIV invades the central nervous system.In the pathogenesis of ADC,the permeability of blood-brain barrier plays an impoartant role.Astrocytes affect the blood-brain barrier mainly through the synthesis of several kinds of cytokines,such as platelet-derived growth factor BB(PDGF-BB),monocyte chemoattractant protein-1(MCP-1),which can impact the tight junction on endothelial cells,and MCP-1 can help HIV-1 infected mononuclear/macrophage pass through blood brain barrier.HIV-1gp120 protein plays an important role in the process of virus infection,especially the 111-383 amino acid fragment,including V1,V2,V3 mutation region,which are located in the external domain,with V3 being the main region of binding receptor,determining virus cell tropism,syncytium inducing etc..In the process of HIV-1 passing through the blood-brain barrier and invading the central nervous system HIV-1gp120 protein can not only produce toxic effect directly on the blood-brain barrier,but also and more importantly affect the permeability of the blood-brain barrier indirectly through other cells such as astrocytes secreting a variety of cytokines.U87 cells secrete many cytokines including MCP-1.Based on the indirect effect,to explore the different effects from the variation among the HIV-1 gp120(111?383aa)derived from different tissues of an ADC patient on the syntheses of PDGF-BB and MCP-1 in U87 cells,this study proposed to express the gp120(111?383aa)derived from different tissues of an ADC patient in U87 cells,detect the level of PDGF-BB and MCP-1,collect the U87 cells supernatnat expressing HIV-1 gp120 protein(containing various cytokines)then incubate with HUVEC,primary mouse brain microvascular endothelial cells and pericytes,tight junction protein and cell activity were detected.In order to study the indirect effects of HIV-1 gp120 protein on the permeability of blood-brain barrier through cytokines produced by glial cells,and explore the mechanism of HIV-1 penetration of blood-brain barrier and invade the central nervous system,to provide a scientific basis for the further investigation of the pathogenesis of AIDS dementia syndrome.METHODS1.Cloning and sequence analysis of HIV-1 gp120(333-1149nt)genesHIV-1 gp120 gene(333-1149nt)of periaortic lymphoide(PL),CNS temporal gray/white matter junction(TGWJ)and periventricular tissue(PV)were amplificated from one ADC patient.PCR products were connected with pMD19-T vector,and the recombinant vector pMD19-T-gp120 were obtained and transformed into Escherichia coli DH5a,go through Blue white screening,ampicillin screening and sequencing,phylogenetic tree analysis using MEGA4 software,and the comparison of amino acid sequences.2.Construction of HIV-1 gp120(333-1149nt)eukaryotic expression vector and its expression in U87 cellsPlasmid pMD19-T-gp120 and expression vector pEGFP-Nl digestion products were identified by agarose gel electrophoresis,purified and T4 connected to obtained the recombinant expression vector pEGFP-N1-gp120 to transform into Escherichia coli DH5?,which is then selected by kanamycin and sequenced correctly.The recombinant plasmid DNA was extracted by alkaline lysis method,transfected with cationic polymer into U87 cells,and the expression of protein was observed by fluorescence microscopy at 24h,36h,48h,60h and 72h.The expression of protein was detected by immunohistochemistry,and the quantitative analysis was performed by Meta Morph software.3.Effects of HIV-1 gpl20 on the synthesis of PDGF-BB and MCP-1 in U87 cellsAfter HIV-1 gp120 derived from three different tissues were successfully expressed in U87 cells,the cell and supernatant were collected at 24h,36h,48h,60h,72h.After cells were freeze-thaw cycle lysed and centrifuged,the PDGF-BB levels in supernatant were determined by ELISA kit.MCP-1 was detected in U87 supernatants by ELISA kit after centrifugation.Cells were collected at 48h then total RNA was extracted,reverse transcripted into cDNA,and MCP-1 mRNA was detected by real-time fluorescent quantitative PCR.4.Culture of blood brain barrier cells in vitro(1)The culture of mouse brain microvascular endothelial cell:brain tissue extracted from 3 to 4 weeks old Kunming mice was homogenized after papain digestion,and then purified via 22%bovine serum albumin(BSA)solution density gradient centrifugation.Cells were resuspended in ECM and cultured in rat tail collagen coated culture flasks,37 C,5%CO2 static culture,the second day change the ECM.Cells were identified by immunofluorescence with specific markers CD31 and VIII.(2)HUVEC cell line:conventional culture.(3)The culture of mouse brain pericytes:according to the primary culture of endothelial cells,the cells were cultured in PM from the third generation,and the specific markers NG2 and PDGFR were identified by immunofluorescence.5.The impact of supernatant of U87 cell expressing gp120 on endothelial cell tight junctionHUVEC cells,primary mouse brain microvascular endothelial cells were incubated respectively with the supernatant of U87 cell expressing gp120 for 24h,then cells are lyzed and centrifuged.The supernatants were quantified by BCA protein quantification method.Tight junction proteins ZO-1,Occludin are detected by Western-Blot,quantified by ImageJ software.Incubated HUVEC cells and primary mouse brain microvascular endothelial cells were obtained and reverse transcriptase as cDNA.The ZO-1 and Occludin mRNA levels in were detected by real-time fluorescence quantitative PCR.6.The impact of supernatant of U87 cell expressing gp120 on the proliferation of HUVEC cells,mouse brain microvascular endothelial cells and pericytesThe supernatant of U87 cells expressing HIV-1 gp120 incubated with HUVEC cells,primary mouse brain microvascular endothelial cells and pericytes respectively,the fluorescence intensity was detected after incubation with 2?8h.7.statistical analysisAll the data were analyzed by SPSS20 software,? =0.05.RESULTS1.Cloning and sequence analysis of HIV-1 gp120(333-1149nt)geneThe three parts of pMD-19T-gp120 cloning vectors were successfully constructed and sequenced.Analysis showed that gp120 genes from TGWJ,PV and the gp120from PL gene were in different clades.The comparison of the amino acid sequence showed that the three parts of the gp120 protein was macrophage tropism,non syncytium inducing type,amino acid sequence of the top four peptide were different.2.Construction of HIV-1 gp120(333-1149nt)eukaryotic expression vector and its expression in U87 cellsThe pEGFP-gp120 eukaryotic expression vector were contructed and transfected into U87 cells,respectively.The green fluorescence was observed in 24h,36h,48h,60h,72h after transfection,and brown particles were visible via immunohistochemical method,which indicated the successful expression of three parts of the source of gp120 protein in U87 cells,the expression of difference had no statistical significance(P>0.05).3.Effects of HIV-1 gp120 on the synthesis of PDGF-BB and MCP-1 in U87 cellsAfter the expression of HIV-1 gp120 in U87 cells for 24h,36h,48h,60h,72h,the concentration of PDGF-BB showed no significant difference in the five time group(P>0.05),and concentration of MCP-1 in PL,TGWJ and PV group compared with the empty vector control group were higher,and the concentration of MCP-1 compared among the three groups at 24h,36h,48h,PL>TGWJ>PV,the differences were statistically significant(P<0.05),especially in the 48h group,MCP-1 concentration of PL was the highest in each time point,the difference between the PL group,TGWJ groupand PV group is more obvious.Real time fluorescent PCR detection of U87 cell MCP-1 mRNA levels showed that U87 cells expressed HIV-1gp 120 protein 48h,mRNA difference in PL group and TGWJ group,PV group MCP-1 was significant(P<0.05),PL group was significantly higher than that of TGWJ group and PV group.4.Culture of blood brain barrier cells in vitroThe mouse brain microvascular endothelial cells and pericytes were cultured successfully.The specific markers of endothelial cells,CD31,VIII and pericyte NG2 and PDGFR ? were detected by immunofluorescence.Human umbilical vein endothelial cell line HUVEC was cultured normally and grew well.5.The impact of supernatant of U87 cell expressing gp120 on endothelial cell tight junctionThe results of Western-blot showed that the expression of tight junction protein ZO-1 and occludin were reduced,and the degree of reduction was PL>TGWJ>PV,and the level of mRNA was detected by real-time fluorescent quantitative PCR.The results showed that there was no significant difference.6.The impact of supernatant of U87 cell expressing gp120 on the proliferation of HUVEC cells,mouse brain microvascular endothelial cells and pericytesAlmar blue showed that the fluorescence intensity of primary endothelial cells in PL group,TGWJ group increased,the difference was statistically significant(P<0.05),there was no significant difference between PV group and the other groups(P>0.05).Compared with pEGFP group,the fluorescence intensity of PL group was higher,the difference was statistically significant(P<0.05),there was no significant difference between TGWJ group and PV group(P>0.05).There was no significant difference in primary pericytes(P>0.05).CONCLUSION1.HIV-1gp120 genes isolated from TGWJ,PV and gp120 genes isolated from PL in one ADC patient are in different clades,HIV-1 represented by the gp120 protein are of macrophage tropism,non syncytium inducing type,the amino acid sequence of the top four peptides are different.2.HIV-1 gp120 protein derived from PL,TGWJ,PV in one ADC protein had no effect on synthesis of PDGF-BB in U87 cells,which could enhance the secretion of MCP-1,and the effect of gp120 protein from PL was the strongest.3.Supernatant of U87 cell expressing gp120 from different tissues decrease the tight junction protein ZO-1 and Occludin of HUVEC cells,primary mouse brain microvascular endothelial cell,the effect of the supernatant corresponding with gp120 proteins of PL was strongest.4.Supernatant of U87 cell expressing gp120 can enhance the activity of HUVEC cells and primary mouse brain microvascular endothelial cells,the effect of the supernatant corresponding with gp120 proteins of PL was strongest,and there was no effect on the primary cells. |
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