ADC病人HIV-1gp120（111-383aa）对血脑屏障相关细胞的影响
发布时间：2018-07-15 20:43
【摘要】：目的人类免疫缺陷病毒Ⅰ型(HIV-1)可入侵中枢神经系统,引起艾滋病痴呆综合征(ADC)。HIV入侵中枢神经系统机制尚不清楚,目前有几种假说,单核/巨噬细胞入侵假说被认为是脑部感染HIV的主要方式。星形胶质细胞对血脑屏障正常结构和功能具有调节、维持作用,当HIV入侵中枢神经系统后,星形胶质细胞可被感染并成为其储存库,此时对血脑屏障通透性的影响在ADC发病中具有重要作用。星形胶质细胞合成的血小板衍生生长因子BB(PDGF-BB)、单核细胞趋化蛋白-1(MCP-1)可对内皮细胞的紧密连接产生影响,同时MCP-1募集外周被HIV-1感染的单核巨噬细胞穿过血脑屏障。HIV-1gp120蛋白茬病毒感染过程中具有重要作用,尤其是gp120的111-383位氨基酸片段,包括变异区V1、V2、V3,位于外部结构域,其中V3是结合受体的主要区域,决定病毒的细胞嗜性、合胞体诱导性等。这个区域氨基酸位点的变异对血脑屏障通透性的影响是否存在差异尚不清楚。在HIV-1突破血脑屏障入侵中枢神经系统过程中,HIV-1gp120蛋白除了直接对血脑屏障有毒性作用,还可以通过影响星形胶质细胞等其他细胞分泌多种细胞因子间接地影响血脑屏障的通透性。U87细胞可分泌MCP-1等多种细胞因子。基于gp 120蛋白的间接作用,为探讨ADC病人体内不同部位HIV-1 gp 120蛋白氨基酸位点的变异对胶质细胞合成PDGF-BB、MCP-1的影响是否存在差异,进而探讨gp120蛋白变异对血脑屏障的间接影响,本研究拟分析一例ADC病人中枢和外周不同部位的HIV-1 gp120(111-383aa)氨基酸位点的变异,并在U87细胞中表达,检测U87细胞合成PDGF-BB、MCP-1因子水平的变化,并收集表达不同部位来源的HIV-1 gp120蛋白的U87细胞上清(含有多种细胞因子),作用于HUVEC(人脐静脉内皮细胞)、原代小鼠脑微血管内皮细胞和周细胞,检测紧密连接蛋白、细胞活性等方面的变化,从而研究HIV-1gp120蛋白通过胶质细胞产生的细胞因子对血脑屏障通透性的间接影响,进而阐明HIV-1 gp120蛋白及其氨基酸位点的变异对血脑屏障通透性的影响,为进一步探讨HIV-1入侵中枢神经系统的机制提供科学依据。方法1.HIV-1gp120(333-1149nt)基因克隆及序列分析扩增1例ADC病人外周主动脉周淋巴结(PL)和CNS颞叶灰白质连接处(TGWJ)、脑室周围组织(PV)的 HIV-1 gp120 基因(333-1149nt),PCR 产物与PMD19-T载体进行TA连接,获得重组载体pMD19-T-gp120,将其转化至大肠杆菌DH5α,经蓝白斑筛选、氨苄青霉素筛选及测序,运用MEGA4软件进行系统进化树分析,并比较氨基酸序列。2.HIV-1gp120(333-1149nt)真核表达载体的构建及在U87细胞中的表达将上述pMD19-T-gp120重组质粒和表达载体pEGFP-N1双酶切,产物经琼脂糖电泳、回收后,T4连接酶连接,获得重组表达载体pEGFP-N1-gp120,将其转化至大肠杆菌DH5α,经卡那霉素筛选,测序正确,碱裂解法提取质粒。阳离子聚合物法转染至U87细胞,在24h、36h、48h、60h、72h荧光显微镜下观察蛋白表达情况,同时免疫组化法检测蛋白表达,Meta Morph软件进行定量分析。3.HIV-1 gp120对U87细胞合成PDGF-BB、MCP-1水平的影响U87细胞成功表达三个部位来源的HIV-1 gp120后,在24h、36h、48h、60h、72h收集细胞及上清。细胞冻融裂解、离心后,ELISA试剂盒检测上清中PDGF-BB水平。细胞上清离心后,ELISA试剂盒检测MCP-1水平。在48h时收集细胞,提取总RNA,逆转录为cDNA为模板,实时荧光定量PCR 检测 MCP-1mRNA 水平。4.血脑屏障相关细胞的体外培养(1)小鼠大脑微血管内皮细胞培养:取3-4周昆明鼠,脑组织制成匀浆,经木瓜蛋白酶消化后,用22%牛血清白蛋白(BSA)溶液密度梯度离心后,弃上清,用内皮培养基悬浮后接种于鼠尾胶原包被的培养瓶中,37℃、5%CO2静置培养,第二天换液并持续观察。传代后固定细胞,免疫荧光法鉴定特异性标记物CD31、Ⅷ 因子。(2)HUVEC传代细胞系:内皮培养基、10%胎牛血清常规培养。(3)小鼠大脑周细胞培养:按上述内皮细胞原代培养方法培养,从第三代开始用周细胞培养基培养,免疫荧光法鉴定特异性标记物NG2、PDGFRβ。5.表达gp120的U87细胞上清对内皮细胞紧密连接蛋白的影响表达gp120的U87细胞上清分别与HUVEC细胞、原代小鼠脑微血管内皮细胞共同孵育,24h后裂解细胞,离心取上清进行BCA蛋白定量,Western-blot检测紧密连接蛋白ZO-1、Occludin,用ImageJ软件定量。提取HUVEC细胞、原代小鼠脑微血管内皮细胞总RNA,逆转录为cDNA为模板,实时荧光定量PCR检测两种细胞的ZO-1、Occludin的mRNA水平。6.表达gp120的U87细胞上清对HUVEC细胞、小鼠脑微血管内废细胞、周细胞增殖活性的影响Almar blue检测方法。表达HIV-1 gp120的U87细胞上清分别与HUVEC细胞、原代小鼠脑微血管内皮细胞、周细胞孵育24h,滴加Almar blue避光培养2-8h后检测荧光强度,荧光强度与细胞活性成正比。7.统计学分析所有数据运用SPSS20软件进行方差分析,a =0.05。结果1.HIV-1 gp120基因克隆及序列分析成功构建了三个部位的pMD-19T-gp120克隆载体,序列分析结果显示来源于TGWJ、PV的gp120基因和分离自PL的gp120基因处在不同进化枝上,氨基酸序列比较显示,三个部位的gp120蛋白均为巨噬细胞嗜性、非合胞体诱导型,顶端四肽的氨基酸序列存在差异。2.HIV-1 gp120真核表达载体的构建及在U87细胞中的表达成功构建了三个部位的pEGFP-gp120真核表达载体,转染进U87细胞后,分别在24h、36h、48h、60h、72h观察到绿色荧光,同时免疫组化法检测可见棕色颗粒,表明三个部位来源的gp120蛋白在U87细胞中成功表达,表达量差异无统计学意义(P0.05)。3.HIV-1 gp120对U87细胞合成PDGF-BB、MCP-1水平的影响U87 细胞表达 HIV-1 gp120 后 24h、36h、48h、60h、72h,PDGF-BB 和 MCP-1检测结果显示,PDGF-BB浓度在五个时间点上各组之间差异无统计学意义(P0.05),MCP-1浓度在PL、TGWJ和PV组相比空载体对照组均有升高,且在24h、36h、48h三个时间点上,三组间两两比较,MCP-1浓度PLTGWJPV组,差异均具有统计学意义(P0.05),特别是在48h,PL组、TGWJ组的MCP-1浓度为各时间点中最高,此时PL组、TGWJ组、PV组的差异也较为明显。实时荧光PCR检测U87细胞MCP-1的mRNA水平结果显示,U87细胞表达HIV-1gp120蛋白48h时,PL组与TGWJ组、PV组MCP-1的mRNA水平差异具有显著性(P0.05),PL组显著高于TGWJ组、PV组。4.血脑屏障相关细胞的培养成功培养小鼠脑微血管内皮细胞、周细胞,免疫荧光法检测内皮细胞的异性标记物CD31、Ⅷ、周细胞特异性标记物NG2、PDGFRβ,荧光清晰可见。人脐静脉内皮细胞系HUVEC正常培养,生长状态良好。5.表达HIV-1 gp120的U87细胞上清对内皮细胞紧密连接蛋白的影响Western-blot检测结果显示,紧密连接蛋白ZO-1、Occludin均出现减少,减少程度PLTGWJPV,同时实时荧光定量PCR检测其mRNA水平,结果显示差异无统计学意义。6.表达HIV-1 gp120的U87细胞上清对HUVEC细胞、原代小鼠脑微血管内皮细胞、周细胞增殖活性的影响Almar blue检测结果显示,与空载体对照组比较原代内皮细胞PL组、TGWJ组荧光强度均有升高,差异均具有统计学意义(P0.05),PV组与之差异无统计学意义(P0.05)。HUVEC细胞与空载体对照组相比,PL组荧光强度升高,其差异具有统计学意义(P0.05),TGWJ组、PV组差异无统计学意义(P0.05)。原代周细胞活性差异无统计学意义(P0.05)。结论1.ADC病人TGWJ、PV来源的gp120基因和PL来源的gp120基因处在不同进化枝上,三个部位的gp120蛋白代表的HIV-1均为巨噬细胞嗜性、非合胞体诱导型,顶端四肽的氨基酸序列存在差异2.ADC病人PL、TGWJ、PV来源的gp120蛋白对U87细胞合成PDGF-BB无影响,能增强MCP-1的分泌,外周PL来源的gp120蛋白作用最强。3.表达ADC病人不同部位gp120蛋白的U87细胞上清使HUVEC细胞、原代小鼠脑微血管内皮细胞的紧密连接蛋白ZO-1、Occludin的含量减少,外周PL来源的gp120蛋白对应上清的影响最强,对血脑屏障的通透性影响最大。4.表达gp120蛋白的U87细胞上清能使HUVEC细胞、原代小鼠脑微血管内皮细胞的活性增强,外周PL来源的gp120蛋白对应上清的影响最强;对原代周细胞活性无影响。
[Abstract]:Objective human immunodeficiency virus type I (HIV-1) can invade the central nervous system and cause the mechanism of the ADC.HIV invasion of the central nervous system. There are several hypotheses that the mononuclear / macrophage invasion hypothesis is considered to be the main mode of brain infection of HIV. The normal structure of the astrocytes on the blood brain barrier and the normal structure of the blood brain barrier. Function has regulatory and maintenance functions. When HIV invade the central nervous system, astrocytes can be infected and become their repositories. At this time the influence of the blood brain barrier permeability plays an important role in the pathogenesis of ADC. Astrocytes synthesize the platelet derived growth factor BB (PDGF-BB) and the monocyte chemoattractant protein -1 (MCP-1). The close connection of the skin cells has an effect, while the MCP-1 infecting mononuclear macrophages infected by HIV-1 have an important role in the virus infection process through the.HIV-1gp120 protein stubble of the blood brain barrier, especially the 111-383 amino acid fragments of the gp120, including the V1, V2, and V3 in the variation region, in the external domain, in which V3 is the main region binding to the receptor. The domain, determining the cell tropism of the virus, the inducibility of the cytosome, and so on. The difference in the effect of the variation of the amino acid site on the permeability of the blood brain barrier is not clear. In the process of HIV-1 breaking through the blood brain barrier to invade the central nervous system, the HIV-1gp120 protein can be directly toxic to the blood brain barrier and can also be affected by the influence of the star. Other cells such as glial cells secrete a variety of cytokines that indirectly affect the permeability of the blood brain barrier (.U87) cells to secrete a variety of cytokines such as MCP-1. Based on the indirect effect of gp 120 protein, the variation of the amino acid loci of the HIV-1 gp 120 protein in different parts of the body of ADC patients affects the synthesis of PDGF-BB and MCP-1 in glial cells. There is no difference, and then to explore the indirect effect of gp120 protein variation on blood brain barrier. This study intends to analyze the variation of the HIV-1 gp120 (111-383aa) amino acid site in the different parts of the central and peripheral parts of the ADC patients, and to express in U87 cells, to detect the changes in the level of PDGF-BB, MCP-1 factor in U87 cells, and to collect and express different parts of the expression. The U87 cell supernatant of the source of HIV-1 gp120 protein (containing a variety of cytokines), acting on HUVEC (human umbilical vein endothelial cells), primary mouse cerebral microvascular endothelial cells and pericytes, detection of close connexin, cell activity and so on, so as to study the blood brain barrier through the cytokines produced by the HIV-1gp120 protein. The influence of the permeability of HIV-1 gp120 protein and its amino acid site on the permeability of blood brain barrier is clarified, and the scientific basis for further exploring the mechanism of HIV-1 invasion of the central nervous system is provided. Method 1.HIV-1gp120 (333-1149nt) gene cloning and sequence analysis of peripheral aorta lymph nodes (PL) of 1 cases of ADC patients were amplified. The HIV-1 gp120 gene (333-1149nt) of the temporal lobe gray and white matter (PV), the HIV-1 gp120 gene (333-1149nt) of the periventricular tissue (PV), the PCR product and the PMD19-T vector were connected to TA, and the recombinant vector pMD19-T-gp120 was obtained. It was converted to the DH5 alpha of Escherichia coli and screened by blue leukoplakia. Ampicillin was screened and sequenced. The phylogenetic tree was used to analyze the phylogenetic tree with MEGA4 software. The construction of a comparative amino acid sequence.2.HIV-1gp120 (333-1149nt) eukaryotic expression vector and the expression in U87 cells cut the pMD19-T-gp120 recombinant plasmid and expression vector pEGFP-N1. After the agarose electrophoresis, the recombinant expression plasmid pEGFP-N1-gp120 was obtained after the recovery of the T4 ligase, and transformed into the Escherichia coli DH5 alpha, and passed through the card. The protein expression was observed under the 24h, 36h, 48h, 60H, 72h fluorescence microscope, and the protein expression was detected by the immunohistochemical method, and the Meta Morph software was used for quantitative analysis of the synthesis PDGF-BB of U87 cells by the Meta Morph software. After a successful expression of HIV-1 gp120 from three sites, cells and supernatants were collected in 24h, 36h, 48h, 60H, 72h. Cells were frozen and thawing. After centrifugation, the PDGF-BB level in the supernatant was detected by ELISA kit. After the cell supernatant was centrifuged, the MCP-1 level was detected by the ELISA reagent box. PCR detection of MCP-1mRNA level.4. blood brain barrier related cells in vitro culture (1) mouse brain microvascular endothelial cells culture: 3-4 weeks from Kunming mice, brain tissue to homogenate, after papain digestion, with 22% bovine serum albumin (BSA) density gradient centrifugation, abandoned supernatant, endothelial culture medium after suspension inoculation of rat tail collagen In the incubated bottle, 37 C, 5%CO2 culture, second days for liquid and continuous observation. After passages, fixed cells were fixed, specific marker CD31, factor VIII. (2) HUVEC passage cell line: endothelial culture medium, 10% fetal bovine serum routine culture. (3) culture of mouse brain pericytes: cultured by the above endothelial cell culture method, From the third generation to the third generation, the specific marker was identified by immunofluorescence. The effect of PDGFR beta.5. on the close connexin of the endothelial cells expressed in the U87 cell of gp120, the U87 cell supernatant of the gp120 was incubated with the original mouse cerebral microvascular endothelial cells, and the lysate cells were centrifuged after 24h. The supernatant was quantified by BCA protein, and Western-blot was used to detect the close connexin ZO-1, Occludin. The HUVEC cells were extracted by ImageJ software. The total RNA of the mouse brain microvascular endothelial cells was extracted. The reverse transcriptase was the template for cDNA, and the real-time fluorescent quantitative PCR was used to detect ZO-1 of two cells. Cell, mouse brain microvascular waste cells and pericytes proliferation activity influence Almar blue detection method. The U87 cell supernatant of HIV-1 gp120 expressed respectively with HUVEC cells, primary mouse brain microvascular endothelial cells, pericytes incubated 24h, and Almar blue avoiding light culture 2-8h to detect fluorescein intensity, fluorescence intensity and cell activity are proportional to.7. statistics The analysis of all data using SPSS20 software for variance analysis, a =0.05. results 1.HIV-1 gp120 gene cloning and sequence analysis successfully constructed the pMD-19T-gp120 cloning vector of three sites. Sequence analysis results show that the gp120 gene of the PV and the gp120 basis of the separation from PL are in different evolutionary branches, the amino acid sequence is more significant. The gp120 protein of three sites were macrophage tropism, non syncytial inducible, and the amino acid sequence of the apical four peptide had different.2.HIV-1 gp120 eukaryotic expression vector, and the expression of the pEGFP-gp120 eukaryotic expression vector in the U87 cells was successfully constructed. After transfection into U87 cells, 24h, 36h, 48h, 60H, 72h, respectively. The green fluorescence was observed and the brown particles were detected by immunohistochemical method. It showed that the expression of gp120 protein in three sites was successfully expressed in U87 cells, and the difference in expression was not statistically significant (P0.05).3.HIV-1 gp120 was used to synthesize PDGF-BB in U87 cells, and MCP-1 level influenced U87 cells to express HIV-1 gp120 24h. The results of MCP-1 detection showed that there was no significant difference in the concentration of PDGF-BB at five time points, and the concentration of MCP-1 was in PL, TGWJ and PV groups were higher than those in the empty carrier control group, and at the three time points of 24h, 36h and 48h, the difference between the three groups was 22, and the difference was statistically significant. The MCP-1 concentration in 48h, PL and TGWJ groups was the highest in each time point. At this time, the difference between the PL group, the TGWJ group and the PV group was also obvious. The culture of.4. blood brain barrier related cells in group V successfully cultured mouse brain microvascular endothelial cells, pericytes and immunofluorescence method to detect CD31, VIII, NG2, PDGFR beta of pericytes specific markers, PDGFR beta, and the normal culture of HUVEC in human umbilical vein endothelial cell line, and the growth state of.5. expressed HIV-1 gp120 U8. The effect of 7 cell supernatant on the close connexin of endothelial cells Western-blot detection results showed that the close connexin ZO-1, Occludin decreased, the degree of PLTGWJPV was reduced, and the real-time fluorescence quantitative PCR was used to detect the mRNA level. The results showed that there was no statistically significant difference in the.6. table with HIV-1 gp120 U87 cell supernatant to HUVEC cells, the primary cells were small The effect of Almar blue on the proliferation activity of rat cerebral microvascular endothelial cells and pericytes showed that compared with the empty carrier control group, the fluorescence intensity of TGWJ group was higher than that of the PL group of the primary endothelial cells, and the difference was statistically significant (P0.05). There was no statistically significant difference between the PV group and the PV group (P0.05), compared with the empty carrier control group, the PL group fluoro The difference of light intensity was statistically significant (P0.05), in group TGWJ, there was no significant difference in group PV (P0.05). There was no significant difference in the activity of primary pericytes (P0.05). Conclusion 1.ADC patient TGWJ, PV source gp120 gene and PL source gp120 gene are on different evolutionary branches, and the three sites of gp120 protein represent macrophages. Cell tropism and non syncytial induction, the amino acid sequence of the apical four peptide has differences in 2.ADC patients PL, TGWJ, and PV derived gp120 protein that does not affect the synthesis of PDGF-BB in U87 cells. It can enhance the secretion of MCP-1. The strongest.3. expression of gp120 protein in the peripheral PL source is the primary expression of the cell supernatant of the different parts of the ADC patients. The close connexin ZO-1, Occludin content of the mouse brain microvascular endothelial cells decreased, and the gp120 protein derived from the peripheral PL had the strongest influence on the supernatant. The U87 cell supernatant with the largest.4. expression of the gp120 protein, which had the largest.4. expression, could enhance the activity of the primary mouse brain microvascular endothelial cells and the peripheral PL source. The gp120 protein had the strongest effect on the supernatant, and had no effect on the viability of primary pericytes.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91;R747.9
[Abstract]:Objective human immunodeficiency virus type I (HIV-1) can invade the central nervous system and cause the mechanism of the ADC.HIV invasion of the central nervous system. There are several hypotheses that the mononuclear / macrophage invasion hypothesis is considered to be the main mode of brain infection of HIV. The normal structure of the astrocytes on the blood brain barrier and the normal structure of the blood brain barrier. Function has regulatory and maintenance functions. When HIV invade the central nervous system, astrocytes can be infected and become their repositories. At this time the influence of the blood brain barrier permeability plays an important role in the pathogenesis of ADC. Astrocytes synthesize the platelet derived growth factor BB (PDGF-BB) and the monocyte chemoattractant protein -1 (MCP-1). The close connection of the skin cells has an effect, while the MCP-1 infecting mononuclear macrophages infected by HIV-1 have an important role in the virus infection process through the.HIV-1gp120 protein stubble of the blood brain barrier, especially the 111-383 amino acid fragments of the gp120, including the V1, V2, and V3 in the variation region, in the external domain, in which V3 is the main region binding to the receptor. The domain, determining the cell tropism of the virus, the inducibility of the cytosome, and so on. The difference in the effect of the variation of the amino acid site on the permeability of the blood brain barrier is not clear. In the process of HIV-1 breaking through the blood brain barrier to invade the central nervous system, the HIV-1gp120 protein can be directly toxic to the blood brain barrier and can also be affected by the influence of the star. Other cells such as glial cells secrete a variety of cytokines that indirectly affect the permeability of the blood brain barrier (.U87) cells to secrete a variety of cytokines such as MCP-1. Based on the indirect effect of gp 120 protein, the variation of the amino acid loci of the HIV-1 gp 120 protein in different parts of the body of ADC patients affects the synthesis of PDGF-BB and MCP-1 in glial cells. There is no difference, and then to explore the indirect effect of gp120 protein variation on blood brain barrier. This study intends to analyze the variation of the HIV-1 gp120 (111-383aa) amino acid site in the different parts of the central and peripheral parts of the ADC patients, and to express in U87 cells, to detect the changes in the level of PDGF-BB, MCP-1 factor in U87 cells, and to collect and express different parts of the expression. The U87 cell supernatant of the source of HIV-1 gp120 protein (containing a variety of cytokines), acting on HUVEC (human umbilical vein endothelial cells), primary mouse cerebral microvascular endothelial cells and pericytes, detection of close connexin, cell activity and so on, so as to study the blood brain barrier through the cytokines produced by the HIV-1gp120 protein. The influence of the permeability of HIV-1 gp120 protein and its amino acid site on the permeability of blood brain barrier is clarified, and the scientific basis for further exploring the mechanism of HIV-1 invasion of the central nervous system is provided. Method 1.HIV-1gp120 (333-1149nt) gene cloning and sequence analysis of peripheral aorta lymph nodes (PL) of 1 cases of ADC patients were amplified. The HIV-1 gp120 gene (333-1149nt) of the temporal lobe gray and white matter (PV), the HIV-1 gp120 gene (333-1149nt) of the periventricular tissue (PV), the PCR product and the PMD19-T vector were connected to TA, and the recombinant vector pMD19-T-gp120 was obtained. It was converted to the DH5 alpha of Escherichia coli and screened by blue leukoplakia. Ampicillin was screened and sequenced. The phylogenetic tree was used to analyze the phylogenetic tree with MEGA4 software. The construction of a comparative amino acid sequence.2.HIV-1gp120 (333-1149nt) eukaryotic expression vector and the expression in U87 cells cut the pMD19-T-gp120 recombinant plasmid and expression vector pEGFP-N1. After the agarose electrophoresis, the recombinant expression plasmid pEGFP-N1-gp120 was obtained after the recovery of the T4 ligase, and transformed into the Escherichia coli DH5 alpha, and passed through the card. The protein expression was observed under the 24h, 36h, 48h, 60H, 72h fluorescence microscope, and the protein expression was detected by the immunohistochemical method, and the Meta Morph software was used for quantitative analysis of the synthesis PDGF-BB of U87 cells by the Meta Morph software. After a successful expression of HIV-1 gp120 from three sites, cells and supernatants were collected in 24h, 36h, 48h, 60H, 72h. Cells were frozen and thawing. After centrifugation, the PDGF-BB level in the supernatant was detected by ELISA kit. After the cell supernatant was centrifuged, the MCP-1 level was detected by the ELISA reagent box. PCR detection of MCP-1mRNA level.4. blood brain barrier related cells in vitro culture (1) mouse brain microvascular endothelial cells culture: 3-4 weeks from Kunming mice, brain tissue to homogenate, after papain digestion, with 22% bovine serum albumin (BSA) density gradient centrifugation, abandoned supernatant, endothelial culture medium after suspension inoculation of rat tail collagen In the incubated bottle, 37 C, 5%CO2 culture, second days for liquid and continuous observation. After passages, fixed cells were fixed, specific marker CD31, factor VIII. (2) HUVEC passage cell line: endothelial culture medium, 10% fetal bovine serum routine culture. (3) culture of mouse brain pericytes: cultured by the above endothelial cell culture method, From the third generation to the third generation, the specific marker was identified by immunofluorescence. The effect of PDGFR beta.5. on the close connexin of the endothelial cells expressed in the U87 cell of gp120, the U87 cell supernatant of the gp120 was incubated with the original mouse cerebral microvascular endothelial cells, and the lysate cells were centrifuged after 24h. The supernatant was quantified by BCA protein, and Western-blot was used to detect the close connexin ZO-1, Occludin. The HUVEC cells were extracted by ImageJ software. The total RNA of the mouse brain microvascular endothelial cells was extracted. The reverse transcriptase was the template for cDNA, and the real-time fluorescent quantitative PCR was used to detect ZO-1 of two cells. Cell, mouse brain microvascular waste cells and pericytes proliferation activity influence Almar blue detection method. The U87 cell supernatant of HIV-1 gp120 expressed respectively with HUVEC cells, primary mouse brain microvascular endothelial cells, pericytes incubated 24h, and Almar blue avoiding light culture 2-8h to detect fluorescein intensity, fluorescence intensity and cell activity are proportional to.7. statistics The analysis of all data using SPSS20 software for variance analysis, a =0.05. results 1.HIV-1 gp120 gene cloning and sequence analysis successfully constructed the pMD-19T-gp120 cloning vector of three sites. Sequence analysis results show that the gp120 gene of the PV and the gp120 basis of the separation from PL are in different evolutionary branches, the amino acid sequence is more significant. The gp120 protein of three sites were macrophage tropism, non syncytial inducible, and the amino acid sequence of the apical four peptide had different.2.HIV-1 gp120 eukaryotic expression vector, and the expression of the pEGFP-gp120 eukaryotic expression vector in the U87 cells was successfully constructed. After transfection into U87 cells, 24h, 36h, 48h, 60H, 72h, respectively. The green fluorescence was observed and the brown particles were detected by immunohistochemical method. It showed that the expression of gp120 protein in three sites was successfully expressed in U87 cells, and the difference in expression was not statistically significant (P0.05).3.HIV-1 gp120 was used to synthesize PDGF-BB in U87 cells, and MCP-1 level influenced U87 cells to express HIV-1 gp120 24h. The results of MCP-1 detection showed that there was no significant difference in the concentration of PDGF-BB at five time points, and the concentration of MCP-1 was in PL, TGWJ and PV groups were higher than those in the empty carrier control group, and at the three time points of 24h, 36h and 48h, the difference between the three groups was 22, and the difference was statistically significant. The MCP-1 concentration in 48h, PL and TGWJ groups was the highest in each time point. At this time, the difference between the PL group, the TGWJ group and the PV group was also obvious. The culture of.4. blood brain barrier related cells in group V successfully cultured mouse brain microvascular endothelial cells, pericytes and immunofluorescence method to detect CD31, VIII, NG2, PDGFR beta of pericytes specific markers, PDGFR beta, and the normal culture of HUVEC in human umbilical vein endothelial cell line, and the growth state of.5. expressed HIV-1 gp120 U8. The effect of 7 cell supernatant on the close connexin of endothelial cells Western-blot detection results showed that the close connexin ZO-1, Occludin decreased, the degree of PLTGWJPV was reduced, and the real-time fluorescence quantitative PCR was used to detect the mRNA level. The results showed that there was no statistically significant difference in the.6. table with HIV-1 gp120 U87 cell supernatant to HUVEC cells, the primary cells were small The effect of Almar blue on the proliferation activity of rat cerebral microvascular endothelial cells and pericytes showed that compared with the empty carrier control group, the fluorescence intensity of TGWJ group was higher than that of the PL group of the primary endothelial cells, and the difference was statistically significant (P0.05). There was no statistically significant difference between the PV group and the PV group (P0.05), compared with the empty carrier control group, the PL group fluoro The difference of light intensity was statistically significant (P0.05), in group TGWJ, there was no significant difference in group PV (P0.05). There was no significant difference in the activity of primary pericytes (P0.05). Conclusion 1.ADC patient TGWJ, PV source gp120 gene and PL source gp120 gene are on different evolutionary branches, and the three sites of gp120 protein represent macrophages. Cell tropism and non syncytial induction, the amino acid sequence of the apical four peptide has differences in 2.ADC patients PL, TGWJ, and PV derived gp120 protein that does not affect the synthesis of PDGF-BB in U87 cells. It can enhance the secretion of MCP-1. The strongest.3. expression of gp120 protein in the peripheral PL source is the primary expression of the cell supernatant of the different parts of the ADC patients. The close connexin ZO-1, Occludin content of the mouse brain microvascular endothelial cells decreased, and the gp120 protein derived from the peripheral PL had the strongest influence on the supernatant. The U87 cell supernatant with the largest.4. expression of the gp120 protein, which had the largest.4. expression, could enhance the activity of the primary mouse brain microvascular endothelial cells and the peripheral PL source. The gp120 protein had the strongest effect on the supernatant, and had no effect on the viability of primary pericytes.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91;R747.9
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