發(fā)布時(shí)間：2018-07-15 20:43

【摘要】：目的人類免疫缺陷病毒Ⅰ型(HIV-1)可入侵中樞神經(jīng)系統(tǒng),引起艾滋病癡呆綜合征(ADC)。HIV入侵中樞神經(jīng)系統(tǒng)機(jī)制尚不清楚,目前有幾種假說,單核/巨噬細(xì)胞入侵假說被認(rèn)為是腦部感染HIV的主要方式。星形膠質(zhì)細(xì)胞對(duì)血腦屏障正常結(jié)構(gòu)和功能具有調(diào)節(jié)、維持作用,當(dāng)HIV入侵中樞神經(jīng)系統(tǒng)后,星形膠質(zhì)細(xì)胞可被感染并成為其儲(chǔ)存庫,此時(shí)對(duì)血腦屏障通透性的影響在ADC發(fā)病中具有重要作用。星形膠質(zhì)細(xì)胞合成的血小板衍生生長(zhǎng)因子BB(PDGF-BB)、單核細(xì)胞趨化蛋白-1(MCP-1)可對(duì)內(nèi)皮細(xì)胞的緊密連接產(chǎn)生影響,同時(shí)MCP-1募集外周被HIV-1感染的單核巨噬細(xì)胞穿過血腦屏障。HIV-1gp120蛋白茬病毒感染過程中具有重要作用,尤其是gp120的111-383位氨基酸片段,包括變異區(qū)V1、V2、V3,位于外部結(jié)構(gòu)域,其中V3是結(jié)合受體的主要區(qū)域,決定病毒的細(xì)胞嗜性、合胞體誘導(dǎo)性等。這個(gè)區(qū)域氨基酸位點(diǎn)的變異對(duì)血腦屏障通透性的影響是否存在差異尚不清楚。在HIV-1突破血腦屏障入侵中樞神經(jīng)系統(tǒng)過程中,HIV-1gp120蛋白除了直接對(duì)血腦屏障有毒性作用,還可以通過影響星形膠質(zhì)細(xì)胞等其他細(xì)胞分泌多種細(xì)胞因子間接地影響血腦屏障的通透性。U87細(xì)胞可分泌MCP-1等多種細(xì)胞因子�；趃p 120蛋白的間接作用,為探討ADC病人體內(nèi)不同部位HIV-1 gp 120蛋白氨基酸位點(diǎn)的變異對(duì)膠質(zhì)細(xì)胞合成PDGF-BB、MCP-1的影響是否存在差異,進(jìn)而探討gp120蛋白變異對(duì)血腦屏障的間接影響,本研究擬分析一例ADC病人中樞和外周不同部位的HIV-1 gp120(111-383aa)氨基酸位點(diǎn)的變異,并在U87細(xì)胞中表達(dá),檢測(cè)U87細(xì)胞合成PDGF-BB、MCP-1因子水平的變化,并收集表達(dá)不同部位來源的HIV-1 gp120蛋白的U87細(xì)胞上清(含有多種細(xì)胞因子),作用于HUVEC(人臍靜脈內(nèi)皮細(xì)胞)、原代小鼠腦微血管內(nèi)皮細(xì)胞和周細(xì)胞,檢測(cè)緊密連接蛋白、細(xì)胞活性等方面的變化,從而研究HIV-1gp120蛋白通過膠質(zhì)細(xì)胞產(chǎn)生的細(xì)胞因子對(duì)血腦屏障通透性的間接影響,進(jìn)而闡明HIV-1 gp120蛋白及其氨基酸位點(diǎn)的變異對(duì)血腦屏障通透性的影響,為進(jìn)一步探討HIV-1入侵中樞神經(jīng)系統(tǒng)的機(jī)制提供科學(xué)依據(jù)。方法1.HIV-1gp120(333-1149nt)基因克隆及序列分析擴(kuò)增1例ADC病人外周主動(dòng)脈周淋巴結(jié)(PL)和CNS顳葉灰白質(zhì)連接處(TGWJ)、腦室周圍組織(PV)的 HIV-1 gp120 基因(333-1149nt),PCR 產(chǎn)物與PMD19-T載體進(jìn)行TA連接,獲得重組載體pMD19-T-gp120,將其轉(zhuǎn)化至大腸桿菌DH5α,經(jīng)藍(lán)白斑篩選、氨芐青霉素篩選及測(cè)序,運(yùn)用MEGA4軟件進(jìn)行系統(tǒng)進(jìn)化樹分析,并比較氨基酸序列。2.HIV-1gp120(333-1149nt)真核表達(dá)載體的構(gòu)建及在U87細(xì)胞中的表達(dá)將上述pMD19-T-gp120重組質(zhì)粒和表達(dá)載體pEGFP-N1雙酶切,產(chǎn)物經(jīng)瓊脂糖電泳、回收后,T4連接酶連接,獲得重組表達(dá)載體pEGFP-N1-gp120,將其轉(zhuǎn)化至大腸桿菌DH5α,經(jīng)卡那霉素篩選,測(cè)序正確,堿裂解法提取質(zhì)粒。陽離子聚合物法轉(zhuǎn)染至U87細(xì)胞,在24h、36h、48h、60h、72h熒光顯微鏡下觀察蛋白表達(dá)情況,同時(shí)免疫組化法檢測(cè)蛋白表達(dá),Meta Morph軟件進(jìn)行定量分析。3.HIV-1 gp120對(duì)U87細(xì)胞合成PDGF-BB、MCP-1水平的影響U87細(xì)胞成功表達(dá)三個(gè)部位來源的HIV-1 gp120后,在24h、36h、48h、60h、72h收集細(xì)胞及上清。細(xì)胞凍融裂解、離心后,ELISA試劑盒檢測(cè)上清中PDGF-BB水平。細(xì)胞上清離心后,ELISA試劑盒檢測(cè)MCP-1水平。在48h時(shí)收集細(xì)胞,提取總RNA,逆轉(zhuǎn)錄為cDNA為模板,實(shí)時(shí)熒光定量PCR 檢測(cè) MCP-1mRNA 水平。4.血腦屏障相關(guān)細(xì)胞的體外培養(yǎng)(1)小鼠大腦微血管內(nèi)皮細(xì)胞培養(yǎng):取3-4周昆明鼠,腦組織制成勻漿,經(jīng)木瓜蛋白酶消化后,用22%牛血清白蛋白(BSA)溶液密度梯度離心后,棄上清,用內(nèi)皮培養(yǎng)基懸浮后接種于鼠尾膠原包被的培養(yǎng)瓶中,37℃、5%CO2靜置培養(yǎng),第二天換液并持續(xù)觀察。傳代后固定細(xì)胞,免疫熒光法鑒定特異性標(biāo)記物CD31、Ⅷ 因子。(2)HUVEC傳代細(xì)胞系:內(nèi)皮培養(yǎng)基、10%胎牛血清常規(guī)培養(yǎng)。(3)小鼠大腦周細(xì)胞培養(yǎng):按上述內(nèi)皮細(xì)胞原代培養(yǎng)方法培養(yǎng),從第三代開始用周細(xì)胞培養(yǎng)基培養(yǎng),免疫熒光法鑒定特異性標(biāo)記物NG2、PDGFRβ。5.表達(dá)gp120的U87細(xì)胞上清對(duì)內(nèi)皮細(xì)胞緊密連接蛋白的影響表達(dá)gp120的U87細(xì)胞上清分別與HUVEC細(xì)胞、原代小鼠腦微血管內(nèi)皮細(xì)胞共同孵育,24h后裂解細(xì)胞,離心取上清進(jìn)行BCA蛋白定量,Western-blot檢測(cè)緊密連接蛋白ZO-1、Occludin,用ImageJ軟件定量。提取HUVEC細(xì)胞、原代小鼠腦微血管內(nèi)皮細(xì)胞總RNA,逆轉(zhuǎn)錄為cDNA為模板,實(shí)時(shí)熒光定量PCR檢測(cè)兩種細(xì)胞的ZO-1、Occludin的mRNA水平。6.表達(dá)gp120的U87細(xì)胞上清對(duì)HUVEC細(xì)胞、小鼠腦微血管內(nèi)廢細(xì)胞、周細(xì)胞增殖活性的影響Almar blue檢測(cè)方法。表達(dá)HIV-1 gp120的U87細(xì)胞上清分別與HUVEC細(xì)胞、原代小鼠腦微血管內(nèi)皮細(xì)胞、周細(xì)胞孵育24h,滴加Almar blue避光培養(yǎng)2-8h后檢測(cè)熒光強(qiáng)度,熒光強(qiáng)度與細(xì)胞活性成正比。7.統(tǒng)計(jì)學(xué)分析所有數(shù)據(jù)運(yùn)用SPSS20軟件進(jìn)行方差分析,a =0.05。結(jié)果1.HIV-1 gp120基因克隆及序列分析成功構(gòu)建了三個(gè)部位的pMD-19T-gp120克隆載體,序列分析結(jié)果顯示來源于TGWJ、PV的gp120基因和分離自PL的gp120基因處在不同進(jìn)化枝上,氨基酸序列比較顯示,三個(gè)部位的gp120蛋白均為巨噬細(xì)胞嗜性、非合胞體誘導(dǎo)型,頂端四肽的氨基酸序列存在差異。2.HIV-1 gp120真核表達(dá)載體的構(gòu)建及在U87細(xì)胞中的表達(dá)成功構(gòu)建了三個(gè)部位的pEGFP-gp120真核表達(dá)載體,轉(zhuǎn)染進(jìn)U87細(xì)胞后,分別在24h、36h、48h、60h、72h觀察到綠色熒光,同時(shí)免疫組化法檢測(cè)可見棕色顆粒,表明三個(gè)部位來源的gp120蛋白在U87細(xì)胞中成功表達(dá),表達(dá)量差異無統(tǒng)計(jì)學(xué)意義(P0.05)。3.HIV-1 gp120對(duì)U87細(xì)胞合成PDGF-BB、MCP-1水平的影響U87 細(xì)胞表達(dá) HIV-1 gp120 后 24h、36h、48h、60h、72h,PDGF-BB 和 MCP-1檢測(cè)結(jié)果顯示,PDGF-BB濃度在五個(gè)時(shí)間點(diǎn)上各組之間差異無統(tǒng)計(jì)學(xué)意義(P0.05),MCP-1濃度在PL、TGWJ和PV組相比空載體對(duì)照組均有升高,且在24h、36h、48h三個(gè)時(shí)間點(diǎn)上,三組間兩兩比較,MCP-1濃度PLTGWJPV組,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),特別是在48h,PL組、TGWJ組的MCP-1濃度為各時(shí)間點(diǎn)中最高,此時(shí)PL組、TGWJ組、PV組的差異也較為明顯。實(shí)時(shí)熒光PCR檢測(cè)U87細(xì)胞MCP-1的mRNA水平結(jié)果顯示,U87細(xì)胞表達(dá)HIV-1gp120蛋白48h時(shí),PL組與TGWJ組、PV組MCP-1的mRNA水平差異具有顯著性(P0.05),PL組顯著高于TGWJ組、PV組。4.血腦屏障相關(guān)細(xì)胞的培養(yǎng)成功培養(yǎng)小鼠腦微血管內(nèi)皮細(xì)胞、周細(xì)胞,免疫熒光法檢測(cè)內(nèi)皮細(xì)胞的異性標(biāo)記物CD31、Ⅷ、周細(xì)胞特異性標(biāo)記物NG2、PDGFRβ,熒光清晰可見。人臍靜脈內(nèi)皮細(xì)胞系HUVEC正常培養(yǎng),生長(zhǎng)狀態(tài)良好。5.表達(dá)HIV-1 gp120的U87細(xì)胞上清對(duì)內(nèi)皮細(xì)胞緊密連接蛋白的影響Western-blot檢測(cè)結(jié)果顯示,緊密連接蛋白ZO-1、Occludin均出現(xiàn)減少,減少程度PLTGWJPV,同時(shí)實(shí)時(shí)熒光定量PCR檢測(cè)其mRNA水平,結(jié)果顯示差異無統(tǒng)計(jì)學(xué)意義。6.表達(dá)HIV-1 gp120的U87細(xì)胞上清對(duì)HUVEC細(xì)胞、原代小鼠腦微血管內(nèi)皮細(xì)胞、周細(xì)胞增殖活性的影響Almar blue檢測(cè)結(jié)果顯示,與空載體對(duì)照組比較原代內(nèi)皮細(xì)胞PL組、TGWJ組熒光強(qiáng)度均有升高,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05),PV組與之差異無統(tǒng)計(jì)學(xué)意義(P0.05)。HUVEC細(xì)胞與空載體對(duì)照組相比,PL組熒光強(qiáng)度升高,其差異具有統(tǒng)計(jì)學(xué)意義(P0.05),TGWJ組、PV組差異無統(tǒng)計(jì)學(xué)意義(P0.05)。原代周細(xì)胞活性差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1.ADC病人TGWJ、PV來源的gp120基因和PL來源的gp120基因處在不同進(jìn)化枝上,三個(gè)部位的gp120蛋白代表的HIV-1均為巨噬細(xì)胞嗜性、非合胞體誘導(dǎo)型,頂端四肽的氨基酸序列存在差異2.ADC病人PL、TGWJ、PV來源的gp120蛋白對(duì)U87細(xì)胞合成PDGF-BB無影響,能增強(qiáng)MCP-1的分泌,外周PL來源的gp120蛋白作用最強(qiáng)。3.表達(dá)ADC病人不同部位gp120蛋白的U87細(xì)胞上清使HUVEC細(xì)胞、原代小鼠腦微血管內(nèi)皮細(xì)胞的緊密連接蛋白ZO-1、Occludin的含量減少,外周PL來源的gp120蛋白對(duì)應(yīng)上清的影響最強(qiáng),對(duì)血腦屏障的通透性影響最大。4.表達(dá)gp120蛋白的U87細(xì)胞上清能使HUVEC細(xì)胞、原代小鼠腦微血管內(nèi)皮細(xì)胞的活性增強(qiáng),外周PL來源的gp120蛋白對(duì)應(yīng)上清的影響最強(qiáng);對(duì)原代周細(xì)胞活性無影響。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R512.91;R747.9

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