CN116286993A - 一种HIV-tat致神经损伤模型的建立方法及其用途 - Google Patents
一种HIV-tat致神经损伤模型的建立方法及其用途 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,具体涉及一种HIV‑tat致神经损伤模型的建立方法及其用途。具体技术方案为:一种HIV‑tat致神经损伤模型的建立方法,将HIV‑1的tat基因序列插入到MLV囊膜基因上游,并在tat后引入一个P2A序列,使tat和ENV同时转录和表达,翻译后经P2A自发切割而形成自由的tat蛋白和ENV蛋白,所构建的病毒MLV‑Tat能正常感染小鼠并表达Tat蛋白,Tat蛋白诱导神经损伤从而成功构建神经损伤模型。模型构建只需将MLV‑tat病毒腹腔注射,对动物损伤较小,操作方便。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种HIV-tat致神经损伤模型的建立方法及其用途。
背景技术
自1996年开始应用联合抗逆转录病毒疗法(combined antiretroviral therapy,cART)后,获得性免疫缺陷综合征(AIDS)从致死性疾病转变为慢性进展且可控状态。因为体内病毒被抑制,艾滋病患者生存时间明显延长。现在预期接受cART的HIV感染者几乎可以与未感染HIV的健康人一样长寿。然而,随着患者生存期的延长,约40%的感染者仍会进一步发展为HIV相关的神经认知功能障碍综合征(HAND)。一项对HAND及非HAND的最新研究显示,经过18个月的抗逆转录病毒治疗后随访,10%的HAND患者认知进一步恶化,4%的非HAND患者进展为HAND。HAND根据临床表现和严重程度包括各种情况,包括无症状的神经认知障碍、中度神经认知障碍和HIV相关的痴呆。
HAND的发病机制涉及因素多且复杂,主要包括HIV和/或病毒蛋白质、神经炎症因子,以及HIV诱导的代谢变化、CNS病毒逃逸、衰老、cARTs药物的神经毒性等。其中,被广泛接受的重要机制为:HIV结构蛋白的直接神经毒性导致神经元损伤和功能障碍。与神经系统有关的HIV结构蛋白主要包括病毒表面糖蛋白gp120,转录反式激活因子(Tat)、病毒蛋白R(Vpr)和负调节因子(Nef)。艾滋病毒在感染的早期阶段就会侵入神经系统,这被认为是通过特洛伊木马机制发生的,在常规免疫监测期间,病毒通过受感染的血液来源的巨噬细胞进入中枢神经系统(CNS)。然而,在高病毒载量的环境中,艾滋病病毒粒子也可能穿过破坏的血脑屏障(BBB)。一旦进入中枢神经系统,这些巨噬细胞,成为长寿细胞,并建立一个持续存在的病毒库。感染的巨噬细胞低水平促进中枢神经系统内其他细胞的感染,包括小胶质细胞、巨噬细胞和星形胶质细胞。HIV感染的单核细胞也可分泌炎性细胞因子和病毒产物,破坏脑微血管内皮细胞,诱导细胞的氧化应激,加速HIV感染的巨噬细胞在血脑屏障中的迁移,直接或者间接引发神经炎症,导致神经元损伤和功能障碍。
HIV是逆转录病毒,编码9种蛋白质,可分为三类:结构蛋白(gag、pol和env),调节蛋白(tat和rev),辅助蛋白(vif、vpr、vpu和nef)。HIV-1反式转录激活因子Tat是第一个从整合的HIV-1前病毒转录和翻译的病毒蛋白,其作为HIV转录的重要激活剂,调节RNA聚合酶II依赖的转录。与典型的转录因子是DNA结合蛋白不同,Tat是一种RNA结合蛋白,可以识别HIV RNA分子中的特定序列转录激活剂反应元件(TAR),对HIV的复制和转录至关重要。Tat蛋白是由86-120个氨基酸组成的小的核蛋白,可以被HIV-1感染的细胞分泌到细胞外、脑脊液以及血清中。
许多研究表明HIV-1tat蛋白参与HAND的发生。当将HIV-1tat蛋白注射进入小鼠大脑中,引起的组织学变化与HAND病人的大脑组织病理学变化非常相似。HIV-1tat的神经毒性机制包括神经元直接去极化、升高包内钙离子浓度、促进炎性因子的产生核分泌、增加单核巨噬细胞的浸润、活化星形胶质细胞核小胶质细胞产生神经炎性因子、激活兴奋性氨基酸受体以及促进细胞凋亡等。
此外,分泌到细胞外的HIV-1tat蛋白,可以破坏突触膜。在小鼠中长期低水平表达HIV-1tat蛋白可损伤突触,表现为神经骨架和突触标记物,例如神经元特异性细胞骨架蛋白βIII-微管蛋白,突触前囊泡蛋白p38(突触素)和突触后密度95蛋白(PSD-95)的表达水平降低。突触损伤也可以由单个HIV蛋白引起,包括HIV tat和gp120。Tat存在于病毒抑制的病人的脑脊液中,具有多种直接和间接神经炎症作用,被认为是HAND神经病理学的关键因素。所以,研究HIV-1tat蛋白在HAND的发生中是如何发挥作用,对于探究HAND的致病机制以及进行治疗均有重要的现实意义。
现有的HIV-1tat蛋白相关HAND动物模型主要为两种,一种是直接给动物注射HIV-1tat蛋白,另一种是使用HIV-1tat转基因动物。
HIV相关的神经认知功能障碍综合征动物模型包括HIV-1病毒蛋白gp120转基因鼠模型、tat转基因鼠(将tat基因包括LTR基因转入小鼠)、LTR转基因鼠(主要用于LTR研究的模型)、nef转基因鼠(将HIV-1Bru的nef基因接在鼠T细胞受体VB8.3的B链增强子/启动子控制之下,得到3个不同的转基因动物鼠系)、全病毒基因转基因鼠等、HIV免疫缺陷病毒感染灵长类动物模型、免疫缺陷病毒(SIV)/猕猴模型、人猴免疫缺陷嵌合病毒(simian/humanimmunodeficiency virus,SHIV)/猕猴模型(含有HIV-1和SIV基因的SHIV)。
将HIV-1病毒基因组直接转入大鼠类动物的疾病模型,使全病毒基因在很多组织器官中表达。这种模型可用于阐明HIV病毒基因产物累积所引起的相关疾病进展的潜在机制。HIV-1转基因(HIV-1Tg)模型是从一个完整的前病毒质粒(pNLS-3)非传染性克隆体发展而来。HIV-1转基因动物均为半合子,将转基因并入其2个等位基因中的1个复本中。因此,当与野生型生育的F344大鼠交配后,其子代是HIV-1Tg大鼠或野生型(Tg-wild type)。将HIV-1病毒蛋白转入小鼠,建立gp120、tat与nef转基因鼠模型,其方法主要是构建受胶质纤维酸性蛋白(GFAP)启动子调控的gp120、tat转基因载体,制备gp120、tat转基因鼠。Gp120转基因鼠也可以神经原纤维素(neurofila-mentlight,NFL)为启动子,由神经细胞特异性分泌gp160,gp160在鼠体内分解出gp120而制备。也可将HIV-1B亚型的nef基因接在鼠T细胞受体VB8.3的B链增强子/启动子控制之下,得到3个不同的转基因动物鼠系。
综上所述,HIV-1相关神经病变动物模型种类较多,其中研究较成熟的有HIV-1Tg大鼠模型及HIV-1周围神经病变动物模型等。这些动物模型对于研究HIV-1引起的认知行为改变、药物依赖提供了重要作用。尽管如此,对于全面深入研究HIV-1相关神经病变的发病机制仍有不足,需要进一步开展HIV动物模型及其应用研究,帮助更好地了解HIV相关神经病变的发病机制。
发明内容
针对现有技术的不足,本发明提供了一种HIV-tat致神经损伤模型的建立方法及其用途。现有技术中,在HIV全序列前病毒DNA转基因小鼠中,已证实在小鼠中表达了完整的病毒基因拷贝,但并非所有小鼠都显示出基因表达或疾病的迹象。由雌性小鼠和表达完整序列基因的正常雄性小鼠有性繁殖产生的F1后代生长非常缓慢。同时,HIV免疫缺陷病毒感染灵长类动物模型中使用的动物的来源和经济成本较高。而且,通过颅内注射手术直接给动物注射HIV-1病毒蛋白,其手术操作风险较大,也可能会损伤动物原本的神经认知功能,且注射的蛋白在小鼠体内不能长时间存留,需要重复注射,操作不便。
为实现以上目的,本发明通过以下技术方案予以实现:
本发明公开了一种HIV-tat致神经损伤模型的建立方法,将HIV-1的tat基因序列插入到MLV囊膜基因上游,并在tat后引入一个P2A序列,使tat和ENV同时转录和表达,翻译后经P2A自发切割而形成自由的tat蛋白和ENV蛋白,所构建的病毒MLV-tat能正常感染小鼠并表达tat蛋白,tat蛋白诱导神经损伤即成功构建模型。
优选的,HIV-1的tat基因序列插入到MLV载体中的过程为:在MLV载体的第6223处TAA后加入起始密码子ATG,后接tat序列,并在tat序列后加入一个P2A序列。
优选的,HIV-tat病毒构建的具体过程为:将MLV质粒用PmlⅠ和SgrA I限制性内切酶进行双酶切,产物经电泳后切胶回收大片段,并在ENV基因前加入ADA tat或NL4-3 tat序列,获得MLV-ADA tat或MLV-NL4-3 tat质粒。
优选的,所述ADA tat的DNA序列如SEQ ID NO.1所示,所述NL4-3 tat的DNA序列如SEQ ID NO.2所示。
相应的,所述的HIV-tat致神经损伤模型在抗HIV-tat相关神经损伤药物的药效测定和药理学研究中的应用。
相应的,所述的HIV-tat致神经损伤模型在筛选抗HIV-tat致神经损伤药物中的应用。
本发明具备以下有益效果:
1.本发明构建了构建两种出可持续表达HIV-1tat蛋白的MLV病毒:MLV-ADA tat、MLV-NL4-3 tat,进而成功构建HIV-tat神经损伤小鼠模型,此神经损伤的动物模型选择使用C57/BL6小鼠,在动物来源以及经济成本也较其他种类灵长类动物和转基因动物的繁育成本更低。而且,建模的时间更快,只需要21天左右,不需要进行长时间的动物繁育,而转基因小鼠的繁育时间远远长于此建模时间。
2.HIV-tat的感染只需要从小鼠的腹腔注射病毒,对动物损伤较小,操作方便。如果通过向动物脑内直接注射HIV-1病毒蛋白构建神经损伤动物模型,手术操作要求较高,且有可能损伤动物大脑,造成人为的神经损伤。
3.本发明除了成功构建HIV-tat神经损伤小鼠模型,还使用RNAseq测出小鼠海马中的大量差异RNA,可为探究HIV相关神经认知功能障碍提供作用机制、信号路径和靶点等关键信息。
附图说明
图1为ADA tat和NL4-3 tat氨基酸比对;
图2为MLV-ADA tat、MLV-NL4-3 tat质粒构建的示意图;
图3为构建的MLV-tat病毒在C57/BL6小鼠体内的感染力;
图4为八臂迷宫检测HIV-tat感染小鼠的学习记忆行为;
图5为条件恐惧性实验检测HIV-tat感染小鼠的恐惧记忆;
图6为HIV-tat感染小鼠海马CA1、CA3区的免疫荧光染色图;
图7为HIV-tat感染小鼠海马区的小胶质细胞被激活;
图8为HIV-tat感染小鼠cortex区的MAP2染色图像;
图9为HIV-tat感染小鼠的海马区H&E染色图像;
图10为HIV-tat感染的小鼠海马CA1区高尔基染色图像;
图11为HIV-tat感染的小鼠海马LTP典型图;
图12为HIV-tat小鼠海马组织中差异RNA的数量。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
若未特别指明,实施举例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1ADA和NL4-3的tat氨基酸序列比对
tat有多种剪接变体,有40%的研究使用了变体tat 1-86,而只有15.5%的研究使用了Tat 1-101,这是HIV-1亚型感染者中最常见的剪接变体。由于tat剪接变体之间存在功能差异,可能促进HAND中的突触损伤。通过使用DNAMAN软件对ADA和NL4-3两种毒株的tat氨基酸序列比对,结果见图1所示,结果显示ADA和NL4-3的tat氨基酸序列存在约25%的差异,为了解不同tat剪接体的功能差异,我们将这两种tat分别构建在MLV载体中,使其能够在小鼠体内持续表达tat蛋白,进而得到不同HIV-tat诱导的神经损伤动物模型。
其中,ADA tat和NL4-3 tat的DNA序列见下表1所示。
表1ADA tat和NL4-3 tat的DNA序列
实施例2构建MLV-ADA tat、MLV-NL4-3 tat质粒
MLV病毒是一种逆转录病毒,在载体克隆过程中放置在两个LTR之间的任何基因都与病毒基因组的其余部分一起永久插入宿主DNA。由于MLV载体缺乏病毒包装和转导所需要的基因,因此以MLV作为载体的病毒具有复制能力较低的特征,也比较安全,且构建在载体上的病毒蛋白可以表达。因此,将HIV-1的tat基因序列插入到MLV囊膜基因上游,并在tat后引入一个P2A序列,使转录tat和ENV同时转录,翻译后经P2A自发切割而形成自由的tat蛋白和ENV蛋白,所构建的病毒MLV-tat能正常感染小鼠并表达Tat蛋白,即成功构建模型。
可表达HIV-tat的MLV病毒构建具体步骤为:将MLV质粒用PmlⅠ和SgrA I限制性内切酶进行双酶切,产物经0.8%琼脂糖凝胶电泳,并切胶回收大片段于Eppendorf管内,用TIANGEN公司的琼脂糖凝胶回收试剂盒回收相应的片段,并测定产物的纯度和浓度。
将上述载体回收片段在ENV基因之前分别加上由生工生物工程(上海)股份有限公司科技公司合成的ADA tat、NL4-3 tat序列。分别将ADA tat、NL4-3 tat序列片段的胶回收产物以等量摩尔比加入Eppendorf管,加入ExnaseⅡ连接酶与同源重组酶5×CEⅡbuffer,37℃反应0.5小时;将同源重组产物取出10μL,加入100μL Stabl3感受态细胞,冰浴30min后42℃热激90s,加入500μL SOC培养基37℃、220rpm培养2小时;2小时后4000g离心1min,移除400μL多余液体。
将剩余液体涂布在含氨苄霉素的LB平板37℃培养12小时;分别在平板上挑取单菌落,接种到5mL LB液体培养基中37℃、220rpm培养12小时。用TIANGEN公司小提试剂盒提取质粒,获得MLV-ADA tat,MLV-NL4-3 tat质粒,并送生工生物工程(上海)股份有限公司科技公司测序验证无误后,即可后续使用。MLV-ADA-tat和MLV-NL4-3-tat质粒构建的示意图如图2所示。
实施例3构建的MLV-tat病毒的感染力验证
可表达HIV-tat的MLV病毒包装:对数生长期的293T细胞(5×106个/10cm皿),共10皿,细胞均在转染前24h已接种至皿、Flask,培养条件为DMEM+10% FBS,37℃,5%CO2;根据慢病毒制备体积及系统需求,计算包装质粒、PEI添加量目的质粒:质粒=3:1,每个10cm皿中添加10μg质粒、30μL PEI。将PEI和质粒混合均匀的DMEM培养基均匀缓慢得添加到293T细胞的10cm皿中,将10cm培养皿置于37℃含5%二氧化碳培养箱培养72h,收集上清病毒原液。再经过高速离心后得到浓缩的病毒。
每只C57/NL6小鼠通过腹腔注射300μL病毒后继续饲养21天,并每天观察小鼠状态(分为对照组、MLV组,MLV-ADA tat组、MLV-NL4-3 tat组,每组6只小鼠)。感染C57/NL6小鼠21天后,取小鼠静脉血,脾脏和大脑组织检测病毒载量。具体如下:
1)设计MLV RU5,mouse GAPDH,mouseβglobin区引物及探针,由上海生物工程有限公司合成,见下表2所示。
表2qPCR引物列表
2)反应体系的配制和加样
反应体系 | 加入量(μL) |
FastStart Universal Probe Master(ROX) | 12.5μL |
F Primer(10μM) | 0.5μL |
MLV 2LTR R Primer(10μM) | 0.5μL |
MLV 2LTR Probe(25μM) | 0.5μL |
Sample | 1μL |
RNase Free ddH2O | 9μL |
Total | 25μL |
3)上机检测,打开Bio-Rad PrimePCR荧光定量PCR仪,按照下表设置程序,放入八联管,在同一板上进行检测,反应条件如下:
4)程序结束下机,PCR程序结束后,仪器已自动记录保存每个循环的荧光信号,通过检测扩增曲线及Ct值进行结果判断与计算,结果见图3所示。结果显示,在小鼠静脉血,脾脏和大脑组织中均检测到MLV RU5。表明我们所构建的MLV病毒已成功感染小鼠,并可以感染小鼠大脑。
实施例4HIV-tat感染小鼠的行为学检测—八臂迷宫
实施例3的实验表明,小鼠大脑中也检测到了病毒,因此通过八臂迷宫实验,检验被感染小鼠在学习记忆认知上是否产生了神经功能损伤。
动物适应实验环境1周后,称重,禁食12~24小时。此后每天训练结束后限制性地给予正常食料(小鼠2~3g),以使体重保持在正常进食小鼠的80~90%。第二天,上午10:00,迷宫各个臂中添加食物(可使用不同食物,此次使用猫粮。每只4~5粒,0.02g)。然后,同时将4~5只动物置于迷宫中央。让其自由摄食、探究10min。下午,14:00重复一次。第三天,上午重复第二天上午的训练。这一过程让动物在没有很强的应激条件下熟悉迷宫环境。下午,八个臂中都添加0.02g猫粮,单只小鼠从中央区域放下,让其自由摄食、探究10min。第四天起,下午14:00小鼠单只进行一次训练:在随机4个臂靠近外端食盒处各放一颗食物,让动物自由摄食。食物吃完或8min后将动物取出。训练结束后限制性地给予正常食料(小鼠2~3g),测量体重,采集数据。(若条件允许,间隔30min后,可进行第两次)第五到十三天,随机重新选择四个臂,重复前一天的训练。训练结束后限制性地给予正常食料(小鼠2~3g),测量体重,采集数据。第十四天,将所有动物放在迷宫中进行8分钟的单次试验,或者直到他们获得所有奖励为止。此时,实验组小鼠的错误次数应接近对照组小鼠,否则延长实验天数。每个试验评估的参数如下:收集所有食物所花费的时间-进入迷宫各臂的时间;工作记忆错误的次数-当动物重新进入该试验中已经被探过的臂时;参考记忆错误数-从未被引诱的臂的进入次数;永久性错误数量-当动物重新进入在该试验中已经探过的未诱饵的臂次数。
记录以下指标:工作记忆错误(working memory errors),即在同一次训练中动物再次进入已经吃过食物的臂;参考记忆错误(reference memory errors),即动物进入不曾放过食物的臂;总的入臂次数;测试时间,即动物吃完所有食物所花的时间。结果见图4所示。
图4的A图为小鼠在八臂迷宫实验过程中的体重记录,T0天小鼠通过禁食18h处理后,在适应性训练的第一天,小鼠的体重发生下降,随着实验天数的增加,小鼠体重逐步回升,感染MLV-NL4-3 tat、MLV-ADA tat的实验组与MLV组以及对照组小鼠的体重趋势一致,没有显著差异,表明MLV病毒不会影响小鼠的生存。
B图为小鼠在八臂迷宫中的总入臂次数、总错误次数以及参考记忆错误次数,在测试的第三天与第四天,感染MLV-NL4-3 tat、MLV-ADA tat组小鼠的总入臂次数、错误次数比空白对照组以及MLV组小鼠高,且有显著性差异,而MLV组与对照组一致。
C图为小鼠在八臂迷宫中的行动轨迹图,可见MLV-NL4-3 tat、MLV-ADA tat组小鼠在吃完所有食物所运动的路程比MLV组和对照组更多,且具有极显著性的差异。(n=6,*P<0.05,**P<0.01)
实施例5HIV-tat感染小鼠的行为学检测—条件恐惧性实验
实施例3的实验表明,含有HIV-tat的MLV病毒感染小鼠后,在小鼠大脑中也检测到了病毒,因此通过条件性恐惧实验,检验被感染小鼠在恐惧认知上是否产生了神经功能损伤。
将单只小鼠放入装在消音盒内的电击室中,小鼠首次进入正方形电击室,先让小鼠自由探索。第一天给予小鼠条件性声音刺激:为training phase,小鼠放入正方形电击室后,关闭消音盒的门。实验方案为无刺激60s、声音刺激7s(80dB,800Hz)、足部电击2s(0.7mA)、无刺激60s。此方案循环4次,记录小鼠运动轨迹、冻结反应(freezing)的次数以及时间等数据。第二天评价小鼠在第一阶段中环境相关的条件性恐惧程度:为contextualphase,小鼠放入正方形电击室后,关闭消音盒的门。实验方案为无刺激120s,循环3次。记录小鼠运动轨迹、冻结反应(freezing)的次数以及时间等数据。第三天观察改变环境信息后小鼠的条件性恐惧程度:为cued phase,提前在消音室内喷适量的非刺激性香水,并将正方形电击室更换为三角形电击室。将小鼠放入电击室后关闭消音盒的门。实验方案为无刺激180s、声音刺激180s(80dB,800Hz)、无刺激90s。记录小鼠运动轨迹、冻结反应(freezing)的次数以及时间等数据。
以上的实验中,第二阶段没有给与任何刺激但是实验动物表现出明显的环境相关性恐惧,表现为活动性明显减少,但是并没有出现freezing反应。在第三阶段,同一只动物在改变了环境后没有表现出任何不动反应(活动性减少),但是在给与声音信号后,可以观察到明显地条件性恐惧freezing反应。结果见图5所示。
图5的A图为第一天训练阶段的条件恐惧性实验过程设计图。小鼠进入实验室后,先有60s的空白刺激时间,之后是7s的声音刺激,在声音刺激的最后2s给予足部电击,此过程循环四次,在最后一个循环完成后,有60s的空白刺激时间。记录下每个时间段下小鼠freezing的时间以及次数。
B、C和D图为条件性恐惧性实验中小鼠在声音刺激前后出现freezing反应的比例。在第一天的训练给予声音以及足部电击的刺激后,小鼠的freezing反应都有明显的上升,但MLV-NL4-3 tat,MLV-ADA tat组小鼠的freezing比例少于MLV组和对照组,且具有极显著性的差异。在训练的第二天contextual phase只有声音刺激的情况下,MLV-NL4-3 tat,MLV-ADA tat组小鼠的freezing比例显著少于MLV组和对照组小鼠的freezing比例,而MLV组与对照组没有显著性差异。在训练的第三天cued phase,更换场景并只给予小鼠声音刺激的情况下,4组小鼠的freezing比例均明显上升;当声音刺激停止后4组小鼠的freezing比例均明显降低,且MLV-NL4-3 tat,MLV-ADA tat组小鼠的freezing比例与MLV组和对照组相比降低得更多。(n=6,*P<0.05,**P<0.01)
实施例4和5的结果都表明对照组和MLV病毒的感染没有使小鼠产生行为学上的差异,而MLV-NL4-3 tat、MLV-ADA tat的感染导致了小鼠学习记忆和恐惧记忆功能的损伤,初步证明本实验成功构建了神经损伤的动物模型。
实施例6免疫荧光检测HIV-tat感染小鼠大脑的突触和神经元数量
实施例5证明构建的表达HIV-tat的MLV病毒可以成功感染小鼠,并使小鼠产生了显著性的行为学差异,出现了明显的神经认知障碍。接下来取HIV-tat感染21天后小鼠大脑,切片后进行免疫荧光染色观察。MAP2(绿色)染成熟神经元的突触,NeuN染神经核。
结果见图6所示,图中,在感染小鼠的海马CA1和CA3区域,四组小鼠的NeuN的染色强度没有显著差异,但是MAP2的染色强度在MLV-NL4-3 tat,MLV-ADA tat组小鼠中与MLV组和对照组相比有明显的减少。HIV-tat蛋白的表达虽然没有造成小鼠神经元细胞数量的明显改变,但减少了海马CA1和CA3区突触的数量。
实施例7检测HIV-tat感染后小鼠海马区的小胶质细胞数量
通过免疫荧光染色观察海马区小胶质细胞的数量是否有变化。DAPI(蓝色)染细胞核,Ki67(绿色)是细胞增殖的标记物,IBA-1(红色)是小胶质细胞的标记物。
结果见图7所示,MLV-NL4-3 tat和MLV-ADA tat组小鼠海马区的小胶质细胞数量与对照组相比明显上升,但是新增的细胞数量无显著变化。在之前研究者的研究中有表明神经损伤的机制之一为小胶质细胞激活引起神经炎症。本实验的结果初步可验证HIV-tat蛋白在小鼠体内表达后激活了海马小胶质细胞,结果与之前的研究一致。
实施例8检测HIV-tat感染小鼠大脑cortex区的突触数量
实施例6和7已经表明我们构建的表达HIV-tat的MLV病毒使小鼠的海马区的突触数量减少,并激活了海马区的小胶质细胞的数量,因此我们同时也检测了小鼠cortex脑区的突触的数量。
结果如图8所示,通过免疫荧光染色的结果显示,HIV-tat感染的小鼠大脑的cortex区的突触标记物MAP2的染色强度与照组相比也有明显的减少。
实施例9HIV-tat感染的小鼠海马生理结构
通过H&E染色观察HIV-tat对小鼠海马CA1区神经细胞结构的影响。如图9所示,4组海马神经元细胞结构完整,染色均一,细胞膜、核膜边界清洗,核仁明显。表明MLV与HIV-tat在小鼠大脑内没有引起海马区结构异常。
综上所述,构建出的两种表达HIV-tat的MLV病毒可成功感染C57/BL6小鼠,并且可以在小鼠脾脏、大脑、血液中感染。在小鼠感染21天后,通过八臂迷宫和条件恐惧测试,HIV-tat感染小鼠的学习记忆和条件恐惧记忆与对照组小组相比呈现出显著性差异。经过小鼠大脑的免疫荧光染色的结果发现,HIV-tat感染小鼠海马CA1和CA3区的MAP2突触标志蛋白的染色强度有显著的降低,而神经元的数量并没有减少。同时被感染小鼠海马区的小胶质细胞也被显著激活。除了海马区突触有明显减少外,被感染小鼠大脑cortex区的突触数量同样也出现了减少。结果均表明,构建出的两种MLV病毒表达的HIV-ADA tat、HIV-NL4-3tat蛋白可以使小鼠产生神经认识损伤,模型构建成功。
实施例10HIV-tat感染的小鼠海马CA1区神经元的树突棘数量变化HIV-tat感染21天后,对小鼠心脏灌洗后取出小鼠完整大脑组织,放在固定液中。用生理盐水将经过4%多聚甲醛固定的小鼠大脑组织轻轻漂洗,置于圆底EP管中,加入高尔基染色液将脑组织完全浸泡,避光处理,浸泡48h后,换一次新染液,之后每隔3天换一次新染液,共计14天。用蒸馏水浸洗小鼠脑组织三次,再用80%冰醋酸浸没脑组织。过夜后用蒸馏水清洗,置于30%的蔗糖溶液中。使用振荡切片机进行切片,厚度为100μm,贴在载玻片上,过夜避光进行晾干处理。将晾干后的组织切片用浓氨水处理15min,然后用蒸馏水洗,再用酸性坚膜定影液处理15min,最后晾干用甘油明胶封片后即可拍照观察。
图10高尔基染色图像显示:MLV-NL4-3 tat组、MLV-ADA tat组小鼠海马CA1区神经元的树突棘数量明显少于对照组和MLV组神经元的树突棘数量,表明HIV-tat的感染使小鼠突触出现了损伤。
实施例11HIV-tat感染的小鼠海马组织的突触可塑性检测
实施例8的免疫荧光染色表明HIV-tat感染的小鼠海马的突触数量减少,实施例10的高尔基染色的结果也表明海马神经元树突棘的数量也明显减少,都提示NL4-3 tat、ADAtat的感染造成了小鼠海马突触的损伤。为了进一步研究NL4-3 tat、ADA tat是否也改变了小鼠海马的突触可塑性,采用电生理检测记录小鼠海马CA3-CA1回路的LTP反应。
提前对切片机的切片槽周围填满碎冰进行降温,切片槽中加满冰冷的切片液并充混合气体(95% O2+5% CO2)。用异氟烷麻醉小鼠,迅速断头取出大脑,同时不断地用冰冷的切片液冲洗大脑(切片液预先充入混合气体30min),然后去除嗅球和小脑。修整脑块后在底座上点少量胶水使大脑组织牢固站立,迅速将底座放在切片机槽里,沿矢状位切成400μm厚的脑片,把脑片放在盛有人工脑脊液(预先充混合气体30min)的玻璃杯中室温下孵育1~2h后备用。将海马切片置于记录电极中,在显微镜下观察并调整,使海马CA1区位于电极位置上。用U型框轻放于脑片上,向脑片通入人工脑脊液和混合气体,排查噪声信号。选择刺激的通道,记录10-70μA电流刺激下的兴奋性突触后电位(fEPSP)斜率,既输入-输出(I-O)曲线。调整电流的大小,使fEPSP的大小在I-O曲线最大值的30-50%,每隔20s给予一个单脉冲的刺激,稳定记录20-30min,既为LTP基线。暂停基线记录,给与2串持续1s的高频刺激,间隔10s,继续记录至少1小时,即为LTP诱导。对高频刺激诱导后1小时的最后5min与诱导前5min的数据进行统计分析。红色为高频刺激前(LTP基线),黑色为高频刺激诱导后。
结果如图11所示,LTP结果显示NL4-3 tat、ADA tat组小鼠海马区经高频刺激诱导产生的LTP幅度明显要弱于对照组小鼠,说明NL4-3 tat、ADA tat的表达可以造成小鼠海马神经元的LTP电生理障碍和突触可塑性损伤。
实施例12HIV-tat感染的小鼠海马组织RNAseq检测
从组织样品中提取total RNA,利用Nanodrop 2000对所提RNA的浓度和纯度进行检测,琼脂糖凝胶电泳检测RNA完整性,Agilent 2100测定RIN值。高质量的RNA是测序成功的基础,为保证测序数据准确性,使用以下方法对样品进行检测,检测结果达到要求后方可进行建库:1)Nanodrop检测RNA的纯度(OD260/280)、浓度是否正常;单次建库要求totalRNA总量2μg,浓度≥300ng/μL,OD260/280介于1.8~2.2之间。2)Agilent 2100精确检测RNA的完整性,检测指标包括:RIN值、28S/18S、图谱基线有无上抬、5S峰;3)电泳检测RNA样品是否弥散或有基因组DNA的污染。RNA样品检测合格后,从总RNA样品中去除rRNA(部分lncRNA具有与mRNA相同的poly A尾结构,用去除rRNA的方法能够最大程度地保留含有poly A尾的lncRNA)。随后向富集得到的RNA中加入fragmentation buffer将RNA打断成小片段,以片段化的RNA为模板,加入6nt随机引物(random hexamers)反转录合成cDNA第一条链,再加入缓冲液、dNTPs(dNTP中的dTTP用dUTP取代)、DNA polymerase I和RNase H合成cDNA第二条链。合成的双链cDNA经纯化、末端修复、加A、连接测序接头处理后,用USER酶降解含有U的cDNA第二链并进行PCR富集,最后用AMPure XP beads纯化PCR产物,得到最终的链特异性文库。
结果如图12所示,其中MLV组(B),MLV-NL4-3 tat组(C),MLV-ADA tat组(D)。
RNA测序结果显示,HIV-tat感染的两组小鼠(MLV-NL4-3 tat组和MLV-ADA tat组)与MLV感染小鼠的共同差异lncRNA有267个,circRNA有6个,mRNA有3个。其中circRNA的变化最多,在图12中列出。MLV组与MLV-NL4-3 tat组、MLV-ADA tat组的共同差异circRNA见下表所示。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (6)
1.一种HIV-tat致神经损伤模型的建立方法,其特征在于:将HIV-1的tat基因序列插入到MLV囊膜基因上游,并在tat后引入一个P2A序列,使tat和ENV同时转录和表达,翻译后经P2A自发切割而形成自由的tat蛋白和ENV蛋白,所构建的病毒MLV-tat能正常感染小鼠并表达tat蛋白,tat蛋白诱导神经损伤即成功构建模型。
2.根据权利要求1所述的一种HIV-tat致神经损伤模型的建立方法,其特征在于:HIV-1的tat基因序列插入到MLV载体中的过程为:在MLV载体的第6223处TAA后加入起始密码子ATG,后接tat序列,并在tat序列后加入一个P2A序列。
3.根据权利要求1所述的一种HIV-tat致神经损伤模型的建立方法,其特征在于:HIV-tat病毒构建的具体过程为:将MLV质粒用PmlⅠ和SgrA I限制性内切酶进行双酶切,产物经电泳后切胶回收大片段,并在ENV基因前加入ADA tat或NL4-3 tat序列,获得MLV-ADA tat或MLV-NL4-3 tat质粒。
4.根据权利要求3所述的一种HIV-tat致神经损伤模型的建立方法,其特征在于:所述ADA tat的DNA序列如SEQ ID NO.1所示,所述NL4-3 tat的DNA序列如SEQ ID NO.2所示。
5.根据权利要求1~4任一项所述的HIV-tat致神经损伤模型在抗HIV-tat相关神经损伤药物的药效测定和药理学研究中的应用。
6.根据权利要求1~4任一项所述的HIV-tat致神经损伤模型在筛选抗HIV-tat致神经损伤药物中的应用。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050241009A1 (en) * | 2004-04-21 | 2005-10-27 | Potash Mary J | Development of a murine model of HIV-1 infection on the basis of construction of EcoHIV, a chimeric, molecular clone of human immunodeficiency virus type 1 and ecotropic moloney murine leukemia virus competent to infect murine cells and mice |
WO2008070385A2 (en) * | 2006-11-08 | 2008-06-12 | The Trustees Of Columbia University In The City Of New York | Uses of a murine model of hiv-1 infection |
CN108138201A (zh) * | 2015-09-04 | 2018-06-08 | 托卡根公司 | 包含2a肽的重组载体 |
CN113143974A (zh) * | 2021-03-03 | 2021-07-23 | 武汉科技大学 | 一种hiv感染致神经损伤动物模型的建立方法及其用途 |
-
2023
- 2023-03-01 CN CN202310206804.6A patent/CN116286993A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050241009A1 (en) * | 2004-04-21 | 2005-10-27 | Potash Mary J | Development of a murine model of HIV-1 infection on the basis of construction of EcoHIV, a chimeric, molecular clone of human immunodeficiency virus type 1 and ecotropic moloney murine leukemia virus competent to infect murine cells and mice |
WO2008070385A2 (en) * | 2006-11-08 | 2008-06-12 | The Trustees Of Columbia University In The City Of New York | Uses of a murine model of hiv-1 infection |
CN108138201A (zh) * | 2015-09-04 | 2018-06-08 | 托卡根公司 | 包含2a肽的重组载体 |
CN113143974A (zh) * | 2021-03-03 | 2021-07-23 | 武汉科技大学 | 一种hiv感染致神经损伤动物模型的建立方法及其用途 |
Non-Patent Citations (5)
Title |
---|
"GenBank: AAA44985.1", GENBANK * |
"GenBank: AAB64167.1", GENBANK * |
CARVALLO, L等: "Murine leukemia virus expressing HIV Tat (MLV-Tat) transactivates HIV LTR in cells in culture and induces neurocognitive impairment in MLV-Tat infected mice", JOURNAL OF NEUROIMMUNE PHARMACOLOGY * |
ELENA IROLLO等: "Mechanisms of neuronal dysfunction in HIV-associated neurocognitive disorders", CELL MOL LIFE SCI., vol. 78, no. 9, pages 1 - 3 * |
余多慰等: "分子生物学", 31 July 2007, 南京:南京师范大学出版社, pages: 156 - 157 * |
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