CN111593075B - 一种系统性红斑狼疮的动物模型的构建方法 - Google Patents
一种系统性红斑狼疮的动物模型的构建方法 Download PDFInfo
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Abstract
本发明公开了本发明属于疾病动物模型构建方法技术领域，具体涉及一种系统性红斑狼疮的动物模型的构建。本发明利用Crispr/Cas9技术，在小鼠谷胱甘肽过氧化物酶4(GPX4)基因2号外显子上游及4号外显子下游插入loxP位点，构建亲代鼠1；亲代鼠2为包含中性粒细胞特异的Cre小鼠；将亲代鼠1和亲代鼠2进行杂交，获得所述杂合的GPX4基因和包含所述Cre基因的小鼠，该小鼠自发出现狼疮表型。本发明最终获得的小鼠，天然发病，3‑4月份开始出现表型，包括皮损，肾炎，自身抗体，低补体，高炎症因子，存在性别差异，雌性小鼠发病更重，更符合人类的发病特点。
Description
技术领域
本发明属于疾病动物模型构建方法技术领域，具体涉及一种系统性红斑狼疮的动物模型的构建。
背景技术
系统性红斑狼疮(SLE)是一种系统性自身免疫疾病，其特征在于炎症因子风暴和自身抗体IgG的产生。中性粒细胞的数量减少是SLE的共同特征。SLE患者的转录组学显示中性粒细胞与肾炎的进展有关，此外，中性粒细胞胞外诱捕网(NETs)与IFNα的产生，狼疮性肾炎，内皮功能障碍有关。然而，SLE患者的血液中性粒细胞数目减少的原因尚不清楚，中性粒细胞死亡方式和背后的分子机制仍然没有系统的阐释。
雌性SPF级MRL/lpr小鼠是目前国际上最为常用的SLE模型，1978年由Murphy等人建立。该鼠由LG/J，AKR/J，C3H、HeDi和C57BL/6J品系小数复杂交配产生，此类小鼠由于Fas基因缺陷，导致T细胞死亡率降低，自身反应性淋巴细胞不能通过凋亡途径清除，导致淋巴结肿大，脾大，并产生自身免疫病症状，特征是大量的anti-dsDNA，ANA抗体，和严重的肾小球肾炎。发病早，无性别差异。
NZB/NZW F1鼠是NZB老鼠和NZW老鼠交配后的第一代老鼠，自发出现狼疮表型，其亲代任一方均不发生病变。由Helyer在1963年发现。该模型小鼠4-5月开始发病，5-6月出现明显的肾小球肾炎的症状，10-12月进展为严重的狼疮，因肾衰竭而死。此类小鼠发病症状与人类相似，性激素对该鼠有明显的影响，雌鼠比雄鼠起病早且严重。特征是高滴度anti-dsDNA，anti-ssDNA抗体，高丙种球蛋白血症。
MRL/lpr鼠模拟的是T细胞凋亡缺陷导致的功能异常，以淋巴、脾脏肿大为特点，这两种器官含有大量淋巴细胞，因此，该模型主要模拟了适应性免疫系统异常。而先天免疫系统正常，例如：IFN-alpha不升高。尽管MRL/lpr鼠表现出anti-dsDNA抗体升高，蛋白尿，和低补体血症，但并不完全符合人类疾病的特点。例如狼疮主要以育龄期女性发病，男女比例为1:9，而MRL/lpr模型没有表现出性别差异。NZB/NZW F1小鼠，表现狼疮表型的原因不明，且发病周期长(6个月)，易受环境因素影响，且实验过程不易控制，为研究疾病的发病机制带来困难。
发明内容
为了解决上述问题，本发明提供一种新的狼疮模型：病因明确，同时能引起先天免疫和后天免疫系统(T、B淋巴细胞为主)异常，且实验周期短，为系统性红斑狼疮的发病机制的研究提供新的动物模型。
本发明提供一种新型系统性红斑狼疮的动物模型的构建方法，其包括如下步骤：
1)利用Crispr/Cas9技术，在小鼠谷胱甘肽过氧化物酶4(GPX4)基因2号外显子上游及4号外显子下游插入loxP位点，构建亲代鼠1；
2)亲代鼠2为包含中性粒细胞特异的Cre小鼠；
3)将亲代鼠1和亲代鼠2进行杂交，获得所述杂合的GPX4基因和包含所述Cre基因的小鼠，该小鼠自发出现狼疮表型。
在本发明一个优选的实施方案中，所述构建方法还包括将获得的所述杂合的GPX4基因和包含所述Cre基因的小鼠再与亲代鼠2进行回交，筛选获得所述杂合的GPX4基因和包含所述Cre基因的小鼠，该小鼠自发出现狼疮表型。
其中，所述的包含中性粒细胞特异的Cre小鼠为Mrp8Cre小鼠或LysMcre小鼠。
其中，亲代鼠的背景为C57/B6。
其中，亲代鼠1具体构建过程为：以sgRNA-Vector为模板PCR扩增出Gpx4-sgRNA的DNA片段，然后胶回收作为sgRNA体外转录的模板，再经过sgRNA体外转录与纯化回收；
根据选定的sgRNA，设计打靶载体，包含同源臂及loxp序列，构建完成后，经酶切、纯化后，与sgRNA、Cas9-mRNA共同显微注射C57小鼠胚胎中，注射后将胚胎移植到代孕受体小鼠的输卵管内。
本发明动物模型利用Crispr/Cas9技术。亲代鼠的一方为谷胱甘肽过氧化物酶4(GPX4)基因外显子2-4旁插入loxP位点。GPX4编码谷胱甘肽过氧化物酶家族的成员，其功能是减少膜脂和脂蛋白中的氢过氧化物，从而保护线粒体功能并抑制细胞铁死亡。杂合和纯合loxp小鼠是活的和可育的，没有报道的异常。当与Cre重组酶小鼠繁殖时，后代可产生可诱导的GPX4基因敲除。
亲代鼠的另一方为LysMCre(又称为Lyz2Cre)鼠，该鼠的溶菌酶2基因(Lyz2)有一个核定位的Cre重组酶，该酶插入了Lyz2的第一个编码ATG中。既消除了内源性Lyz2基因的功能，又将Cre表达置于内源性Lyz2启动子、增强子元件的控制之下。该小鼠是活的和可育的，大小正常，没有表现出任何明显的身体或行为异常。这些LysMCre小鼠可用于Cre-loxp研究骨髓细胞系(粒细胞，单核细胞)和先天免疫应答。
两种鼠的背景均为C57/B6，杂交和回交后，获得GPX4fl/wtLysMCre+小鼠。该小鼠自发出现狼疮表型。
本发明最终获得的小鼠，天然发病，3-4月份开始出现表型，包括皮损，肾炎，自身抗体，低补体，高炎症因子，存在性别差异，雌性小鼠发病更重，更符合人类的发病特点。
值得注意的是，本发明获得的小鼠中性粒细胞GPX4降低，与人类狼疮患者中性粒细胞一致。由于是特定基因在特定细胞的敲低，因此模型发病的病因明确，由天然免疫系统异常，引发适应性免疫改变，识别自身抗原，最终导致狼疮发病。
附图说明
图1所示为用免疫印迹的方法，检测健康人(HCs)和狼疮患者治疗前(Pre)和治疗后(Treat)的中性粒细胞GPX4含量的变化，反映的是蛋白水平GPX4的变化。可以看到狼疮患者GPX4含量明显低于健康人，在治疗后含量上升。
图2所示为用流式细胞术的方法标记中性粒细胞内的GPX4，A图根据荧光值反应中性粒细胞内GPX4含量的高低，反映了蛋白水平GPX4的变化。可以看出狼疮患者的GPX4含量明显低于健康人。B图所示为收集A图中12位狼疮患者的临床资料，并根据SLEDAI评分准则进行打分，将得分与GPX4的荧光值做相关分析。SLEDAI得分越高，提示疾病越严重。可以看出，疾病越严重，中性粒细胞内的GPX4含量越低。
图3所示为用qPCR的方法检测中性粒细胞内的GPX4，反映了GPX4在转录水平的变化。可以看出狼疮患者中性粒细胞的GPX4转录水平明显低于正常人。
图4所示为Gpx4基因条件性敲除小鼠基因打靶，以及条件性敲除示意图。
图5所示为GPX4fl/wtLysMcre+小鼠杂交过程示意图。
图6A所示为髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)，用流式细胞术的方法检测中性粒细胞GPX4含量。图6B图所示为在外周血中除了中性粒细胞以外其他有核细胞的GPX4含量。可以看出，GPX4只在GPX4fl/wtLysMcre+小鼠的中性粒细胞中表达下降，表明该模型符合设计要求。
图7所示为髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)的中性粒细胞Lipid-ROS含量对比。Lipid-ROS是铁死亡的标志物，铁死亡发生时Lipid-ROS水平增高。可以看出GPX4fl/wtLysMcre+鼠的Lipid-ROS水平上升。
图8所示为髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)的外周血中性粒细胞比例对比。可以看出，GPX4fl/ wtLysMcre+小鼠的中性粒细胞比例明显降低。
图9所示为髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)的皮损对比。GPX4fl/fl小鼠皮毛一直完好(图中为6月大小)，而GPX4fl/wtLysMcre+小鼠在三月大小时已出现明显皮损，这些小鼠在六月大小时皮损面积进一步扩大。
图10所示为髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)的肾脏免疫复合物沉积的情况。处死4月大小的小鼠，取其中一个肾脏，按照纵切面切出6微米厚的片子，进行染色，粉色代表IgG，黄色代表IgM，绿色代表C1q，蓝色代表DNA，Merge代表将所有颜色重叠在一起。可以看出，GPX4fl/wtLysMcre+小鼠明显增加了肾小球IgG、IgM、C1q的沉积。
图11所示为在髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl),在4月大小时，肾小球的HE染色代表图。GPX4fl/ wtLysMcre+小鼠肾小球内细胞增生，结构紊乱。
图12所示为在髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl),在4月大小时，尿蛋白的检测。并区分雌鼠和雄鼠。纵坐标为BCA蛋白定量法在562nm测出的OD值，数值越大代表蛋白含量越多。可以看出，GPX4fl/wtLysMcre+小鼠蛋白尿升高。(GPX4fl/fl:雌鼠＝15只,雄鼠＝4只,GPX4fl/wtLysMCre+:雌鼠＝20只,雄鼠＝11只)。
图13所示为在髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)血浆中抗自身双链DNA抗体(anti-dsDNA)含量的对比。收集3月大小和6月大小GPX4fl/wtLysMcre+小鼠内眦血，并提取血浆，以6月大小的GPX4fl /fl小鼠作为对照。通过ELISA的方法检测抗自身双链DNA抗体。并区分了雌鼠和雄鼠。可以看到GPX4fl/wtLysMcre+小鼠抗自身双链DNA抗体不断增加。(GPX4fl/fl:雌鼠＝6,雄鼠＝6,GPX4fl/wtLysMCre+:雌鼠(3月/6月)＝16只/29只,雄鼠(3月/6月)＝7只/7只)。
图14所示为在髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)血浆中补体C3(Complement 3)含量的对比。收集3月大小和6月大小GPX4fl/wtLysMcre+小鼠内眦血，并提取血浆，以6月大小的GPX4fl/fl小鼠作为对照。通过ELISA的方法检测补体C3，并区分了雌鼠和雄鼠，可以看到GPX4fl/wtLysMcre+小鼠补体C3不断下降(GPX4fl/fl:雌鼠＝6,雄鼠＝6,GPX4fl/wtLysMCre+:雌鼠(3月/6月)＝16只/29只,雄鼠(3月/6月)＝7只/7只)。
图15所示为在髓系来源的中性粒细胞中条件性敲低GPX4的小鼠(GPX4fl/wtLysMcre+)和没有敲低GPX4的对照鼠(GPX4fl/fl)血浆中炎症因子含量的对比。收集3月大小和6月大小GPX4fl/wtLysMcre+小鼠内眦血，并提取血浆，以6月大小的GPX4fl/fl小鼠作为对照。通过多因子试剂盒的方法检测炎症因子一型干扰素α(IFN-α)，白介素6(IL-6)，二型干扰素γ(IFN-γ)，白介素10(IL-10)。并区分了雌鼠和雄鼠。可以看到GPX4fl/wtLysMcre+小鼠补体炎症因子不断增加(GPX4fl/fl:雌鼠＝6,雄鼠＝6,GPX4fl/wtLysMCre+:雌鼠(3月/6月)＝16只/29只,雄鼠(3月/6月)＝7只/7只)。所有散点图的数据通过平均值±SD的方式显示，*p<0.05,**p<0.01,***p<0.001,****p<0.0001,ns p>0.05。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1 GPX4蛋白在狼疮患者中性粒细胞中的表达含量鉴定
中性粒细胞的分离：使用K2E(EDTA)管(BD Vacutainer公司)收集健康对照和SLE患者的外周血。为了分离中性粒细胞，先用磷酸缓冲盐溶液将外周血等比稀释备用，在15毫升离心管中倒入适量Ficoll-Hypaqu分离液，再将稀释的外周血平铺在分离液上层，注意不破坏分离液界面，然后进行离心分离。转速500G，升速最大，降速最小，离心20分钟，室温。离心完成后，用吸管吸取红细胞上层的白色沉淀，即为中性粒细胞。下一步用裂解缓冲液(BD)裂解红细胞，再次离心，转速500G，10分钟，保留离心管底部的中性粒细胞。使用CD16(Biosciences公司)和CD11b(Biolegend公司)抗体通过流式细胞术对该方法进行验证，中性粒细胞的纯度大于98％。取分离好的中性粒细胞立即进行流式细胞术的染色分析，或者储存于-80度冰箱保存。
免疫印记：使用RIPA缓冲液(Huaxingbio公司)配合蛋白酶抑制剂和磷酸酶抑制剂混合物(Thermo Fisher Scientific公司)在冰上裂解中性粒细胞。使用BCA蛋白质测定试剂盒(Thermo Fisher Scientific公司)确定裂解物的蛋白质浓度。将RIPA裂解液与loading buffer在95℃煮沸10分钟，然后进行电泳，再转移至PVDF膜(Biorad公司)。封闭膜，然后与抗GPX4(1：1000)抗体在4℃下过夜。洗涤膜，并与抗兔IgG-HRP(1：5000)一起温育1小时。用蛋白质印迹检测系统Tanon-5200(Bio-Tanon，公司)将蛋白条带可视化。灰度值分析通过ImageJ(1.50g，NIH)软件进行。根据制造商的说明，通过细胞质和核提取试剂盒(SC-003，Invent)分离并确定细胞的核含量。结果显示，狼疮中性粒细胞GPX4表达下降，在治疗后回复(图1)。
流式细胞术：用Cytofix/Cytoperm(BD公司)溶液固定中性粒细胞以检测细胞内GPX4的表达，用抗人GPX4抗体(Abcam公司)对细胞进行染色。孵育2小时后，用PBS将细胞清洗，再用驴抗兔IgG Alexa Fluor 647(ab150075，Abcam)染色半小时，继续清洗细胞，最后用流式机器检测。
SLEDAI评分：如患者出现以下症状，每一条记8分：癫痫发作，精神症状，器质性脑病，视觉受损，颅神经异常，狼疮性头痛，脑血管意外，脉管炎；如患者出现以下症状，每一条记4分：关节炎，肌炎，管型尿，血尿，蛋白尿，脓尿；如患者出现以下症状，每一条记2分：脱发，新出现皮疹，粘膜溃疡，胸膜炎；如患者出现以下症状，每一条记1分：发热，血小板降低，白细胞减少。将得分累积，0-4分代表病情基本无活动，5-9分代表轻度活动，10-14分代表中度活动，≥15分代表重度活动。结果显示，狼疮患者中性粒细胞GPX4表达下降，GPX4含量与SLEDAI评分负相关(图2)。
实时定量PCR：用TRIzol(Invitrogen公司)提取中性粒细胞RNA，并使用PrimeScriptTM RT Master Mix(Takara Bio公司)转化为cDNA。使用SYBR Premix Ex TaqTMII(Takara Bio公司)进行实时定量PCR。根据△△Ct计算方法将相对RNA表达水平相对于GAPDH mRNA标准化。
使用的引物如下：
GAPDH
正向：5'-TCAACGACCACTTTGTCAAGCTCA-3'
反向：5'-GCTGGTGGTCCAGGGGTCTTACT-3'，
GPX4
正向：5'-AAGTGGATGAAGATCCAACCCAAG-3'
反向：5'-GGGGCAGGTCCTTCTCTATCA-3'，
结果显示，狼疮患者中性粒细胞GPX4在转录水平下降(图3)。
通过对中性粒细胞GPX4的鉴定，我们发现狼疮患者GPX4在蛋白水平和转录水平都低于正常人，并且这种降低与疾病活动度SLEDAI评分负相关，即：GPX4表达量越低，SLEDAI评分越高，狼疮病情越重。因此，推测GPX4下降可能是参与狼疮发病的重要因素，下一步，我们通过构建在中性粒细胞条件性敲低GPX4的小鼠模型来验证这种推测。
实施例2 GPX4基因在中性粒细胞条件性敲低小鼠制作实验方案
基因信息:GPX4基因位于小鼠第10号染色体，GPX4基因编码蛋白的主要有两个剪接体：GPX4-201、GPX4-202。这两个剪接体都有7个外显子，二者仅1号外显子不同。
制作原理:使用cas9/gRNA系统将2个loxp元件分别敲入到Gpx4基因的2号外显子上游及4号外显子下游，即用2个loxp锚定Gpx4基因的2～4号外显子。该小鼠与组织特异表达cre的小鼠杂交，可以在表达cre酶的组织器官特异性删除Gpx4基因的2～4号外显子，这3个外显子长度一共392bp，非3的整数倍，删除后将造成Gpx4基因2种剪接体都发生移码突变，导致Gpx4基因敲除，从而得到条件性敲除Gpx4基因的小鼠。
Gpx4基因条件性敲除小鼠基因打靶，以及条件性敲除示意图如下图4所示。
gRNA的设计、体外转录和纯化选择脱靶概率低的sgRNA。通过序列比对在预置loxp的靶点附近设计2～3条gRNA。测定切割效率后，选择切割活性最高的gRNA，用于后续的实验中。以sgRNA-Vector为模板PCR扩增出Gpx4-sgRNA的DNA片段，然后胶回收作为sgRNA体外转录的模板。再经过sgRNA体外转录与纯化回收，分装保存于-80℃冰箱备用。
打靶载体的设计和构建根据选定的sgRNA，设计打靶载体，包含同源臂及loxp序列。构建完成后，经酶切、纯化后，与sgRNA、Cas9-mRNA共同显微注射受精卵。
显微注射将纯化好的sgRNA、Cas9-mRNA共同注射到C57小鼠胚胎中，注射后将胚胎移植到代孕受体小鼠的输卵管内。
小鼠鉴定:胚胎移植后21天小鼠出生，出生2周左右完成基因型鉴定，鉴定出GPX4突变型。
中性粒细胞Cre
用于中性粒细胞条件敲除的cre鼠有Mrp8Cre和LysMcre，本实验选用LysMcre，购买自Jackson lab。LysMcre敲入等位基因具有一个核定位的Cre重组酶，该酶插入了溶菌酶2基因(Lyz2)的第一个编码ATG中。既消除了内源性Lyz2基因的功能，又将NLS-Cre表达置于内源性Lyz2启动子/增强子元件的控制之下。这些LysMcre小鼠可用于Cre-lox研究骨髓细胞谱系(粒细胞，单核细胞)和先天免疫应答。
杂交策略如图5所示。通过对回交后的小鼠进行鉴定，鉴定出GPX4是杂合，LysmCre是纯合的GPX4fl/wt LysMcre+/+小鼠，最终获得GPX4在髓系来源中性粒细胞单倍体缺陷的小鼠。该小鼠在髓系来源的中性粒细胞中敲低了GPX4的表达，促使中性粒细胞发生铁死亡。
实施例3中性粒细胞条件性敲低小鼠表型鉴定
GPX4fl/fl和Gpx4fl/wtLysmCre+小鼠无需额外干预即可。每月一次收集尿液，收集尾静脉血，以及拍照。可以看到，Gpx4fl/wtLysmCre+小鼠三月开始出现严重的皮损。图中所示小鼠均为雌鼠，Gpx4fl/wtLysmCre+雌鼠出现皮损的概率为88.9％，雄鼠为5.6％，明显存在性别差异(图9)。
流式细胞术：通过PE偶联抗小鼠Ly-6G(Biolegend)，APC偶联抗小鼠/人CD11b(Biolegend)和FITC偶联抗小鼠CD45(103108，Biolegend)来鉴定小鼠外周血中性粒细胞比例。CD45在所有免疫细胞表面表达，而中性粒细胞同时还表达Ly-6G和CD11b，因此，CD45+Ly-6G+CD11b+即为中性粒细胞。结果显示，Gpx4fl/wtLysmCre+小鼠外周血中性粒细胞比例下降(图8)。为了检测细胞内GPX4的表达，用Cytofix/Cytoperm(BD)固定细胞，用FITC偶联抗小鼠Ly-6G&Ly-6C(BD)和抗人GPX4抗体(ab125066，Abcam)对细胞进行染色。温育2小时后，洗涤细胞，然后用驴抗兔IgG Alexa Fluor 647(ab150075，Abcam)染色，在清洗细胞并上机分析。Ly-6G&Ly-6C可以标记出小鼠中性粒细胞，通过流式圈门，分析这群细胞的GPX4荧光强度。结果显示，Gpx4fl/wtLysMCre+小鼠外周血中性粒细胞GPX4表达下降(图6)。为了测量脂质ROS，将新鲜分离的小鼠中性粒细胞洗涤两次，并与1mM C11-BODIPY(581/591)(Invitrogen)孵育15分钟。然后，中性粒细胞洗涤两次并重悬于磷酸盐缓冲盐水(PBS)中。通过使用流式细胞术测量C11-BODIPY在488nm的荧光变化来评估脂质-ROS。结果显示，Gpx4fl/wtLysMCre+小鼠外周血中性粒细胞lipid-ROS增加(图7)。
酶联免疫吸附测定：用测定缓冲液将小鼠血清以1：100稀释用于检测抗dsDNA抗体，并用1：25000稀释以检测补体C3。按照试剂制造商提供的说明书，使用小鼠抗dsDNA IgGELISA试剂盒(5120，Alpha Diagnostic公司)和补体3ELISA试剂盒(6270，AlphaDiagnostic公司)。)进行测定。结果表明，Gpx4fl/wtLysMCre+小鼠出现抗dsDNA IgG阳性(图13)和补体降低(图14)。
免疫组化：肾脏取自小鼠，固定在4％多聚甲醛中，包埋在石蜡中，并用苏木精和曙红(H&E)染色。结果表明，Gpx4fl/wtLysMCre+小鼠出现肾小球肾炎(图11)。
蛋白尿测定：使用BCA蛋白质测定试剂盒(23225，赛默飞世尔科技)检测尿液中蛋白质的浓度。结果表明，Gpx4fl/wtLysMCre+小鼠出现蛋白尿升高(图12)。
免疫荧光：小鼠的第二个肾脏在-80℃下冷冻在OCT化合物(Tissue-Tek)中，用于评估免疫复合物的沉积。先将冷冻的肾脏切出6μm厚的纵切面，再用封闭缓冲液(100mMTris-HCl，pH 8.0、0.3％Triton X-100、2％BSA和50μg/ml山羊非特异性IgG)处理6μm速冻肾组织切片1小时。然后，将组织用Alexa Fluor 594山羊抗小鼠IgG(1：200)，山羊抗小鼠IgM Alexa Fluor 647(1：200)或兔抗C1q抗体(1：200)加山羊抗兔IgG Alexa Fluor 488(1：500)进行染色，洗片三次，晾干。在切片上滴加含有抗猝灭剂的DAPI试剂(Solarbio公司)中，最后盖玻片封片备用。用A1 HD25/A1R HD25尼康共聚焦激光显微镜(日本尼康)观察所有载玻片。使用Photoshop CC 2017和Illustrator CC 2017(Adobe)优化图像。结果表明，Gpx4fl/wtLysMCre+小鼠出现肾脏免疫复合物IgG、IgM、C1q的沉积(图10)。
多因子检测：按照制造商的说明，使用LEGENDplexTM小鼠Th细胞因子检测仪(Biolegend公司)测量小鼠血浆中的细胞因子。使用LEGENDplexTM数据分析软件(8.0版)进行数据分析。结果表明，Gpx4fl/wtLysMCre+小鼠出现炎性因子增加(图15)。
总结，GPX4fl/wtLysMCre+小鼠的中性粒细胞中表现出GPX4蛋白降低和lipid-ROS升高，以及外周血中性粒细胞的百分比明显降低。值得注意的是，小鼠在第3个月出现了典型的狼疮皮肤病变(雌性小鼠出现概率为88.9％，雄性小鼠为5.6％)，第4个月出现严重肾小球肾炎伴有明显的蛋白尿以及IgG，IgM和C3的肾小球沉积，以及在6个月内抗dsDNA滴度逐渐升高和血清C3逐渐降低。此外，在Gpx4fl/wtLysMCre+小鼠中发现包括IFN-α和IL-6在内的血清炎症因子水平明显高于对照组，这表明在Gpx4fl/wt LysMCre+小鼠体内存在炎症水平增高的情况，以上特点类似于人类SLE的自然过程。综上所述，这些结果表明中性粒细胞的GPX4单倍体不足会诱发狼疮的发生，因此是一种新型狼疮鼠模型。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国医学科学院北京协和医院
<120> 一种系统性红斑狼疮的动物模型的构建方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213> 小鼠(Mus musculus)
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tcaacgacca ctttgtcaag ctca 24
<210> 2
<211> 23
<212> DNA
<213> 小鼠(Mus musculus)
<400> 2
gctggtggtc caggggtctt act 23
<210> 3
<211> 24
<212> DNA
<213> 小鼠(Mus musculus)
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<211> 21
<212> DNA
<213> 小鼠(Mus musculus)
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ggggcaggtc cttctctatc a 21
Claims (3)
1.一种新型系统性红斑狼疮的动物模型的构建方法，其包括如下步骤：
1）利用Crispr/Cas9技术，在小鼠谷胱甘肽过氧化物酶4（GPX4）基因2号外显子上游及4号外显子下游插入loxP位点，构建亲代鼠1，所述亲代鼠1为C57/B6小鼠；
2）亲代鼠2为包含中性粒细胞特异的Cre小鼠，所述的包含中性粒细胞特异的Cre小鼠为LysMcre小鼠；
3）将亲代鼠1和亲代鼠2进行杂交，获得所述杂合的GPX4基因和包含所述Cre基因的小鼠，该小鼠自发出现狼疮表型。
2.如权利要求了1所述的构建方法，其特征在于，还包括将获得的所述杂合的GPX4基因和包含所述Cre基因的小鼠再与亲代鼠2进行回交，筛选获得所述杂合的GPX4基因和包含所述Cre基因的小鼠，该小鼠自发出现狼疮表型。
3.如权利要求1或2所述的构建方法，其特征在于，亲代鼠1具体构建过程为：以sgRNA-Vector为模板PCR扩增出Gpx4-sgRNA的DNA片段，然后胶回收作为sgRNA体外转录的模板，再经过sgRNA体外转录与纯化回收；
根据选定的sgRNA，设计打靶载体，包含同源臂及loxp序列，构建完成后，经酶切、纯化后，与sgRNA、Cas9-mRNA共同显微注射C57小鼠胚胎中，注射后将胚胎移植到代孕受体小鼠的输卵管内。
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