CN114788835B - Foxp3阳性巨噬细胞在制备治疗急性脑梗死药物中的应用 - Google Patents
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Abstract
本发明公开了Foxp3阳性巨噬细胞在制备治疗急性脑梗死药物中的应用。本发明首次通过单细胞测序、免疫荧光、WB、流式细胞术等多重方法在脑卒中的病灶中发现Foxp3阳性的巨噬细胞。对Foxp3阳性的巨噬细胞进行功能分析我们发现Foxp3阳性的巨噬细胞吞噬死亡神经元、受损的髓鞘以及浸润的中性粒细胞的能力增强。在巨噬细胞中特异性地敲除Foxp3将下调巨噬细胞对死亡神经元、受损髓鞘的吞噬，下调巨噬细胞清除神经炎症的能力，最终加重急性脑卒中病情。表明Foxp3阳性巨噬细胞可应用于制备治疗急性脑梗死和/或急性无菌性炎症的药物。
Description
技术领域
本发明属于生物医学技术领域，具体涉及Foxp3阳性巨噬细胞在制备治疗急性脑梗死药物中的应用。
背景技术
作为调节性T细胞的特异性生物学标志物，Foxp3在调节性T细胞发育与机体免疫抑制中发挥不可忽视的关键作用。既往的研究报道Foxp3阳性的调节性T细胞参与炎症抑制、免疫耐受、肿瘤转移、自身免疫性疾病等病理生理过程。
在脑血管疾病中，急性缺血性脑卒中(acute ischemic stroke,AIS)是一种备受关注的疾病，也是造成我国致死致残率极高的一种疾病。从病理生理学角度上看，脑部的急性缺血可以诱导神经元死亡并继发导致缺血半暗带神经组织损伤和病变扩大的炎症反应。既往研究表明卒中后5-7天Foxp3阳性的调节性T细胞从外周浸润到卒中病灶，发挥抗炎与组织修复的作用。同时，卒中后静脉输注Foxp3阳性的调节性T细胞可以减小卒中病灶，促进白质修复。
越来越多的证据表明，Foxp3的表达不仅限于T细胞，并在可以存在于疾病背景下的髓系细胞(包括树突状细胞和嗜酸性粒细胞)。然而，对于Foxp3在巨噬细胞中的表达，自2011年首次报告以来一直存在争议。一些学者认为，采用流式细胞术在巨噬细胞中检测到的Foxp3蛋白信号是“粘性”细胞的自发荧光干扰造成的。另一方面，最新的研究也报道了通过RNA测序证实RENCA肿瘤中Foxp3阳性的巨噬细胞的存在，并且该研究小组还进一步在小鼠中鉴定了Foxp3的第四个转录变体。目前，关于Foxp3在巨噬细胞中的功能仍有待进一步确定。
发明内容
我们首次发现急性缺血性脑卒中的病灶中Foxp3阳性的巨噬细胞浸润。进一步对Foxp3阳性的巨噬细胞进行功能分析我们发现Foxp3阳性的巨噬细胞的吞噬死亡神经元、受损的髓鞘以及浸润的中性粒细胞的能力增强。在巨噬细胞中特异性地敲除Foxp3将下调巨噬细胞对死亡神经元、受损髓鞘的吞噬，下调巨噬细胞清除神经炎症的能力，最终加重急性缺血性脑卒中病情。
因此，本发明的第一个目的是提供Foxp3阳性巨噬细胞在制备治疗急性脑梗死药物中的应用。
本发明的第二个目的是提供Foxp3阳性巨噬细胞在制备治疗急性无菌性炎症药物中的应用。
本发明的第三个目的是提供表达Foxp3的巨噬细胞作为靶点用于筛选治疗或预防急性脑梗死药物的用途。
本发明的第四个目的是提供表达Foxp3的巨噬细胞作为靶点用于筛选治疗或预防急性无菌性炎症药物的用途。
本发明的第五个目的是提供促进巨噬细胞表达Foxp3的试剂在制备治疗急性脑梗死药物中的应用。
本发明的第六个目的是提供促进巨噬细胞表达Foxp3的试剂在制备治疗急性无菌性炎症药物中的应用。
本发明的第七个目的是提供一种治疗急性脑梗死药物，该药物包含Foxp3阳性巨噬细胞和/或促进巨噬细胞表达Foxp3的试剂。
本发明的第八个目的是提供一种治疗急性无菌性炎症药物，该药物包含Foxp3阳性巨噬细胞和/或促进巨噬细胞表达Foxp3的试剂。
本发明的有益效果：
1、本发明首次通过单细胞测序、免疫荧光、WB(Western blotting)、流式细胞术等多重方法在脑卒中的病灶中发现Foxp3阳性的巨噬细胞，排除既往单纯流式细胞术检测巨噬细胞Foxp3蛋白的自发荧光干扰，填补了以往研究对髓系细胞Foxp3的表达及功能研究的缺陷，为急性脑梗死及其他急性无菌性炎症的治疗提供了新靶点。
2、本发明首次在体内实验中通过在巨噬细胞中特异性敲除Foxp3，从而验证Foxp3阳性巨噬细胞的抗炎、强吞噬能力与神经修复功能。
附图说明
图1是Foxp3阳性巨噬细胞改善中风后神经炎症。
(A)WT C57/BL6雄性小鼠接受60分钟的tMCAO。收集中风小鼠的冠状脑切片用于免疫荧光染色和共聚焦显微镜分析。显示tMCAO后3d梗塞周围区域中用Foxp3(绿色)、Iba1(小胶质细胞/巨噬细胞标记物，红色)和CD3(T细胞标记物，蓝色)标记的切片的代表性图像。白色虚线勾勒出Iba1+细胞聚集的中风梗塞。实验重复3次。(B)在tMCAO后1-14天处死具有C57/BL6背景的Foxp3-Egfp小鼠。收集中风小鼠的冠状脑切片用于免疫荧光染色和共聚焦显微镜分析。显示梗塞周围区域用Iba1(小胶质细胞/巨噬细胞标记物，红色)标记的切片的代表性图像。(C)通过流式细胞术检测CD11b+F4/80+巨噬细胞中Foxp3的表达。荧光色素减一(FMO，无Foxp3染色)染色用作Foxp3+巨噬细胞门控的阴性对照。CD11b+F4/80+巨噬细胞中Foxp3+细胞的百分比(平均值±标准偏差)显示在流式图的右侧。
图2是Foxp3阳性巨噬细胞吞噬死亡神经元、受损的髓鞘以及浸润的中性粒细胞的能力增强。
(A-C)在tMCAO后第5天处死6只Foxp3-Egfp小鼠。来自同侧大脑的CD45+CD11b+CD3-F4/80+巨噬细胞用流式细胞术分离，并进行10×单细胞RNA测序(scRNAseq)。总共扫描了22908个细胞，鉴定了1474个表达Foxp3 mRNA的巨噬细胞。(A)单独的巨噬细胞中Foxp3表达的tSNE图。(B)比较了具(+)或不具(-)Foxp3 mRNA表达的巨噬细胞的基因表达谱。左图：火山图显示与没有Foxp3 mRNA表达的巨噬细胞相比，表达Foxp3的巨噬细胞中上调和下调的基因。部分吞噬作用相关基因被指定。右图：指示的吞噬作用相关基因的定量。与Foxp3-巨噬细胞相比，**P<0.01，***P<0.001；通过t检验(中位数和四分位数与小提琴图一起显示)。(C)表达Foxp3的巨噬细胞中上调基因的GO-BP富集。(D)在tMCAO后第5天处死野生型C57/BL6小鼠并进行流式细胞术分析CD45+CD11b+CD3-F4/80+巨噬细胞中的NeuN+、O4+和Ly6G+细胞，分别被鉴定为吞噬死亡神经元、髓鞘碎片和死亡中性粒细胞的巨噬细胞。N＝7。与Foxp3-巨噬细胞相比，*P<0.05，***P<0.001；通过t检验(平均值±标准差)。(E-F)Foxp3-Egfp小鼠在tMCAO后第5天处死。对冠状脑切片进行Iba1(红色)和NeuN(蓝色，E)或MBP(蓝色，F)的免疫标记。白色箭头强调Iba1+细胞吞噬了表达Foxp3-EGFP的神经元(E)或髓鞘碎片(F)。白色箭头显示Iba1+细胞没有Foxp3-EGFP表达，也没有死亡神经元(E)或髓鞘碎片(F)的吞噬作用。(G)Foxp3 CKO小鼠和Foxp3 WT同窝小鼠在最后一次他莫昔芬注射后5-7天接受tMCAO，并在中风后5天处死。用流式细胞术分析脑细胞。CD45+F4/80+巨噬细胞中的NeuN+、O4+和Ly6G+细胞分别被鉴定为吞噬死亡神经元、髓鞘碎片或死亡中性粒细胞的巨噬细胞。每组N＝4。*P<0.05，***P<0.001，与Foxp3 WT相比；通过t检验(平均值±标准差)进行统计分析。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1、急性缺血性脑卒中的病灶中Foxp3阳性的巨噬细胞浸润
为了确定AIS后巨噬细胞中Foxp3的表达，我们对雄性C57/BL6野生型(WT)小鼠进行60分钟的短暂大脑中动脉闭塞(tMCAO)造模，具体方法如下：取9周龄正常饲养的C57/BL6雄性小鼠以5％的异氟烷诱导麻醉后，1.5％的异氟烷维持麻醉，将小鼠固定于手术台上，消毒后颈前正中切开皮肤，显微镜下暴露右侧颈总动脉、颈内动脉和颈外动脉，结扎颈外动脉、颈总动脉，在颈外动脉距颈总动脉分叉部约1-2mm处剪一小口，经该切口将特制硅胶线栓插入颈内动脉，阻塞大脑中动脉起始部。1h后拔出线栓，恢复脑血流灌注。麻醉期间保持小鼠体温在37.0℃，术后单笼喂养，自由进水进食。假手术组(Sham)小鼠仅在显微镜下暴露颈总动脉、颈内动脉和颈外动脉后缝合皮肤，术后处理同tMCAO小鼠。在造模3天后以CO2窒息法处死小鼠后，用0.9％NaCl溶液从左心室行全身灌注以去除血液，再以4％多聚甲醛灌注小鼠，取脑，以4％多聚甲醛浸泡脑组织过夜，再以30％蔗糖的PBS溶液脱水，直至脑组织沉至糖溶液底部。取固定脱水后脑组织，行25μm冰冻切片，浸泡于冰冻切片固定液，储存于-20℃。将冰冻切片以1％Triton的PBS溶液在室温破膜20min，以PBS洗涤后，用5％正常驴血清+0.3％Triton+PBS溶液室温孵育1h，再予兔抗小鼠CD3单克隆抗体(1:500)+山羊抗小鼠Iba1单克隆抗体(1:500)+小鼠抗小鼠Foxp3单克隆抗体(1:500)+2％驴血清+0.3％Triton+PBS溶液4℃孵育过夜。经PBS洗涤后，用驴抗兔的Alexa Fluor 405荧光二抗(1:1000)+驴抗山羊的Cy3荧光二抗(1:1000)+驴抗小鼠的Alexa Fluor 488的荧光二抗(1:1000)+0.3％Triton+PBS溶液室温孵育1h，经PBS洗涤后用封片液封片。使用共聚焦显微镜在大脑半球缺血半暗带观察到Foxp3表达(图1A)。我们注意到Foxp3主要与Iba1(小胶质细胞和巨噬细胞的标志物)共定位，但不与CD3(T细胞标志物)共定位。在Iba1+Foxp3+细胞中，Foxp3蛋白有的聚集成细胞器样结构(图1A，细胞a)，有的分散在细胞质(图1A，细胞b)和细胞核(图1A，细胞c)中。
为了确定Foxp3阳性的巨噬细胞在急性缺血性脑卒中后浸润到脑实质的时间，我们对具有C57/BL6背景的Foxp3-Egfp(绿色)(The Jackson Laboratory,006772)小鼠进行tMCAO术，并在1-14天的不同时间点处死。对小鼠脑片进行免疫荧光染色(Iba1，红色)，步骤同前，并用共聚焦显微镜分析。我们发现Foxp3-EGFP+巨噬细胞主要在中风后5-7d浸润到梗塞周围区域(图1B)。
同时，我们通过流式细胞术进一步证实Foxp3-EGFP+阳性的巨噬细胞在急性缺血性卒中后浸润到脑实质的情况。在MCAO术后1天、3天、5天、7天及14天，取各组小鼠脑组织，分为梗死侧和非梗死侧大脑半球，剪碎后的脑组织置于1mL的0.25％胰酶中，在37℃消化20min，获得脑组织匀浆，进而用密度梯度离心法分离脑髓鞘，具体方法为：在1mL脑组织匀浆中加入6mL的0.15M NaCl溶液及3mL 100％Percoll，配置成30％Percoll的脑组织匀浆；另以100％Percoll及0.15M NaCl溶液配制成70％Percoll，在15mL EP管内将30％Percoll的脑组织匀浆置于3mL 70％Percoll上，形成明显的密度梯度分层，于水平离心机上18℃中以800g(低升降速)离心25min，将最上层髓鞘去除后，得到脑组织单细胞悬液。在得到单细胞悬液后，进行以下染色步骤：1)以PBS洗涤单细胞(x2)；2)对细胞进行Fc段抗体阻断(室温，15min)；3)用PBS溶解针对表面抗原的荧光抗体混合液，4℃染色30min；4)以PBS洗涤细胞(x2)；5)用细胞破膜固定液对细胞进行固定并破膜(室温，30min)6)用破膜液洗涤细胞(x2)；7)用破膜液溶解针对胞内及核内抗原的荧光抗体混合液，室温染色20min；8)用破膜液洗涤细胞(x2)；9)用PBS重悬细胞，以FACSCalibur BD Bioscience流式细胞仪检测样品，并以Flow Jo 10.0v软件进行数据分析。同样的，我们发现Foxp3-EGFP+巨噬细胞主要在中风后5-7d浸润到梗塞周围区域(图1C)。
实施例2、Foxp3信号定义了一个独特的巨噬细胞亚群，在缺血性脑梗死病灶中具有超强的细胞吞噬能力
为了探索Foxp3+巨噬细胞是否代表AIS中功能不同的巨噬细胞亚群，我们对雄性C57/BL6野生型(WT)小鼠进行60分钟的短暂大脑中动脉闭塞(tMCAO)造模，并在造模5天后以CO2窒息法处死小鼠，用0.9％NaCl溶液从左心室行全身灌注以去除血液，分离梗死侧大脑半球并制备单细胞悬液(方法同前)，进行流式表面抗体染色(方法同前)，通过流式细胞分选仪分选出CD45+CD11b+CD3-F4/80+的巨噬细胞，并进行单细胞RNA测序(scRNAseq)。如图2A所示，在扫描到的22908个巨噬细胞中，1474个细胞(确定为Foxp3+巨噬细胞)中检测到Foxp3 mRNA表达。我们关注到Foxp3+巨噬细胞的转录谱，发现吞噬相关基因显着上调，包括Trem2、Fcgr1、Fcgr2b、Fcgr3、Rock1、Mertk、Cd14和Lrp1(图2B)。基因本体(GO)分析进一步表明差异表达的基因在吞噬作用(如调节吞噬作用，GO：0050764)和内吞物分解代谢过程(如调节自噬，GO：0010506)中富集，表明Foxp3+巨噬细胞可能积极参与清除中风病变中的死细胞和碎片(图2C)。
为了研究Foxp3在巨噬细胞吞噬中的作用，我们对雄性C57/BL6野生型(WT)小鼠进行60分钟的短暂大脑中动脉闭塞(tMCAO)造模，并在造模5天后以CO2窒息法处死小鼠，用0.9％NaCl溶液从左心室行全身灌注以去除血液，分离梗死侧大脑半球并制备单细胞悬液(方法同前)，进行流式抗体染色(方法同前)，以CD45+CD11b+CD3-F4/80+标记浸润到脑实质的巨噬细胞，并分为Foxp3+和Foxp3-的细胞，然后进一步分析这两群细胞对死亡神经元(标记为NeuN+)、髓鞘(标记为O4+)和中性粒细胞(标记为Ly6G+)的吞噬情况。我们发现Foxp3+巨噬细胞内的NeuN+、O4+或Ly6G+细胞数量明显高于Foxp3-的巨噬细胞(图2D)。同时，我们对大脑中动脉闭塞(tMCAO)造模的雄性C57/BL6野生型小鼠和具有C57/BL6背景的Foxp3-Egfp(绿色)小鼠进行免疫荧光染色进一步证实Foxp3+巨噬细胞的清除能力是否增强。我们对雄性C57/BL6野生型(WT)小鼠进行60分钟的短暂大脑中动脉闭塞(tMCAO)造模，并在造模5天后以CO2窒息法处死小鼠，对小鼠脑片进行免疫荧光染色(Iba1，红色；Foxp3，绿色；NeuN，MBP，蓝色)，步骤同前，并用共聚焦显微镜分析。我们发现与Foxp3-巨噬细胞(白色箭头)相比，在Foxp3+巨噬细胞(白色箭头)中可以看到与死亡神经元(NeuN+)或髓鞘碎片(MBP+)的共定位增加(图2E-F)，说明Foxp3+巨噬细胞的清除能力较Foxp3-巨噬细胞强。
为了探讨Foxp3对巨噬细胞清除能力的影响，我们构建了Foxp3特异性敲除小鼠。为了有条件地敲除位于X染色质中的Foxp3基因，首先构建Foxp3fl/fl突变小鼠：在Foxp3-204(ENSMUST00000115740.8)的转录本中，外显子II-X包含翻译起始位点ATG。因此，LoxP点被设计为位于Foxp3基因的外显子II-(floxed,fl)的侧翼以破坏Foxp3蛋白质功能。采用成簇的规则间隔短回文重复序列(CRISPR)相关系统(Cas)9(CRISPR/Cas9)来修饰Foxp3基因。简而言之，转录引导RNA(gRNA)并首先在体外构建供体载体。接下来，将Cas9-gRNA供体质粒微注射到C57/BL6小鼠的受精卵中，然后将其移植到替代雌性小鼠(C57/BL6中。Foxp3fl/flF0小鼠根据基因分型与PCR和测序进行选择。然后将Foxp3fl/flF0小鼠与野生型C57/BL6小鼠杂交以产生稳定的Foxp3fl/+F1小鼠。后代在使用前与健康的野生型C57/BL6小鼠回交六代，以避免遗传异质性对动物脑缺血易感性的潜在影响。然后将Foxp3fl/fl小鼠培育成半合子LyzmCre-ERT2小鼠(C57/BL6背景；江苏集萃药康公司提供)以产生携带半合子LyzmCre-ERT2转基因和floxed Foxp3等位基因(LyzmCre-ERT2Foxp3fl/Y)的雄性小鼠——他莫昔芬诱导的Foxp3条件性敲除(CKO)小鼠并在研究中使用。Foxp3 CKO小鼠直到他莫昔芬给药才显示出明显的表型。当暴露于他莫昔芬时，可以预期在所有表达Cre的细胞中Cre诱导的Foxp3丢失。同时，基因型为LyzmCre-ERT2Foxp3fl/Y的Foxp3 CKO小鼠的同窝小鼠注射他莫昔芬并设置为Foxp3WT对照。为了诱导Cre表达，对8-12周龄的小鼠进行了五次腹膜内他莫昔芬注射(75mg/kg，每天)。在最后一次他莫昔芬注射后5-7天对小鼠进行脑缺血建模。
我们对雄性Foxp3 CKO小鼠及Foxp3 WT对照小鼠进行60分钟的短暂大脑中动脉闭塞(tMCAO)造模，并在造模5天后以CO2窒息法处死小鼠，用0.9％NaCl溶液从左心室行全身灌注以去除血液，分离梗死侧大脑半球并制备单细胞悬液(方法同前)，进行流式抗体染色(方法同前)，以CD45+F4/80+标记浸润到脑实质的巨噬细胞，然后进一步分析两组巨噬细胞对髓鞘(标记为O4+或MBP+)和中性粒细胞(标记为Ly6G+)的吞噬情况。我们发现在tMCAO第5天，Foxp3 CKO小鼠大脑中巨噬细胞对髓鞘碎片(O4+或MBP+)和中性粒细胞(LyG+)的吞噬能力受损(图2G)。因此，这些结果表明Foxp3+巨噬细胞的吞噬能力增强。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，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。
Claims (6)
1.Foxp3阳性巨噬细胞在制备治疗急性脑梗死药物中的应用。
2.Foxp3阳性巨噬细胞在制备治疗急性脑梗死导致的无菌性炎症药物中的应用。
3.表达Foxp3的巨噬细胞作为靶点用于筛选治疗或预防急性脑梗死药物的用途。
4.表达Foxp3的巨噬细胞作为靶点用于筛选治疗或预防急性脑梗死导致的无菌性炎症药物的用途。
5.促进巨噬细胞表达Foxp3的试剂在制备治疗急性脑梗死药物中的应用。
6.促进巨噬细胞表达Foxp3的试剂在制备治疗急性脑梗死导致的无菌性炎症药物中的应用。
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