CN114010658B - TREM2hi巨噬细胞在制备治疗心脏功能障碍药物中的应用 - Google Patents
TREM2hi巨噬细胞在制备治疗心脏功能障碍药物中的应用 Download PDFInfo
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- CN114010658B CN114010658B CN202111411702.5A CN202111411702A CN114010658B CN 114010658 B CN114010658 B CN 114010658B CN 202111411702 A CN202111411702 A CN 202111411702A CN 114010658 B CN114010658 B CN 114010658B
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Abstract
本发明公开了TREM2hi巨噬细胞在制备治疗心脏功能障碍药物中的应用,属于医药技术领域。本发明首次公开心脏来源的巨噬细胞中存在一类高表达髓系细胞触发受体2的巨噬细胞亚群,其具有强大的识别并减少线粒体损伤的功能,在维持心脏代谢平衡中起到了重要的作用。因此,TREM2hi巨噬细胞在治疗心脏功能障碍中具有重要的应用价值。
Description
技术领域
本发明涉及医药技术领域,具体涉及TREM2hi巨噬细胞在制备治疗心脏功能障碍药物中的应用。
背景技术
心脏功能障碍是全球范围内一个紧迫的公共健康问题。虽然有相应的治疗方法,但是预后依旧非常差,现有的治疗方法可改善临床表现出的症状,但不能完全解决心肌细胞的功能异常。通过改善心脏微环境,促进心肌细胞修复,治疗心功能障碍或使心功能恢复正常是可能的。因此探究心脏功能障碍的病理生理机制,探寻有效、靶向防治心脏功能障碍的治疗策略是极为关键的。
心脏作为人体新陈代谢最活跃的器官,在所有组织中,其线粒体的含量是最高的,以满足心肌细胞收缩和舒张的巨大能量需求。心脏功能障碍时,线粒体异常不仅使ATP产生减少,还可直接导致心肌细胞损伤和死亡,从而导致疾病进展。异常线粒体是活性氧(ROS)产生的主要来源,可诱导心肌细胞损伤;异常线粒体通过释放细胞色素c到细胞质中,激活半胱天冬酶促进细胞程序性死亡;损伤线粒体促进局部炎症反应,导致心肌细胞炎症损伤。线粒体损伤也与异常的细胞钙稳态、血管平滑肌病理、肌纤维分裂和细胞分化有关,这些都是心功能不全的重要发病机制。因此,维持心肌线粒体数量和质量稳定,对于心脏组织稳态的维持及心脏功能障碍的恢复至关重要。
众所周知,巨噬细胞在心脏功能障碍的发生发展中发挥着重要的作用。心脏损伤初期,大量巨噬细胞等免疫细胞在心肌组织浸润、活化,发挥促炎、吞噬作用。随着病程进展,浸润的巨噬细胞从促炎型巨噬细胞转化为促修复作用的巨噬细胞,修复损伤的心肌组织。但是目前对心脏损伤修复的细胞和分子基础认识还存在一定的盲区。
最新研究发现,在正常情况下,代谢旺盛的心肌细胞通过囊泡的形式排出损伤的线粒体,损伤线粒体在心脏中的堆积及其释放的活性氧、mtDNA 等会加重心肌损伤,而心脏巨噬细胞则通过清除这些含有损伤线粒体的囊泡来维持心脏组织微环境的稳定(José ANicolás-at el.A Network of Macrophage Supports MitochondrialHomeostasis in the Heart,Cell.2020Oct 1;183(1):94-109.e23.)。
巨噬细胞亚群在生理和病理状态下发挥着不同的功能,包括参与炎症调节、病原体清除、免疫监视、心血管发育、电传导和心肌修复等。心脏单细胞测序实验结果揭示了心脏巨噬细胞亚群存在转录差异,证实了它们具有高度异质性和不同的表型及功能。但是,在心脏功能障碍后心脏组织中浸润的不同免疫细胞亚群中,哪些巨噬细胞亚群与心脏功能的恢复和保护密切相关呢?目前尚未有研究报道。
发明内容
本发明的目的在于探究在心脏功能障碍后可用于修复损伤心肌细胞的巨噬细胞亚群,并将其应用到心脏功能障碍的治疗当中。
为实现上述目的,本发明采用如下技术方案:
本发明提供了TREM2hi巨噬细胞在制备治疗心脏功能障碍药物中的应用,所述TREM2hi巨噬细胞为表达髓系细胞触发受体2的巨噬细胞。
本发明研究发现,心脏巨噬细胞中存在一类高表达髓系细胞触发受体2(TREM2)的巨噬细胞亚群,可显著改善心脏功能障碍,实现改善心脏微环境与保护心功能。
具体的,所述TREM2hi巨噬细胞为心脏来源的高表达髓系细胞触发受体2的巨噬细胞。
进一步的,所述TREM2hi巨噬细胞为心脏免疫细胞中一类同时表达CD45、CD11b、F4/80、CD163和TREM2的细胞亚群。
所述TREM2hi巨噬细胞的提取方法包括:首先取供体心脏心室组织,匀浆消化后制得细胞悬液,再利用Percoll法分离得到非心肌细胞混合液(包括免疫细胞、内皮细胞和成纤维细胞),然后往非心肌细胞混合液中加入TREM2一抗,孵育后加入CD45抗体、CD11b抗体、F4/80抗体、CD163抗体和TREM2二抗,孵育,最后利用细胞分选仪分选出活的有核CD45+CD11b+F4/80+TREM2+CD163+细胞,即为所述TREM2hi巨噬细胞。
研究表明,在病理情况下,小鼠心肌细胞释放的损伤线粒体增多,并在心肌间质中大量堆积;TREM2基因缺失的小鼠表现出巨噬细胞吞噬损伤线粒体功能障碍。本发明从健康小鼠心脏中分离提取TREM2hi巨噬细胞,并通过心包腔内注射到心脏功能障碍模型中,对TREM2hi巨噬细胞治疗心脏功能障碍的效果进行了研究,证实其具有较好的治疗效果。
具体的,所述心脏功能障碍表现为心肌细胞释放的损伤线粒体增多。
进一步的,所述心脏功能障碍为脓毒症或心肌梗死导致的心脏功能障碍。
进一步的,所述心脏功能障碍为心力衰竭。
具体的,所述药物通过减少心肌细胞中损伤的线粒体达到改善心脏功能障碍的目的。
研究表明,经TREM2hi巨噬细胞治疗,可以降低心脏功能障碍模型小鼠血清中的肌钙蛋白、乳酸脱氢酶和乳酸水平;显著降低心脏组织中炎症因子如白介素IL-1β、白介素IL-6、肿瘤坏死因子-α(TNF-α)的表达水平以及心房钠尿肽(ANP)和脑钠肽(BNP)水平。
进一步的,所述药物的给药方式为心包腔内注射。研究表明,通过心包腔内注射TREM2hi巨噬细胞,TREM2hi巨噬细胞浸润在心肌细胞周围,对心肌细胞起到保护作用。
进一步的,所述药物包括有效剂量的TREM2hi巨噬细胞和药学上可接受的载体。所述的药学上可接受的载体可以为Matrigel。
本发明具备的有益效果:
本发明首次公开心脏来源的巨噬细胞中存在一类高表达髓系细胞触发受体2的巨噬细胞亚群,其具有强大的识别并清除损伤线粒体的功能,在维持心脏代谢平衡中起到了重要的作用。因此,TREM2hi巨噬细胞在治疗心脏功能障碍中具有重要的应用价值。
附图说明
图1为实施例1中小鼠CLP(盲肠结扎穿孔术)心功能障碍模型后不同时间点心脏指标变化,其中(A)为超声心动图,(B)为心脏射血分数(EF),(C)为心脏缩短分数(FS),(D)为心输出量(CO),(E)为模型后血清肌钙蛋白(cTNI)变化;(F)为血清乳酸脱氢酶(LDH)变化;(G)为心脏组织心房钠尿肽(ANP)mRNA相对变化;(H)为心脏组织脑钠肽(BNP)mRNA相对变化。*表示P<0.05;**表示P<0.01;***表示P<0.001,下同。
图2为实施例1中CLP模型3d后和对照组小鼠心肌间质中损伤线粒体的数量,(A)为电镜下线粒体数量改变,(B)为统计图。
图3为实施例2中野生型C57BL/6和TREM2-/-小鼠的心脏中巨噬细胞吞噬线粒体囊泡的电镜图片。
图4为实施例2中野生型C57BL/6和TREM2-/-小鼠脓毒症后心脏中损伤线粒体清除障碍,(A)为电镜下线粒体数量改变,(B)为统计图。
图5为实施例5中小鼠心脏来源TREM2hi巨噬细胞在注射3d后的心脏组织切片荧光图,蓝色为DAPI,绿色为注射TREM2hi巨噬细胞,左图的标尺为20μm,右图为左图中方框部分放大图,标尺为10μm。
图6为实施例6中CLP(盲肠结扎穿孔术)心功能障碍模型下注射TREM2hi巨噬细胞组和对照组小鼠的超声心动图,其中(A)为未进行心包腔内注射的CLP模型组心超图(Control组);(B)为注射TREM2hi巨噬细胞组小鼠心超图(+TREM2hi);(C)为注射非TREM2hi巨噬细胞组小鼠心超图(+Non-TREM2)。
图7为实施例6中CLP模型下注射TREM2hi巨噬细胞组和对照组小鼠的超声指标分析,其中(A)为心脏射血分数(EF),(B)为心脏缩短分数(FS),(C)为心输出量(CO)。
图8为实施例6中CLP模型下注射TREM2hi巨噬细胞小鼠和对照组小鼠的血清肌钙蛋白(cTNI)、乳酸脱氢酶(LDH)和乳酸水平分析,其中(A)为cTNI,(B)为LDH,(C)为乳酸。
图9为实施例6中CLP模型下注射TREM2hi巨噬细胞小鼠和对照组小鼠的炎症因子mRNA分析,其中(A)为白介素IL-1β,(B)为白介素IL-6,(C)为肿瘤坏死因子-α(TNF-α)。
图10为实施例6中CLP模型下注射TREM2hi巨噬细胞小鼠和对照组小鼠的心脏损伤指标mRNA水平,其中(A)为心房钠尿肽(ANP),(B)为脑钠肽(BNP)。
图11为实施例6中CLP模型下注射TREM2hi巨噬细胞小鼠和对照组小鼠的心脏ATP含量。
图12为实施例6中CLP模型下注射TREM2hi巨噬细胞小鼠和对照组小鼠的心脏电镜下线粒体形态。
图13为实施例7中小鼠心肌梗死模型下注射TREM2hi巨噬细胞组和对照组小鼠的超声心动图,其中(A)为未进行心包腔内注射的小鼠心肌梗死模型组心超图(Control组);(B)为注射TREM2hi巨噬细胞组小鼠心超图(+TREM2hi);(C)为注射非TREM2hi巨噬细胞组小鼠心超图(+Non-TREM2)。
图14为实施例7中小鼠心肌梗死模型下注射TREM2hi巨噬细胞组和Non-TREM2组小鼠的超声指标分析,其中(A)为心脏射血分数(EF),(B)为心脏缩短分数(FS),(C)为心输出量(CO)。
具体实施方式
下面结合具体实施例对本发明做进一步说明。以下实施例仅用于说明本发明,不用来限制本发明的适用范围。在不背离本发明精神和本质的情况下,对本发明方法、步骤或条件所做的修改或替换,均属于本发明的范围。
下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
野生型C57BL/6购于上海斯莱克实验动物公司;
TREM2-/-小鼠由圣路易斯华盛顿大学,Marco Colonna教授赠送;
TREM2一抗:Rat anti Human/Mouse TREM2 Antibody(R&D Systems);
TREM2二抗:Donkey anti-Rat IgG(H+L)Highly Cross-Adsorbed SecondaryAntibody,Alexa Fluor Plus 647(Thermo Fisher Scientific);
CD45:Rat anti-Mouse CD45 Antibody,AF700(BD Biosciences);
CD11b:Rat anti-mouse/human CD11b antibody(BioLegend);
F4/80:Rat anti-mouse F4/80Antibody,PE(BioLegend);
CD163:Rat anti-mouse CD163 Antibody,Super Bright 600(Thermo FisherScientific);
Percoll:上海翌圣生物科技有限公司;
DPBS:Dulbecco's Phosphate Buffered Saline(DPBS),without Calcium andMagnesium(Biological Industries);
LiberaseTL:LiberaseTMTL Research Grade(Sigma-Aldrich);
DnaseⅠ:国药集团化学试剂有限公司;
HEPES:Sigma-Aldrich;
EDTA:国药集团化学试剂有限公司;
FBS:Certified Fetal Bovine Serum(Biological Industries);
HBSS:1X Hanks’Balanced Salt Solution(HBSS),Sterile(Sangon Biotech);
Matrigel:Corning。
实施例1全身炎症打击下,小鼠心功能受损,同时心肌细胞释放的损伤线粒体增多,并在心肌间质中大量堆积。
雄性小鼠腹腔注射氯胺酮(50mg/kg)和咪唑安定(5mg/kg)麻醉后,通过腹部下部1.5cm的纵向切口暴露盲肠。盲肠远端四分之三用4-0丝线结扎,用22G针头穿刺。将盲肠纳入腹膜腔内,用4-0丝线缝合切口。术后,小鼠皮下注射1ml无菌生理盐水,苏醒后可自由进食和饮水,即为脓毒症盲肠穿孔(CLP)模型。同时设假手术组(仅暴露盲肠,不进行结扎穿孔)。
术后不同时间点对小鼠进行心脏超声检测,收集血清和心脏组织,检测血清cTNI和LDH水平,qPCR检测心脏损伤指标ANP、BNP。
结果如图1所示,心脏超声结果显示脓毒症导致的心脏功能障碍模型造模成功。脓毒症后小鼠心脏功能EF、FS和CO明显受损,小鼠心肌损伤指标血清cTNI、LDH升高,心脏衰竭指标ANP、BNP表达升高。
由图2显示,在脓毒症后3d心肌间损伤线粒体堆积。
实施例2TREM2+巨噬细胞吞噬清除损伤心脏中的异常线粒体。
雄性野生型C57BL/6小鼠和TREM2-/-小鼠,麻醉后分别建立脓毒症盲肠穿孔(CLP)模型,术后7d电镜检测巨噬细胞吞噬损伤线粒体及心肌间损伤线粒体堆积的情况。
由图3显示,野生型C57BL/6小鼠心脏中巨噬细胞吞噬损伤线粒体。TREM2-/-小鼠中表现出巨噬细胞吞噬损伤线粒体功能障碍。
由图4显示,脓毒症心功能障碍模型后,TREM2-/-小鼠心肌间损伤线粒体大量堆积,提示巨噬细胞TREM2基因缺失影响其清理吞噬损伤线粒体。
实施例3提取分选出心脏来源的TREM2hi巨噬细胞
野生型C57BL/6小鼠醉后打开胸腔,用15mL DPBS灌注心脏。随后,取下心脏,去除周围脂肪组织和心房,将心室切碎成大约1mm3的碎片。在含有0.25mg/mL Liberase TL、20μg/mL Dnase I、10mM HEPES的无血清1640培养基中,37℃消化15min。用1000μL微量移液器对组织悬液进行吹打。细胞悬液用70μm的滤器过滤,去除残留的未消化组织碎片,然后在1mL HBSS中稀释。
将percoll原液与10×DPBS按1:9配置等渗percoll液。用PBS分别配置60%percoll液和15%percoll液,取3mL 60%percoll加入15mL离心管。倾斜离心管,沿管壁加入5mL 15%percoll,形成梯度界面。
将细胞悬液铺在Percoll液梯度界面上层,以400g、4℃离心20min。收集液面之间的细胞层,10mL PBS清洗两次,用200μL DPBS重悬,即为小鼠心脏单细胞悬液,主要为非心肌细胞,包括免疫细胞、内皮细胞和成纤维细胞。
往心脏单细胞悬液中加入TREM2一抗,4℃孵育30min,用DPBS清洗后,加入200μLDPBS重悬。再加入CD45抗体、CD11b抗体、F4/80抗体、CD163抗体和TREM2二抗,4℃孵育30min,用DPBS清洗后,加入200μL DPBS重悬。活力染料(Calcein和/或DyeCycleTMRuby)在4℃下孵育10min,孵育后,在Beckman mofloAstrios EQ仪器上用100μm的喷嘴分选出活的有核CD45+CD11b+F4/80+TREM2+CD163+细胞。即,TREM2hi巨噬细胞。将单细胞分选到含5%FBS的PBS中,显微镜下计数,PBS调整细胞悬液密度为8×106个/ml,置于冰上。
通过细胞计数分析得出,CD45+免疫细胞约占心脏非心肌细胞的10%,而TREM2hi巨噬细胞约占免疫细胞的40%。
实施例4心包腔内细胞注射
麻醉小鼠,并行气管插管呼吸机通气,常规消毒,铺巾。以小鼠第3-4肋间隙和左侧腋窝与胸骨下端连线的交点为中点,沿左侧腋窝与胸骨下端连线方向剪2cm的切口。固定暴露心脏,用50μL微量注射器,30G胰岛素注射针头,缓慢匀速将实施例1分选得到的含有2×105个细胞的25μLMatrigel(Corning),分3点注射入心包腔。用4-0丝线缝合切口并抽出胸腔气体,小鼠皮下注射1mL无菌生理盐水,苏醒后撤除呼吸机,可自由进食和饮水。
实施例5心包腔注射心脏来源TREM2hi巨噬细胞浸润入心脏的情况
取实施例3提取WT小鼠的TREM2hi巨噬细胞,转染带绿色荧光蛋白的腺病毒,用绿色荧光蛋白标记TREM2hi巨噬细胞,按实施例4的步骤注射到受体WT小鼠心包腔内。3d后麻醉小鼠取心脏组织,冰冻切片后,DAPI染色,共聚焦镜下观察。
由图5显示,3d后受体小鼠心脏中仍存在TREM2hi巨噬细胞。
实施例6心脏来源TREM2hi巨噬细胞改善脓毒症心脏功能障碍小鼠的心脏功能
WT小鼠进行心脏超声检测,选取健康的小鼠分别进行心包腔内注射实施例3分选得到的TREM2hi巨噬细胞和非TREM2hi巨噬细胞(CD45+免疫细胞中除去TREM2hi巨噬细胞的其他细胞),不关腹,暴露盲肠。盲肠远端四分之三用4-0丝线结扎,用22G针头穿刺。将盲肠纳入腹膜腔内,用4-0丝线缝合切口。术后,小鼠皮下注射1mL无菌生理盐水,即为脓毒症盲肠穿孔(CLP)模型。同时设Control组(不注射细胞,仅进行CLP)。
3d后对小鼠再次进行心脏超声检测,收集血清和心脏组织,检测血清cTNI、LDH和乳酸水平,qPCR检测心脏炎症因子水平及心脏损伤指标,同时试剂盒检测心脏ATP含量,电镜检测心脏线粒体损伤情况。
结果如图6和图7所示,Control组结果显示,脓毒症导致的心脏功能障碍模型造模成功。相较于注射Non-TREM2组,注射TREM2hi巨噬细胞的小鼠EF、FS和CO明显改善,说明TREM2hi巨噬细胞挽救部分心脏功能。
由图8显示,在脓毒症时,与注射Non-TREM2hi巨噬细胞的小鼠相比,注射TREM2hi巨噬细胞的小鼠血清cTNI、LDH和乳酸水平更低。
由图9显示,在脓毒症时,注射TREM2hi巨噬细胞的小鼠心脏组织中炎症因子mRNA水平低于注射Non-TREM2hi巨噬细胞组。
由图10显示,在脓毒症时,注射TREM2hi巨噬细胞的小鼠心脏组织中心脏损伤指标水平低于注射Non-TREM2hi巨噬细胞组。
由图11和图12显示,在脓毒症时,注射TREM2hi巨噬细胞的小鼠心脏组织中ATP含量显著高于注射Non-TREM2hi巨噬细胞组,电镜提示心脏线粒体损伤情况更轻。
实施例7心包腔注射心脏来源TREM2hi巨噬细胞改善心肌梗死小鼠的心脏功能
麻醉小鼠,并行气管插管呼吸机通气,碘伏消毒小鼠心前区。以小鼠第3-4肋间隙和左侧腋窝与胸骨下端连线的交点为中点,沿左侧腋窝与胸骨下端连线方向剪开2cm的切口。钝性分离胸大肌及肋骨外肌。于第3-4肋间拉开心腔,固定暴露心脏。在左心耳下缘水平线下2mm处以7-0尼龙线结扎冠状动脉左前降支。结扎成功后,分别进行心包腔内注射实施例3分选得到的TREM2hi巨噬细胞和非TREM2hi巨噬细胞(CD45+免疫细胞中除去TREM2hi巨噬细胞的其他细胞),关胸并抽出胸内气体,待小鼠恢复意识,撤除呼吸机。同时设Control组(不注射细胞,仅进行小鼠心肌梗死造模)。
一周后对小鼠进行心脏超声检测,评价心功能改变。结果如图13和图14所示,Control组结果显示,心梗导致的心脏功能障碍模型造模成功。相较于注射Non-TREM2组,注射TREM2hi巨噬细胞的小鼠EF、FS和CO明显改善,说明TREM2hi巨噬细胞挽救心梗后部分心脏功能。
Claims (6)
1.TREM2hi巨噬细胞在制备治疗心脏功能障碍药物中的应用,其特征在于,所述TREM2hi巨噬细胞为心脏免疫细胞中一类同时表达CD45、CD11b、F4/80、CD163和TREM2的细胞亚群;所述心脏功能障碍表现为心肌细胞释放的损伤线粒体增多,所述药物通过减少心肌细胞中损伤的线粒体达到改善心脏功能障碍的目的。
2.如权利要求1所述的应用,其特征在于,所述TREM2hi巨噬细胞的制备方法包括:首先取供体心脏心室组织,匀浆消化后制得细胞悬液,再利用Percoll法分离得到非心肌细胞混合液,然后往非心肌细胞混合液中加入TREM2一抗,孵育后加入CD45抗体、CD11b抗体、F4/80抗体、CD163抗体和TREM2二抗,孵育,最后利用细胞分选仪分选出活的有核CD45+CD11b+F4/80+TREM2+CD163+细胞,即为所述TREM2hi巨噬细胞。
3.如权利要求1所述的应用,其特征在于,所述心脏功能障碍为脓毒症导致的心脏功能障碍。
4.如权利要求1所述的应用,其特征在于,所述心脏功能障碍为心肌梗死导致的心脏功能障碍。
5.如权利要求1所述的应用,其特征在于,所述药物的给药方式为心包腔内注射。
6.如权利要求1所述的应用,其特征在于,所述药物包括有效剂量的TREM2hi巨噬细胞和药学上可接受的载体。
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