CN117264880A - 一种射血分数保留型心力衰竭细胞模型 - Google Patents
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Abstract
本发明公开了一种射血分数保留型心力衰竭细胞模型。本发明的心肌细胞模型是由动物心肌细胞经过高糖高脂培养基共同孵育培养后得到的心肌细胞,培养后所述心肌细胞出现收缩功能保留,而舒张功能下降,并在达到50%最大舒张用时(TR50)开始出现舒张功能的下降,所述高糖高脂培养基为含有12.5mmol/L葡萄糖和0.2mmol/L棕榈酸的DMEM培养基。本发明的本发明的心肌细胞模型,可以应用于射血分数保留型心力衰竭(HFpEF)的发病机制探索,也可以为HFpEF的药物靶点研究提供新的方案。
Description
技术领域
本发明涉及一种射血分数保留型心力衰竭细胞模型,属于生物技术领域。
背景技术
心力衰竭是各种心血管疾病最常见且严重的终末阶段心血管综合征,可分为射血分数降低型心力衰竭(Heart Failure with Reduced Ejection Fraction,HFrEF)和射血分数保留型心力衰竭(Heart Failure with Preserved Ejection Fraction,HFpEF,LVEF≧50%)。其中,HFpEF发病机制仍不明确、诊断困难、预后差,且尚无可靠的治疗方案,已成为困扰临床医生的难题,明确HFpEF的发病机制是解决难题的关键点。HFpEF的特征表现包括左心室舒张功能障碍、充盈压增高伴生物学标志物升高等。近年来越来越多的基础研究不断揭示HFpEF的发病机制,其中明确导致HFpEF心脏损伤的诱因很多,包括糖尿病、高血压、肥胖、高脂血症及微循环障碍等。近年来随着对HFpEF的认知不断深入,发病机制有了新的进展。
HFpEF是一种异质性疾病,表现为多种表型且很难识别并诊断。在2020年葛均波院士等学者提出最新分类:根据病因学将HFpEF分为5型,其中代谢相关性HFpEF也越来越引起大家的重视。代谢相关性HFpEF合并更多的糖尿病、肥胖、脂质代谢紊乱,也有证据表明糖尿病、肥胖合并HFpEF患者预后更差,越早出现心血管各类并发症。在发病机制上目前认为糖基化终产物、脂质沉积会引起的心肌细胞直接损伤,最终引起心脏舒张功能减退及活动耐量的下降。目前由于HFpEF的多种表型及诊断困难等难题,亟待构建体外模型用于验证其机制。
HFpEF的主要病理生理学过程包括系统性炎症、心外膜脂肪组织炎症与堆积、脂肪炎性因子分泌、冠状动脉微循环血管功能障碍、心肌纤维化、心室-动脉僵硬度增加等导致左心室舒张功能障碍、左室腔减少(向心性重构)伴左室充盈压升高、内皮及冠状动脉微血管功能障碍。钙离子是心肌细胞收缩、舒张的核心环节。肌钙蛋白与钙离子结合后其构象发生改变,暴露肌动蛋白与肌球蛋白结合位点,促发肌丝滑动,心肌进而发生收缩;而在舒张期,钙离子与肌钙蛋白解离,被转运回肌浆网。在心脏收缩及舒张的过程中,细胞内钙离子水平也能反应舒张功能的改变。那么,在构建HFpEF细胞模型中也检测到单细胞收缩及舒张功能,同时也对细胞内钙瞬变做了分析,这些指标可以很好的反映心肌细胞舒张功能。
综上,根据代谢相关性HFpEF的发病特点、病理生理学改变及表型特征,构建新型的体外细胞模型具有重要的意义,能够更好的为HFpEF的发病机制以及药物靶点的验证提供新的工具。
发明内容
本发明的目的是:根据代谢相关性HFpEF的发病特点、病理生理学改变及表型特征,构建一种HFpEF型体外细胞模型,为HFpEF的发病机制以及药物靶点的验证提供新的工具。
为了实现上述目的,本发明提供了一种射血分数保留型心力衰竭细胞模型,所述细胞模型是由成年鼠分离的心肌细胞经过高糖、高脂(“双重打击”)诱导后得到的细胞模型,培养后所述心肌细胞呈现收缩功能保留,而舒张功能下降的表型,并在达到50%最大舒张时间(Time-to 50%relengthening,TR50)开始出现舒张功能的下降;所述高糖、高脂诱导的条件为:与含有12.5mmol/L葡萄糖和0.2mmol/L棕榈酸的DMEM培养基共同孵育。
优选地,所述共同孵育的时间为12h。
本发明还提供了上述HFpEF细胞模型在研究HFpEF中的应用。
本发明还提供了上述的HFpEF细胞模型在筛选和制备治疗HFpEF药物中的应用。
本发明与现有技术相比,具有如下有益效果:
本发明通过尝试高血糖、高脂不同浓度、时间诱导的方案,通过在细胞水平测定心肌细胞收缩及舒张功能的方法,多次验证高糖、高脂“双重打击”刺激后心肌细胞呈现出射血分数保留但舒张功能下降的表型,结果表明,本发明的细胞模型达到了HFpEF的表型特点;本发明的HFpEF细胞模型,可以应用于HFpEF的发病机制探索,也可以为HFpEF的药物靶点研究提供新的方案。
附图说明
图1为分离成年小鼠的心肌细胞测定心肌细胞收缩、舒张功能;
图2为高糖、高脂同时-“双重打击”孵育心肌细胞12小时后测定收缩及舒张功能;
图3为心肌细胞分别给予高糖、高脂、“双重打击”、以及甘露醇孵育12小时后收缩、舒张功能检测;
图4为“双重打击”细胞模型中细胞内钙瞬变检测结果。
具体实施方式
为使本发明更明显易懂,兹以优选实施例,并配合附图作详细说明如下。
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。
实施例
1、细胞来源:8-12周龄C57/BL雄性鼠分离得到心肌细胞,在显微镜下呈现长杆状(杆状细胞的比例要达到≥85%以上),所述心肌细胞的提取步骤如下:
(1)分离成年鼠心肌组织:首先,成年鼠用4%水合氯醛腹腔注射:0.5ml/只,等待3分钟后小鼠麻醉完成;分离小鼠心肌组织。
(2)用EDTA缓冲液(见表1)和Perfusion缓冲液(见表2)分别灌注心肌组织5分钟/次。结束后用温热的(±35℃)胶原蛋白缓冲液灌注、消化心肌组织,灌注时间至少持续5分钟/次。灌注2-3次后可以观察到心脏缓慢溶解呈胶冻状。
表1:EDTA缓冲液配方
注释:HEPES:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;BDM:2,3-butanedione monoxime;EDTA-2Na:ethylenediaminetetracetic acid disodium。上述剂量为配置1L的溶液需要试剂的总量;所有药品溶解后调整pH值到7.45。
表2:Perfusion缓冲液配方
试剂名称 | 总用量 | 公司名称 | 货号 |
氯化钠 | 7.59g | 1st Base,Singapore | BIO-1111 |
氯化钾 | 0.37g | Sigma-Aldrich | P9541 |
磷酸氢二钠 | 0.059g | Sigma-Aldrich | S8282 |
HEPES | 2.383g | 1st Base,Singapore | BIO-1825 |
葡萄糖 | 1.801g | Sigma-Aldrich | G8270 |
BDM | 1.011g | Sigma-Aldrich | B0753 |
牛磺酸 | 1.251g | Sigma-Aldrich | T8691 |
MgCl2 | 1.861g | Sigma-Aldrich | M8266 |
注释:HEPES:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;BDM:2,3-butanedione monoxime;所有药品溶解后调整pH值到7.45。
(3)取出1滴灌注液在显微镜下观察心肌细胞状态,如果达到杆状细胞分布≥85%,则进行下一步操作;如果未达到此标准,则重复以上操作直到杆状的心肌细胞达标为止。
(4)加入当天配置好的Stop缓冲液终止消化,将心肌细胞悬液转移到50ml离心管中,用70μm的滤器过滤;其中,Stop缓冲液配方为:在95ml的Perfusion缓冲液中加入5mlFBS最终配置成100ml的溶液,pH值7.45。
(5)按照顺序逐次加入:吸入2ml钙刺激液1#、2#、3#(见表3)依次加入离心管,混合后离心200rpm 3分钟,吸取上清液弃去,每次间隔时长为15分钟。
表3:钙刺激液1#/2#/3#配方
注释:两种缓冲液根据以上剂量混合最终配置成8ml的钙刺激液;其中,生长培养基的配方如下列表4所示:
表4:生长培养基配方
试剂名称 | 总用量 | 公司名称 | 货号 |
M199 | 97ml | Gibco | M4530 |
BSA | 0.1g | Thermo fisher | A11133 |
ITS | 1ml | Thermo fisher | 230900 |
CD lipid | 1ml | Thermo fisher | 11905031 |
P/S | 1ml | Thermo fisher | 15140163 |
注释:BSA:bovine serum albumin;ITS:insulin-transferrin-selenium,CDLipid:chemically defined lipid concentrate;P/S:penicillin-streptomycinsolution。
此时已经完成小鼠心肌细胞的分离,管底部白色沉积物即为分离后的成年鼠心肌细胞。
2、构建“双重打击”(“Double Damage”,DD)细胞模型:
(1)配置三组DMEM培养基
①高糖培养基:葡萄糖终浓度为12.5mmol/L的DMEM培养基;
②高脂培养基:棕榈酸(Palmitic acid,PA)终浓度为0.2mmol/L的DMEM培养基;
③“双重打击”细胞培养基:同时加入高糖及高脂,其中含有终浓度为12.5mmol/L葡萄糖和0.2mmol/L棕榈酸DMEM培养基。
(2)细胞刺激:
①将分离的小鼠心肌细胞分别加入上述的DMEM培养基,调整到密度为10000/mL,然后加入6孔板中,每个孔加入4mL心肌细胞悬液;
②按顺序,分别加入三组培养基:高糖、高脂、“双重打击”;
③放入37℃培养箱,孵育时间为:12h;
④完成心肌细胞刺激。
3、采用IonOptix soft-edge(IonOptix公司,Milton,MA)显微镜,在0.5Hz条件下,检测上述分离得到的成年鼠心肌细胞的收缩、舒张功能。
检测方法如下:
(1)用显微镜检测心肌细胞的收缩舒张功能的变化,典型指标包括:
①最大收缩值/细胞长度:peak shortening%cell length;
②最大细胞收缩速度:+dl/dt;
③最大细胞舒张速度:-dl/dt;
④达到10%,50%,90%最大收缩用时:Time-to-10%,50%and 90%shortening(TP10、50、90);
⑤达到10%,50%,90%最大舒张用时:Time-to-10%,50%,and 90%relengthening(TR10、50、90)。
分离的心肌细胞的收缩及舒张功能测定结果如图1所示。
不同浓度、时间刺激后观察细胞的活性,测定心肌细胞收缩、舒张功能后确认最终的条件(图2)。
这组结果说明单纯给予高糖或者高脂刺激12h后心肌细胞呈收缩、舒张功能均正常。给予“双重打击”刺激12h后心肌细胞出现收缩功能保留,而舒张功能下降,并在达到50%最大舒张用时(TR50)开始出现舒张功能的下降。根据实验结果可以得出:“双重打击”的刺激药物的浓度及时间,分别为高糖:12.5mmol/L,高脂:0.2mmol/L,同时加入细胞皿中孵育12h。
心肌舒张功能受损,与细胞内钙调控密切相关,细胞内钙的水平增高提示肌浆网钙泵功能减弱,最终引起舒张减退,故测定细胞内钙的改变是反映舒张功能的重要指标,图4的结果显示了“双重打击”细胞模型呈现出细胞内钙增高,清除钙的能力下降。这些结果均提示“双重打击”细胞的舒张功能减退。
以上所述,仅为本发明的较佳实施例,并非对本发明任何形式上和实质上的限制,应当指出,对于本技术领域的普通技术人员,在不脱离本发明的前提下,还将可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (5)
1.一种射血分数保留型心力衰竭细胞模型,其特征在于,所述细胞模型是由动物心肌细胞与高糖、高脂培养基共同孵育培养后得到的细胞模型,培养后所述心肌细胞呈现收缩功能保留,而舒张功能下降的表型,并在达到50%最大舒张用时(TR50)开始出现舒张功能的下降,所述高糖、高脂培养基为含有12.5mmol/L葡萄糖和0.2mmol/L棕榈酸的DMEM培养基。
2.如权利要求1所述的射血分数保留型心力衰竭细胞模型,其特征在于,所述动物心肌细胞为成年鼠心肌细胞。
3.如权利要求1所述的射血分数保留型心力衰竭细胞模型,其特征在于,所述共同孵育的时间为12h。
4.权利要求1所述的射血分数保留型心力衰竭细胞模型在研究射血分数保留的心力衰竭中的应用。
5.权利要求1所述的射血分数保留型心力衰竭细胞模型在筛选和制备治疗射血分数保留的心力衰竭药物中的应用。
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