CN115369077B - Meflc细胞株及其构建方法和应用 - Google Patents
Meflc细胞株及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种MEFLC细胞株及其构建方法和应用,该细胞株于2022年4月23日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO:62409。其构建方法包括1)构建pCW‑MYOG‑T2A‑Puro慢病毒;2)慢病毒感染人iPSc细胞株,诱导MYOG基因表达,并筛选阳性单克隆细胞株;3)将阳性的单克隆扩大培养,同时持续诱导MYOG的表达;4)更换培养条件,并持续诱导MYOG表达,同时筛选阳性单克隆细胞株保证MYOG阳性细胞的纯度,待细胞形态明显发生变化,呈成纤维细胞状,即得MEFLC。本发明的MEFLC细胞株在心肌细胞共培养中发挥着重要作用,因此,可参与治疗心肌细胞凋亡引起的心血管疾病。
Description
技术领域
本发明涉及一种MEFLC细胞株,以及其构建方法和应用。
背景技术
心肌梗死(myocardial infarction,MI)是冠状动脉发生急性闭塞,由此造成闭塞血管相应供血区域的心肌组织细胞发生不可逆的坏死。心肌细胞作为终末分化细胞,在受到缺血缺氧损伤而坏死之后无法像其他可再生组织细胞一样发生细胞再生而自行修复,取而代之的是发生纤维化而形成瘢痕组织,随后出现心脏重塑和逐步出现心功能不全。利用干细胞通过心肌再生,替代心梗后损失的心肌细胞,可以促进瘢痕修复,避免心脏重构发展成为心衰,但胚胎干细胞的应用存在道德和伦理方面的问题。
近年来,iPSc(诱导多能干细胞)技术的发展为整个干细胞生物学领域和临床再生医学提供了新的研究方向。iPSc具有类似胚胎干细胞的全能性,又避免了道德和伦理的争议,iPSc技术的发展、心肌细胞分化及纯化方法的建立,使得体外制备及培养人心肌细胞成为可能。目前,iPSc技术在心血管疾病方面的研究和应用体现在两个方面,一是将心血管病病人的体细胞编程为iPSc后在体外建立疾病模型,可以在细胞水平研究发病机制及有效药物,二是将iPSc或iPSc分化的心肌细胞移植到病人体内,来治疗心肌梗死、心力衰竭等疾病。
已有体内实验证实未分化的iPSc细胞可以在体内分化成为心肌细胞,但这类细胞由于具有胚胎干细胞特征而具有致瘤风险。因此,在体外诱导iPSc细胞分化为心肌谱系细胞,然后再移植注入心脏可能是规避这一风险更佳的选择。已有研究证实,在MI小鼠心梗区注射iPSc-CM能够明显改善心梗小鼠的心功能,在MI大鼠心梗部位移植iPSc-CM和内皮细胞共培养的细胞薄片也能改善心脏功能,但移植到局部的细胞存活率低一直是细胞移植疗法共同存在的问题,优化体内生存时间是临床转化的关键问题。
转录因子基因MYOG编码肌细胞生成素(Myogenin)蛋白,是生肌调节因子(MRFs)基因家族的成员之一。MRFs转录因子家族(包括Myod、Myf5、Mrf4和MYOG)在骨骼肌发生的每一个阶段都起着关键作用。该家族所有成员共同含有一个保守的螺旋-环-螺旋(bHLH)基序,可以与下游基因的E盒结合,从而激活下游肌肉特异性基因的表达。研究表明,MYOG通过控制、启动成肌细胞的融合和肌纤维的形成,而在肌肉分化过程中起关键作用。在小鼠中的研究显示,MYOG基因缺失会导致严重的肌肉分化缺陷,从而造成围产期死亡。因此,MYOG是骨骼肌发育过程中必需的调控因子,并且是不可替代的。目前,已有研究证实,MYOG基因在小鼠、鸭、草鱼、京海黄鸡等动物的心脏中都有表达,可能与心肌的生长发育相关。但MYOG基因在人心脏组织中的表达及在心脏发育中的作用尚未见报道。
发明内容
本发明所要解决的技术问题,就是提出一种MEFLC细胞株,以及其构建方法和应用。
为解决上述技术问题,本发明采用以下技术方案予以实现:
一种MEFLC细胞株,为人心肌成纤维样细胞,于2022年4月23日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCCNO.62409。
MEFLC细胞株的培养基的组成为高糖DMEM培养液(Gibco,C11995500BT)+体积含量10%的FBS(Excell BIO,FSP500)+体积含量1%的NEAA(Solarbio,P1400)+体积含量1%的PS(Solarbio,N1250)+Dox(生工,A600889-0025),Dox在培养液中的终浓度是2μg/mL。
MEFLC细胞株的构建方法,包括如下步骤:
MEFLC细胞株的构建方法,其特征在于:包括如下步骤:
1)构建含MYOG基因的慢病毒;慢病毒为具备四环素诱导系统的慢病毒,除此之外也可以是一些其他诱导系统的慢病毒,只是在诱导MYOG基因表达时对应的诱导试剂需要改变。
2)慢病毒感染人iPSc细胞株,诱导MYOG基因表达,并筛选阳性单克隆细胞株;
3)将阳性的单克隆扩大培养,同时持续诱导MYOG的表达;
4)更换培养条件,并持续诱导MYOG表达,同时筛选阳性单克隆细胞株保证MYOG阳性细胞的纯度,待细胞形态明显发生变化,呈成纤维细胞状,即得MEFLC。
本发明通过试验探究了MEFLC的功能。
MEFLC抵抗缺氧(CoCl2)、活性氧(H2O2)、抗肿瘤药物(阿霉素)的细胞毒性。MEFLC细胞株可在复苏冻存心肌细胞时作为降低细胞凋亡比例的添加剂。
MEFLC与人诱导多能干细胞来源的心肌细胞(hiPSc-CM)共培养,能够对hiPSc-CM在缺氧(CoCl2)、活性氧(H2O2)、抗肿瘤药物(阿霉素)处理下具有保护作用。MEFLC细胞株可在心肌细胞回输治疗心肌细胞凋亡引起的心血管疾病中作为支持型细胞药物,心肌细胞与MEFLC细胞混合注射,可降低心肌细胞凋亡从而保证治疗效果。
MEFLC降低冻存的hiPSc-CM在复苏后的凋亡比例。MEFLC细胞株可在缺氧/活性氧/阿霉素的环境中作为降低细胞凋亡比例的添加剂。
MEFLC提高hiPSc-CM对病毒感染的抵抗能力。
MEFLC促进hiPSc-CM的肌节发育和细胞体积增加。
MEFLC可以显著促进hiPSc-CM在小鼠心脏组织中的存活。
MEFLC可以显著降低心梗(myocardialinfarction,MI)小鼠心脏组织中的胶原容积分数以及防止心梗后室壁变薄。MEFLC细胞株可制备治疗心梗的注射药物。
附图说明
图1为HCFC与MEFLC分别在不同浓度的CoCl2、H2O2、阿霉素条件下表现出的相对细胞活力曲线;
图2为MEFLC与hiPSc-CM共培养可以减少由CoCl2、H2O2、阿霉素造成的心肌细胞凋亡增加;
图3为MEFLC降低冻存的hiPSc-CM在复苏后的凋亡比例;
图4为MEFLC提高hiPSc-CM对病毒感染的抵抗能力;
图5为MEFLC促进hiPSc-CM的肌节发育和细胞体积增加;
图6为MEFLC可以显著延长hiPSc-CM在小鼠心脏组织中的存活时间;
图7为注射MEFLC显著降低MI小鼠心脏胶原容积分数;
图8为注射MEFLC显著增加MI小鼠心脏室壁厚度。
具体实施方式
为让本领域的技术人员更加清晰直观的了解本发明,下面将结合附图,对本发明作进一步的说明。
1.MEFLC构建步骤:
1.1构建pCW-MYOG-T2A-Puro慢病毒载体:用常规分子克隆方法将MYOG cDNA和一个嘌呤霉素抗性基因亚克隆到pCW-Cas9-Blast载体中(Addgene,83481),取代原载体中的Cas9和Blast基因,得到pCW-MYOG。
1.2慢病毒包装:
1.2.1接种HEK293T细胞到6孔板中,用D10培养液(DMEM培养液+体积含量10%的胎牛血清)进行培养,待细胞汇合度达到70%-80%时准备进行转染;
1.2.2在转染前1h,弃去原来培养液,加入2mL/孔预热的无血清OptiMEM培养液;
1.2.3按照产品说明书用Lipofectamine 2000试剂进行转染。将pCW-MYOG(20μg)、pVSVg(10μg)(Addgene)、psPAX2(15μg)(Addgene)共转染HEK293T细胞;
1.2.4在6h后,将培养液更换成DMEM培养液+体积含量10%的胎牛血清+体积含量1%的BSA(牛血清白蛋白);
1.2.5继续培养约60h后,取培养液于3000rpm、4℃离心10min,去除细胞残渣。
1.2.6用0.45μm低蛋白结合滤膜(Millipore Steriflip HV/PVDF)过滤上清液,去除细胞残渣。
1.2.7将含病毒的培养液按照体积比4:1与质量百分含量10%的蔗糖缓冲液混合(50mM Tris-Hcl,pH 7.4,100mM NaCl,0.5mM EDTA)混合,10000g、4℃离心4h。小心弃去上清液,离心管在吸水纸上沥干3min,加入PBS缓冲液重悬,-80℃保存。
1.3pCW-MYOG-T2A-Puro慢病毒感染人iPSc细胞株(DYR0100):
1.3.1hiPSc培养:将人诱导多能干细胞(hiPSc)DYR0100(ATCC)接种于Matrigel基质(康宁,354277)包被的平板上,然后用STEMUP(Nissan Chemical Corporation)进行培养。STEMUP培养基每两天更换一次。iPSc每隔3天传代一次,或在细胞培养达到80-90%汇合度时传代。传代过程中用1×DPBS(Gibco,14040133)冲洗1次,然后在室温下用使用1×DPBS(Gibco,14190144)稀释的0.5mM EDTA(Invitrogen,15575020)处理10min。传代比为1:3-1:6。
1.3.2转染:待hiPSc细胞汇合度达到70%-80%时进行转染。感染复数(MOI)约为0.3-0.5。转染24h后,将培养液更换成新鲜STEMUP(含终浓度为2μg/mL的盐酸多四环素(Dox))。2天后,将培养液更换成STEMUP(含Dox 2μg/mL+嘌呤霉素(puromycin)(InvivoGen))进行筛选。筛选2-3天后,能够得到约30%的转化效率。挑取单个的克隆接种到不同的皿中培养,即得hiPSc-MYOG细胞株。24小时后加入Dox开始诱导MYOG表达,48小时后加入Puromycin(终浓度2μg/mL)进行筛选。筛选过程持续48-72小时,挑取存活的单克隆进行扩大培养。
1.4利用实时定量PCR技术鉴定单克隆是否表达MYOG,将阳性的单克隆扩大培养(命名为hiPSc-MYOG),同时在培养液中加入Dox持续诱导MYOG的表达,将此时定为第1天。培养液:StemUp培养液。此时的培养条件为:①StemUp培养液+Dox(终浓度2μg/mL)。
1.5使用培养条件①培养hiPSc-MYOG15-20天后,换培养条件②持续培养hiPSc-MYOG。培养条件②的组成为:高糖DMEM培养液+体积含量10%的FBS+体积含量1%的NEAA+体积含量1%的PS+Dox(终浓度2μg/mL)。每10-14天使用Puromycin(终浓度2μg/mL)筛选3-4天,保证MYOG阳性细胞的纯度。在30-40天,细胞形态明显发生变化,呈成纤维细胞状,我们将这种细胞命名为MEFLC(Myogenin-ExpressingFibroblast-LikeCells)。
1.6 MEFLC使用培养条件②进行培养。
2 MEFLC的功能分析
2.1 MEFLC抵抗缺氧(CoCl2)、活性氧(H2O2)、抗肿瘤药物(阿霉素)的细胞毒性。
与人心脏成纤维细胞系(HCFC)相比,MEFLC展示出对缺氧(CoCl2)、活性氧(H2O2)、抗肿瘤药物(阿霉素)更强的耐受能力。可用于心梗、心衰、冠心病等疾病的治疗。
2.1.1实验材料准备:MEFLC使用培养条件②进行培养;HCFC用培养条件③进行培养。培养条件③的组成为:高糖DMEM培养液+体积含量10%的FBS+体积含量1%的NEAA+体积含量1%的PS。
2.1.2细胞接种:MEFLC和HCFC汇合度约80%时,用0.05%胰蛋白酶消化,于200g离心5min收集细胞,再用适当体积培养液重悬细胞并计数。按照10000cell/孔接种MEFLC和HCFC到96孔板。
2.1.3药物处理:接种24h后,开始加入Doxo、CoCl2、H2O2、LPS进行药物处理。每种药物每个处理浓度均做8个重复。
Doxo浓度梯度为:0μM、0.004μM、0.01μM、0.04μM、0.13μM、0.41μM、1.23μM、3.7μM、11.1μM、33.3μM、100μM、300μM,处理时间24h。
CoCl2浓度梯度为:0μM、1.33μM、4μM、13μM、41μM、123μM、370μM、1.11mM、3.33mM、10mM、30mM,处理时间为24h。
H2O2浓度梯度为:0mM、0.004mM、0.01mM、0.04mM、0.13mM、0.41mM、1.23mM、3.7mM、11.1mM、33.3mM、100mM、300mM,处理时间为4h。
LPS浓度梯度为:0μg/mL、2μg/mL、10μg/mL、50μg/mL、100μg/mL、150μg/mL、200μg/mL,处理时间为48h。
2.1.4细胞活性检测:用PrestoBlue细胞活性检测试剂(Invitrogen,A13261)检测细胞活性。
2.1.5实验结果与分析:与HCFC相比,MEFLC在高浓度CoCl2(123μM~30mM)、高浓度Doxo(3.7μM~300μM)、中等浓度H2O2(0.04mM~11.1mM)和150μg/mLLPS处理后,细胞活性显著高于HCFC。
如图1所示,MEFLC表现出对CoCl2、H2O2、Doxo更强的耐受能力。
2.2 MEFLC与人诱导多能干细胞来源的心肌细胞(hiPSc-CM)共培养,能够对hiPSc-CM在缺氧(CoCl2)、活性氧(H2O2)、抗肿瘤药物(阿霉素)处理下具有保护作用
对照组:hiPSc-CM与人心脏成纤维细胞系共培养(CM+HCFC),hiPSc-CM单独培养(CM)。
实验组:hiPSc-CM与MEFLC共培养(CM+MEFLC)。
与对照组相比,CM+MEFLC组在CoCl2、H2O2、阿霉素处理的情况下,凋亡细胞的比例显著降低。
2.2.1实验材料准备:MEFLC使用培养条件②进行培养至汇合度约80%,使用丝裂霉素(mitomycin,20μg/mL)处理5h,之后继续使用培养条件②培养,标记为MEFLC-Mito-C;HCFC用培养条件③培养至汇合度约80%,同样使用丝裂霉素(mitomycin,20μg/mL)处理5h,之后继续使用培养条件③培养,标记为HCFC-Mito-C;为了方便后续的流式细胞分析,我们使用绿色荧光蛋白标记hiPSc-CM:hiPSc-CM用AAV-EGFP感染(MOI=2),感染效率>98%,之后使用培养条件④继续培养,命名为hiPSc-CM-EGFP。培养条件④的组成为:高糖DMEM培养液+体积含量3%的KOSR+体积含量1%的NEAA+体积含量1%的PS。
2.2.2细胞共培养:按照HCFC/MEFLC:hiPSc-CM=7:3和HCFC/MEFLC:hiPSc-CM=5:5用培养条件③进行共培养,同时设置一组只有hiPSc-CM作为对照组,每组细胞总数相同。具体方法为:CM组:12孔板每孔铺2.5×105个hiPSc-CM-EGFP;CM+HCFC(7:3)组:12孔板每孔铺7.5×104个iPSc-CM-EGFP以及1.75×105个HCFC-Mito-C;CM+MEFLC(7:3)组:12孔板每孔铺7.5×104个iPSc-CM-EGFP以及1.75×105个MEFLC-Mito-C;CM+HCFC(5:5)组:12孔板每孔铺iPSc-CM-EGFP和HCFC-Mito-C各1.25×105个;CM+MEFLC(5:5)组:12孔板每孔铺iPSc-CM-EGFP和MEFLC-Mito-C各1.25×105个。细胞每2-3天换液。
2.2.3药物处理:共培养4天后,加入不同的药物处理。其中Doxo浓度为1μM,处理时间24h。CoCl2浓度为1mM,处理时间为24h。H2O2浓度为1mM,处理时间为4h。每组设3-4个生物学重复。对照组无任何药物处理。
2.2.4细胞凋亡检测:使用APCAnnexinV细胞凋亡检测试剂盒(Biolegend,640920)检测不同药物处理之后各组心肌细胞(GFP阳性)凋亡比例(APC阳性)。
2.2.5实验结果与分析:如图2所示,与CM组和CM+HCFC组相比,无论是7:3还是5:5组中,CM+MEFLC组在Doxo、CoCl2、H2O2处理后,细胞凋亡比例都有显著下降。说明在缺氧(CoCl2)、活性氧(H2O2)、抗肿瘤药物(阿霉素)处理下,MEFLC对hiPSc-CM具有保护作用。
2.3 MEFLC降低冻存的hiPSc-CM在复苏后的凋亡比例
2.3.1实验材料准备:MEFLC使用培养条件②进行培养至汇合度约80%,使用丝裂霉素(mitomycin,20μg/mL)处理5h,之后继续使用培养条件②培养,标记为MEFLC-Mito-C;HCFC用培养条件③培养至汇合度约80%,同样使用丝裂霉素(mitomycin,20μg/mL)处理5h,之后继续使用培养条件③培养,标记为HCFC-Mito-C;hiPSc-CM用AAV-EGFP感染(MOI=2),感染效率>98%,命名为hiPSc-CM-EGFP,之后使用培养条件④恢复培养3天后,用0.05%胰蛋白酶消化细胞,用细胞冻存液STEMdiff冻存细胞,液氮中保存。
2.3.2细胞复苏与共培养:hiPSc-CM-EGFP快速从液氮中取出,在37℃水浴条件下融化,快速加入等体积培养液,于200g离心5min收集细胞,再用适当体积培养液重悬细胞并计数。同时用0.05%胰蛋白酶消化MEFLC-Mito-C和HCFC-Mito-C,于200g离心5min收集细胞,再用适量培养液重悬细胞并计数。按照HCFC/MEFLC:iPSc-CM=7:3使用培养条件③进行共培养,同时设置一组只有hiPSc-CM作为对照组,每组细胞总数相同。具体方法为:CM组:12孔板每孔铺2.5×105个hiPSc-CM-EGFP;CM+HCFC组:12孔板每孔铺7.5×104个hiPSc-CM-EGFP以及1.75×105个HCFC-Mito-C;CM+MEFLC组:12孔板每孔铺7.5×104个iPSc-CM-EGFP以及1.75×105个MEFLC-Mito-C。每组设3-4个生物学重复。培养2天后,更换培养液。培养4天后,检测心肌细胞凋亡比例。
2.3.3细胞凋亡检测:使用APCAnnexinV细胞凋亡检测试剂盒(Biolegend,640920)检测三组细胞中心肌细胞(EGFP阳性)的凋亡比例(APC阳性)。
2.3.4实验结果与分析:如图3所示,CM+MEFLC组与CM组相比,心肌细胞凋亡比例显著下降(7.8%),而CM+HCFC组心肌细胞凋亡比例却有显著增加。说明MEFLC与hiPSc-CM共培养能够降低冻存心肌细胞复苏后的凋亡比例,提高复苏存活率。
图3中柱形图CM、CM+HCFC、CM+MEFLC对应的数据:
CM | CM+HCFC | CM+MEFLC | |
ave | 42.57 | 59.09 | 34.81666667 |
2.4 MEFLC提高hiPSc-CM对病毒感染的抵抗能力
2.4.1实验材料准备:MEFLC使用培养条件②进行培养至汇合度约80%,使用丝裂霉素(mitomycin,20μg/mL)处理5h,之后继续使用培养条件②培养,标记为MEFLC-Mito-C;HCFC用培养条件③培养至汇合度约80%,同样使用丝裂霉素(mitomycin,20μg/mL)处理5h,之后继续使用培养条件③培养,标记为HCFC-Mito-C;hiPSc-CM用AAV-mcherry感染(MOI=2),显微镜下观察红光细胞比例为>98%,之后使用培养液培养条件④进行培养,标记为hiPSc-CM-mcherry。
2.4.2细胞共培养:按照HCFC/MEFLC:hiPSc-CM=7:3用培养条件③进行共培养,同时设置一组只有hiPSc-CM作为对照组,每组细胞总数相同。具体方法为:CM组:12孔板每孔铺2.5×105个hiPSc-CM-mcherry;CM+HCFC组:12孔板每孔铺7.5×104个hiPSc-CM-mcherry以及1.75×105个HCFC-Mito-C;CM+MEFLC组:12孔板每孔铺7.5×104个hiPSc-CM-mcherry以及1.75×105个MEFLC-Mito-C。每组设3-4个生物学重复。
2.4.3病毒感染:细胞培养3天后,使用腺病毒AAV-EGFP按照MOI=0.5感染共培养细胞(三组:CM、CM+HCFC、CM+MEFLC)。病毒感染24h后,吸走含有病毒的培养液,用PBS清洗1次,换回培养条件③。继续培养48h后,使用流式细胞仪检测GFP阳性(GFP阳性表征细胞被腺病毒AAV-EGFP感染)的心肌细胞比例。
2.4.4病毒感染比例测定:使用0.05%胰蛋白酶消化细胞,于200g离心5min收集细胞,用PBS重悬制成细胞悬液,用流式细胞仪分析各组心肌细胞的病毒感染比例(即mcherry阳性细胞中GFP阳性细胞比例)。
2.4.5实验结果与分析:如图4所示,CM+MEFLC组与CM组相比,GFP阳性心肌细胞比例下降22.0%,而CM+HCFC组与CM组GFP阳性心肌细胞的比例无显著差异。说明MEFLC与CM共培养可以显著提高hiPSc-CM抵抗病毒感染的能力。
图4中柱形图CM、CM+HCFC、CM+MEFLC对应的数据:
CM | CM+HCFC | CM+MEFLC | |
Ave | 31.11 | 26.74333333 | 9.09 |
2.5 MEFLC促进hiPSc-CM的肌节发育和细胞体积增加
如图5所示,MEFLC与hiPSc-CM(CM+MEFLC组)共培养1周后,与对照组(HCFC与hiPSc-CM共培养,CM+HCFC组)相比,hiPSc-CM面积显著增加(p<0.001),具有有序肌节结构的hiPSc-CM的比例显著上升(p<0.05)。这说明MEFLC与hiPSc-CM共培养,可以促进hiPSc-CM的肌节发育和体积增加。
2.6 MEFLC可以显著促进hiPSc-CM在小鼠心脏组织中的存活
【实验目的】
检测带有荧光标记的MEFLC和hiPSc-CM在小鼠心肌多点注射后的荧光强度的变化,以此检测各组细胞在小鼠心肌组织中的存活时间。
【实验材料】
实验细胞:hiPSc-CM使用慢病毒HBLV-ZsGreen-Puro感染后,发出绿色荧光;MEFLC细胞用慢病毒HBLV-mCherry-Puro感染后,发出红色荧光。
实验动物:C57BL/6J小鼠,雄性,20g±2g,18只。
【实验方案】
(1)实验分组:
实验组1心肌多点注射MEFLC+hiPSc-CM,n=6
实验组2心肌多点注射hiPSc-CM,n=6
对照组:心肌多点注射PBS(对照组),n=3
(2)细胞数量:
实验组1:每只小鼠注射细胞总量是,MEFLC(7x105cells)+hiPSc-CM(3x105cells),分3个位点注射。
实验组2:每只小鼠注射细胞总量是,hiPSc-CM(3x105cells),分3个位点注射。
(3)心肌注射步骤:a.小鼠腹腔注射三溴乙醇(1.25%,0.1ml/10g)进行麻醉,手术区脱毛,进行气管插管;b.进行左心室上中下三点(呈三角形)注射,每只小鼠注射30μL生理盐水或细胞;c.关胸,动物保温状态等待。
(4)活体成像步骤:活体成像步骤:
分别在注射后1天,3天,7天,14天,21天,28天利用SIimaging小动物活体成像系统进行活体成像检测小鼠心肌荧光表达。
检测成像前,小鼠采用腹腔注射三溴乙醇(1.25%,0.1ml/10g)进行麻醉,然后置入小动物活体成像仪
检测zs-Green荧光:
激发波长465nm,发射波长510nm,曝光时间60s。
【实验结果】
小鼠心肌组织zs-Green荧光强度-时间对照图如图6所示,在注射后21天和28天,MEFLC+hiPSc-CM组的荧光强度要显著高于hiPSc-CM组的荧光强度,说明MEFLC+hiPSc-CM组存活的hiPSc-CM要显著多于hiPSc-CM组,即MEFLC可以促进hiPSc-CM在小鼠心脏组织中的存活。
2.7MEFLC可以显著降低心梗(myocardialinfarction,MI)小鼠心脏组织中的胶原容积分数以及防止心梗后室壁变薄。
【实验目的】
研究MEFLC是否对MI小鼠有治疗效果。
【实验材料】
实验动物:C57BL/6J小鼠,雄性,20g±2g,12只。
【实验方案】
(1)实验分组:
实验组:MI组+心肌注射MEFLC(n=6);
对照组:MI组+心肌注射PBS(n=6)。
(2)细胞数量:
实验组中每只小鼠注射MEFLC 1×106cells,分3个位点注射。
(3)小鼠MI手术建模:腹腔注射三溴乙醇(1.25%,0.1ml/10g)进行麻醉,手术区脱毛,进行气管插管;左胸部切口,肌肉分离以暴露肋骨,第四和第五肋骨之肋间隙切口。然后打开胸部露出心脏,通过缝合线将左冠状动脉前降支永久性结扎形成心肌梗死模型。在左冠状动脉前降支周围注射PBS或MEFLC(30μL)。关闭胸腔后,动物保温状态等待苏醒。
(4)术后第7及28天搜集小鼠心脏组织进行切片及HE、Masson染色。
【实验结果】
(1)如图7所示,对术后7天的实验组和对照组的MI小鼠心脏组织切片进行Masson染色,获得病理切片扫描图像,利用ImageJ软件分析各组的胶原容积分数。实验组的平均胶原容积分数(21.51%)显著低于对照组的平均胶原容积分数(41.14%),n=6,p=0.0065。
(2)如图8所示,对术后28天的实验组和对照组的MI小鼠心脏组织切片进行HE染色,获得病理切片扫描图像,与实验组相比,对照组的室壁明显变薄(黑色箭头指示的是室壁变薄处)。我们认为单独注射MEFLC可以有抑制心肌细胞凋亡的作用,图8的结果也显示,MEFLC单独注射MI小鼠,减少了心室壁的变薄,意味着心肌细胞在心梗后死亡的数量是减少的。
Claims (7)
1.一种MEFLC细胞株,该细胞株于2022年4月23日保藏于广东省微生物菌种保藏中心,保藏编号为GDMCC NO:62409。
2.一种如权利要求1所述的MEFLC细胞株在制备治疗心肌细胞凋亡引起的心血管疾病的药物中的应用,其特征在于,所述心肌细胞凋亡引起的心血管疾病为心梗。
3.如权利要求2所述的应用,其特征在于,所述药物的剂型为注射液。
4.如权利要求2所述的应用,其特征在于,MEFLC细胞株作为心肌细胞回输的支持型细胞药物。
5.如权利要求3所述的应用,其特征在于,MEFLC细胞与心肌细胞混合注射或MEFLC细胞单独注射。
6.一种如权利要求1所述的MEFLC细胞株在复苏冻存心肌细胞时或在心肌细胞处于缺氧/活性氧/阿霉素的环境中作为降低细胞凋亡比例的添加剂的应用。
7.一种如权利要求1所述的MEFLC细胞株在制备治疗心梗的注射药物中的应用。
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