CN115851595B - 过表达myog的细胞株在调控巨噬细胞极化方向中的应用 - Google Patents
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Abstract
本发明公开了一种过表达MYOG的细胞株在调控巨噬细胞极化方向中的应用，属于生物医药技术领域。本发明通过过表达MYOG基因，构建了一种能够调控巨噬细胞极化的细胞株，这种细胞株可以使巨噬细胞向M2方向极化，可以用于调节心肌梗死后早期的过度的炎症反应，促进心梗后的组织修复，改善心功能。
Description
技术领域
本发明属于生物医药技术领域，具体来说，涉及一种过表达MYOG的细胞株在调控巨噬细胞极化方向中的应用。
背景技术
心血管疾病(cardiovascular disease，CVD)对人类健康构成危害。心肌梗死(myocardial infarction，MI)简称心梗，是一种常见的急性心血管系统疾病，主要由于供应心脏血液流动的主要血管闭塞，血流中断，从而导致心肌的缺血性坏死。心肌梗死后心脏重构将可能引起心力衰竭、心脏破裂等严重后果。目前临床上由于经皮冠状动脉介入术(PCI)等治疗手段的广泛应用，急性心肌梗死早期病死率较前明显降低，而晚期由于心脏重构引起的心力衰竭及心脏破裂等病死率明显提高。
炎症和免疫应答是心肌损伤后的主要反应，巨噬细胞作为重要的天然免疫细胞，在心肌梗死后炎症反应及心脏重构中起重要作用。巨噬细胞源自外周血中的单核细胞，在不同微环境下可活化为2种巨噬细胞类型：经典活化(classical activation，M1型)巨噬细胞和替代活化(alternative activation，M2型)巨噬细胞。在心肌梗死后1～3d，即急性炎症期，心脏原位巨噬细胞和心肌细胞的大量死亡会招募血液中的中性粒细胞和促炎单核细胞向心脏聚集，此时M1型巨噬细胞数量增加，吞噬心梗部位的细胞碎片，同时分泌多种促炎细胞因子和趋化因子(如IL-1β、IL-6、IL-12、TNF-α等)，协助吞噬坏死细胞碎片。心肌梗死后3～7d，炎症阶段逐步转变为修复阶段，此时M2型巨噬细胞数量增加，并分泌转化生长因子-β(TGF-β)、血管内皮生长因子(VEGF)和IL-10等，促进损伤修复和瘢痕形成。虽然炎症反应对于清除坏死组织是必须的，但过度的炎症反应会导致梗死区室壁变薄，心肌梗死灶愈合不良，以致心功能受损。在心梗早期，促进巨噬细胞向M2型极化对于心梗修复具有保护作用，靶向巨噬细胞的治疗方案有巨大的应用前景。目前的研究已经报道的增加心肌M2型巨噬细胞的方法有心肌内注射成纤维细胞生长因子-2、肝细胞生长因子-12、FGF-9，静脉注射磷脂酰丝氨酸脂质体、心肌内间充质干细胞移植等，但这些技术扔处于研究阶段，尚未在临床上得到应用。
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。本实验室前期报道了过表达MYOG基因能够抑制由血管紧张素II诱导的心肌细胞凋亡，MYOG基因与巨噬细胞极化的关系尚未见报道。
发明内容
本发明所要解决的技术问题，就是提出一种过表达MYOG的细胞株在调控巨噬细胞极化方向中的应用。
本发明通过过表达MYOG基因，构建了一种能够调控巨噬细胞极化的细胞株-MEFLC细胞株，该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。这种细胞株可以用于调节心肌梗死后早期的过度的炎症反应，促进心梗后的组织修复，改善心功能。
附图说明
图1为本发明的技术路线图；
图2为流式细胞仪测定巨噬细胞标志物CD68的结果图；
图3为MEFLC与M1或M2巨噬细胞共培养的示意图；
图4为流式细胞仪检测常氧和缺氧条件下各组中HLA-DR和CD206的阳性细胞比例柱形图。
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细胞株构建(与CN115369077A专利申请相同)
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)。
1.6MEFLC使用培养条件②进行培养。
2.MEFLC调控巨噬细胞极化的研究，如图1所示，为本发明的技术流程示意图。
2.1外周血单核细胞获取：采用密度梯度离心法获取外周血单个核细胞(Peripheral Blood Mononuclear Cell，PBMC)，随后按照MojoSortTM Human CD14Nanobeads(Biolend，480093)产品说明书中记载的步骤分离出其中的CD14+单核细胞。
2.2单核细胞向M0型巨噬细胞极化：CD14+单核细胞用高糖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+M-CSF(25μg/mL)培养7-10天，可观察到细胞由悬浮状态转变为贴壁细胞，此时将细胞用0.05％胰蛋白酶消化后，使用流式细胞仪测定巨噬细胞标志物CD68比例大于98％(图2)，说明单核细胞成功极化为M0型巨噬细胞。
2.3M0型巨噬细胞向M1型巨噬细胞极化：M0型巨噬细胞用培养液高糖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+M-CSF(25μg/mL)+TNF-α(20μg/mL)培养1天后，更换培养液为高糖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+M-CSF(25μg/mL)+IFN-γ(20μg/mL)+LPS(50μg/mL)继续极化1天，即得M1型巨噬细胞。
2.4M0型巨噬细胞向M2型巨噬细胞极化：M0型巨噬细胞用培养液高糖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+M-CSF(25μg/mL)+IL-4(20μg/mL)+IL-10(20μg/mL)极化2天，即得M2型巨噬细胞。
2.4MEFLC与M1/M2型巨噬细胞共培养：以图3所示的方式将MEFLC与M1/M2巨噬细胞共培养。共培养前MEFLC接种在用培养条件②培养。共培养开始后培养液更换为高糖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，共培养时间为48小时。实验分为8组：①常氧-M1+MEFLC，②常氧-M1，③常氧-M2+MEFLC，④常氧-M2，⑤缺氧-M1+MEFLC，⑥缺氧-M1，⑦缺氧-M2+MEFLC，⑧缺氧-M2。用三气培养箱模拟缺氧环境，缺氧条件为94％ N2，1％ O2，5％ CO2。
2.5MEFLC培养上清对M1/M2巨噬细胞的影响：
2.5.1MEFLC培养上清制备：使用培养条件②培养MEFLC至汇合度约80％，更换培养液为高糖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继续培养48h，之后收集培养上清液用于下一步实验。
2.5.3MEFLC培养上清作用于M1/M2型巨噬细胞：将已完成极化的M1/M2型巨噬细胞更换含50％体积分数的MEFLC上清+50％体积分数的10％的FBS+体积含量1％的NEAA+体积含量1％的PS。上清处理时间为48h。实验分组为：①常氧-M1+MEFLC上清，②常氧-M2+MEFLC上清，③缺氧-M1+MEFLC上清，④缺氧-M2+MEFLC上清。同样用三气培养箱模拟缺氧环境，缺氧条件为94％ N2，1％ O2，5％ CO2。
2.6流式细胞术检测M1型巨噬细胞标志物(HLA-DR)和M2型巨噬细胞标志物(CD206)：按照Biolegend细胞表面染色方法对各组细胞进行染色，用流式细胞仪进行分析，结果如图4所示。
2.7实验结果与分析：如表1和图4所示，在常氧和缺氧环境下，与MEFLC共培养或用MEFLC培养上清处理可以降低M1和M2型巨噬细胞HLA-DR水平，增加M1型巨噬细胞CD206阳性比例，说明MEFLC能使巨噬细胞向M2方向极化。
表1 常氧和缺氧条件下MEFLC及培养上清对M1/M2型巨噬细胞极化的影响
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于这里的实施例，本领域技术人员根据本发明的揭示，对于本发明做出的改进和修改都应该在本发明的保护范围之内。
Claims (5)
1.过表达MYOG的细胞株或其培养上清液在制备调控巨噬细胞极化方向的产品中的应用，其特征在于，所述过表达MYOG的细胞株为MEFLC细胞株，该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。
2.如权利要求1所述的应用，其特征在于，细胞株调控巨噬细胞向M2型转化。
3.如权利要求2所述的应用，其特征在于，细胞株调控巨噬细胞由M1型向M2型转化。
4.如权利要求1所述的应用，其特征在于，所述产品为注射剂。
5.过表达MYOG的细胞株或其培养上清液在制备治疗心肌梗死的炎症性并发症的药物中的应用，其特征在于，所述过表达MYOG的细胞株为MEFLC细胞株，该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。
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