与胚胎心肌共培养通过Cx43影响ESC源细胞间信号传导的研究
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摘要
背景：心血管疾病是一类严重威胁人类健康、危及人类生命的疾病。近年来干细胞移植疗法治疗缺血性心肌病成为研究的热点。许多种干细胞或干细胞分化而来的细胞都可作为移植的种子细胞。尽管移植细胞后心肌功能有所改善,但移植细胞的存活率极低,及与宿主细胞间整合不良,可能导致移植后心律失常的发生,远期预后不佳。这仍是一个有待解决的重要问题。
目的：研究小鼠胚胎心室肌细胞与小鼠胚胎干细胞之间的相互作用以及共培养对小鼠胚胎干细胞分化细胞间的缝隙连接形成、分布、功能方面的影响。
方法：本实验中,我们采用经典的悬滴—悬浮—贴壁法共培养小鼠胚胎干细胞(mES)与小鼠胚胎心室肌细胞(mEVs),在共培养期间显微镜下观察细胞生长情况、拟胚体(EBs)形态以及EBs的跳动情况；划痕负载染料迁移实验(SLDT)检测细胞间缝隙连接传导功能；从mRNA及蛋白水平研究心肌及缝隙连接相关基因的表达。免疫荧光染色确定连接蛋白的分布情况。从而研究细胞间相互作用以及mES分化而来的心肌细胞的功能。
结果：(1)在mEVs与mES细胞的共培养体系中,mES形成的拟胚体自发搏动的比率在分化中期较对照组明显增加；(2)共培养组与对照组间心肌标志物cTNT在mRNA水平上无统计学差异；(3)划痕实验显示共培养组中荧光黄染料在单位时间内沿划痕向两侧迁移距离较对照组远,说明共培养组细胞间缝隙连接传导作用增强；(4)分化中期,共培养组的连接蛋白Connexin43(Cx43) mRNA水平及总蛋白水平均较对照组有明显升高；(5)免疫荧光染色提示共培养组细胞间Cx43表达丰富,较对照组分布更规则。
结论：我们的研究结果发现与mEVs共培养能够促进mES分化而来的心肌细胞表达更多的Cx43,分布倾向于细胞两极,细胞间分布更加规则,组成的缝隙连接能够形成更为有效的信号通路。这可为心肌细胞移植疗法提供重要的研究基础,对改善移植细胞与宿主细胞间的整合状态,形成更具功能性的连接具有重要意义。
Background:Cardiovascular diseases is one of the most serious and life threatened diseases for human beings. Stem cell therapy for ischemic cardiomyopathy has been a hot research topic in recent years. A variety of stem cells or stem cell-derived cells can be used for transplantation as potential candidates. Despite improved cardiac function after transplantation, one of the major problems is still remained-the poor integration between host tissue and donor cells which can lead to serious post-transplantation arrhythmia and poor long-term outcome. Objective:To evaluate the cellular interaction between mES and mEVs, the formation and function of gap junction in mES-derived cardiomyocytes under cocultured conditions.
Methods:In the present study, we cocultured murine embryonic stem cells (mES) with murine embryonic ventricular myocytes (mEVs) byhanging drop method and investigate the cellular morphology, spontaneous beating status by microscopeobservation; intercellular gap junctional communication by scrape loading dye transfer (SLDT); the related genes and protein expression by PCR and Western blot; and connexin location by immunofluorescence staining. Thereby to study the cellular interactions between mES and mEVs and the function of mES-derived cardiomyocytes.
Results:We found that (1) whenmEVs are added to a culture system of embryonic stem cells, the number of spontaneously beating areas in embryonic bodies (EBs) increases at mid-developmentalstage;(2) mRNA level of cardiac marker cTNT shows no significant difference between coculture group and control group;(3) SLDT showsintercellular gap junction communication enhanced in coculture group;(4) Cx43expression upregulated at mid-developmental stage both at mRNA and total protein level;(5) and Cx43is distributed more orderly between cardiomyocytes in coculture group.
Conclusions:Our findings suggest mES-derived cardiomyocytes are able to express more Cx43and form effective signaling pathwayswhen coculture with mEVs which is importantfor providing more functional grafts for cardiac stem cells therapy byimproving the integration between transplanted and host cells.
目的：研究小鼠胚胎心室肌细胞与小鼠胚胎干细胞之间的相互作用以及共培养对小鼠胚胎干细胞分化细胞间的缝隙连接形成、分布、功能方面的影响。
方法：本实验中,我们采用经典的悬滴—悬浮—贴壁法共培养小鼠胚胎干细胞(mES)与小鼠胚胎心室肌细胞(mEVs),在共培养期间显微镜下观察细胞生长情况、拟胚体(EBs)形态以及EBs的跳动情况；划痕负载染料迁移实验(SLDT)检测细胞间缝隙连接传导功能；从mRNA及蛋白水平研究心肌及缝隙连接相关基因的表达。免疫荧光染色确定连接蛋白的分布情况。从而研究细胞间相互作用以及mES分化而来的心肌细胞的功能。
结果：(1)在mEVs与mES细胞的共培养体系中,mES形成的拟胚体自发搏动的比率在分化中期较对照组明显增加；(2)共培养组与对照组间心肌标志物cTNT在mRNA水平上无统计学差异；(3)划痕实验显示共培养组中荧光黄染料在单位时间内沿划痕向两侧迁移距离较对照组远,说明共培养组细胞间缝隙连接传导作用增强；(4)分化中期,共培养组的连接蛋白Connexin43(Cx43) mRNA水平及总蛋白水平均较对照组有明显升高；(5)免疫荧光染色提示共培养组细胞间Cx43表达丰富,较对照组分布更规则。
结论：我们的研究结果发现与mEVs共培养能够促进mES分化而来的心肌细胞表达更多的Cx43,分布倾向于细胞两极,细胞间分布更加规则,组成的缝隙连接能够形成更为有效的信号通路。这可为心肌细胞移植疗法提供重要的研究基础,对改善移植细胞与宿主细胞间的整合状态,形成更具功能性的连接具有重要意义。
Background:Cardiovascular diseases is one of the most serious and life threatened diseases for human beings. Stem cell therapy for ischemic cardiomyopathy has been a hot research topic in recent years. A variety of stem cells or stem cell-derived cells can be used for transplantation as potential candidates. Despite improved cardiac function after transplantation, one of the major problems is still remained-the poor integration between host tissue and donor cells which can lead to serious post-transplantation arrhythmia and poor long-term outcome. Objective:To evaluate the cellular interaction between mES and mEVs, the formation and function of gap junction in mES-derived cardiomyocytes under cocultured conditions.
Methods:In the present study, we cocultured murine embryonic stem cells (mES) with murine embryonic ventricular myocytes (mEVs) byhanging drop method and investigate the cellular morphology, spontaneous beating status by microscopeobservation; intercellular gap junctional communication by scrape loading dye transfer (SLDT); the related genes and protein expression by PCR and Western blot; and connexin location by immunofluorescence staining. Thereby to study the cellular interactions between mES and mEVs and the function of mES-derived cardiomyocytes.
Results:We found that (1) whenmEVs are added to a culture system of embryonic stem cells, the number of spontaneously beating areas in embryonic bodies (EBs) increases at mid-developmentalstage;(2) mRNA level of cardiac marker cTNT shows no significant difference between coculture group and control group;(3) SLDT showsintercellular gap junction communication enhanced in coculture group;(4) Cx43expression upregulated at mid-developmental stage both at mRNA and total protein level;(5) and Cx43is distributed more orderly between cardiomyocytes in coculture group.
Conclusions:Our findings suggest mES-derived cardiomyocytes are able to express more Cx43and form effective signaling pathwayswhen coculture with mEVs which is importantfor providing more functional grafts for cardiac stem cells therapy byimproving the integration between transplanted and host cells.
引文
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