小鼠胚胎心脏心外膜细胞体外培养模型的建立

doi:10.3969/j.issn.2095-4344.2013.44.001

摘要/Abstract

摘要：

背景：胚胎心脏心外膜细胞是最近被发现的具有分化为心肌细胞和血管平滑肌细胞潜能的心脏干细胞，可分化为心脏三系细胞，为心脏损伤的再生提供了新的细胞来源，但其定向分化机制及调控因素仍不清楚。

目的：体外建立小鼠胚胎心脏心外膜细胞培养模型。

方法：解剖显微镜下分离11.5-12.5 d小鼠胚胎心脏，剪除肺静脉血管、心房组织及左心室，移至6孔板(或35 mm培养皿)培养，24 h后移走胚胎心脏组织继续培养。相差显微镜观察胚胎心外膜细胞生长特点；使用免疫荧光技术对细胞进行胚胎心外膜细胞特异性抗体Wt-1、Tbx18染色。

结果与结论：胚胎心外膜单层细胞从组织块边缘长出，呈鹅卵石样，并围绕组织块向外延伸。移去胚胎心脏后细胞继续生长，且增殖迅速，三四天后长至融合。所有细胞均强阳性表达胚胎心脏心外膜细胞特异性抗体Wt-1及Tbx18。结果表明，胚胎心脏心外膜细胞生长迅速、形态单一，均表达心外膜细胞特异因子，细胞纯度高，成功构建了体外胚胎心脏心外膜细胞培养模型，为研究其定向分化的分子机制提供了新的思路。

关键词: 器官移植, 心肺移植, 心脏干细胞, 心外膜细胞, 细胞培养, 分化, 再生, 增殖, 信号通路, Wt-1, Tbx-18, 国家自然科学基金

Abstract:

BACKGROUND:The embryonic epicardium can differentiate into myocardial cells and the cardiac stem cells with the potential of vascular smooth muscle cells, and it can differentiate into cardiac three-line cells which provide a new cell source for the regeneration of cardiac injury. But the directed differentiation mechanisms and regulatory factors are still unclear.

OBJECTIVE: To establish the in vitro culture model of epicardial cells of mouse embryonic heart.

METHODS: Embryonic hearts were dissected from the mice at pregnant 10.5-11.5 days, and the pulmonary veins, atrial tissue and left ventricle were cut off, then the embryonic hearts were transplanted into the 6-well plates (or 35 mm dish) for culture. After cultured for 24 hours, the embryonic heart tissues were removed and cultured continuously. Phase contrast microscope was used to observe the growth characteristics of embryonic epicardial cells; the immunofluorescence technique was used to stain the specific antibody Wt-1 and Tbx18 ofembryonic epicardial cells.

RESULTS AND CONCLUSION: The embryonic epicardial cell monolayers grew from tissue block edge with cobblestone-like and extended outwardly around the tissue blocks. After removed the embryonic heart, the cells grew continuously with rapid proliferation, and got fusion at 34 days. All the embryonic epicardial cells could positively express the specific antibody Wt-1 and Tbx18 of embryonic epicardial cells. The results indicate that embryonic epicardial cells have the characteristics of rapidly growth and uniform morphology, and can express the embryonic epicardial cell specific antibody with high purity. The successfully constructed in vitro culture models of embryonic epicardial cells provide new idea for the molecular mechanisms of directed differentiation.

Key words: embryonic stem cells, cell culture techniques, regeneration, cell proliferation

中图分类号:

R318

曾彬，王艾丽，胡冬冬，李昌. 小鼠胚胎心脏心外膜细胞体外培养模型的建立[J]. 中国组织工程研究, 2013, 17(44): 7661-7666.

Zeng Bin, Wang Ai-li, Hu Dong-dong, Li Chang. Establishment of in vitro culture model of mouse embryonic heart epicardial cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(44): 7661-7666.

图/表（结果） 3

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引言

长期以来，成体哺乳动物心脏被认为是终末分化器官，心脏细胞的损伤坏死将由心肌细胞的肥大及瘢痕组织所取代^[1-2]。近年来，心肌梗死的年轻化和猝死令人吃惊。目前治疗主要是药物、介入治疗和搭桥手术，虽能改善症状，甚至使闭塞血管再通，却不能替代坏死心肌，心脏移植能彻底改善心脏状态，但临床开展难度很大。自从20世纪70年代末受损的蝾螈心脏可以由绞碎的心肌碎片成功修补以来，细胞移植治疗心脏疾病备受关注。近年来，新兴的细胞移植技术将自体或异体的心肌细胞移植入心肌梗死后的心肌瘢痕组织中，代替不可复生的坏死心肌，改善心脏功能。目前干细胞再生治疗成为研究热点，种子细胞的寻找限制着干细胞的运用^[3-4]。骨髓源成体干细胞、胚胎干细胞、诱导多能干细胞的研究虽然取得重大进展，但因其各自的局限性限制了临床运用^[5-7]；心脏干细胞优越的生物学特性逐渐吸引了研究者的兴趣^[8-9]。随着研究的进展，发现哺乳动物心脏存在具有内在再生和修复潜能的心脏干细胞；这些细胞在体内、外心脏环境下可被诱导分化为3种主要的心系细胞：心肌细胞、血管平滑肌细胞和血管内皮细胞，而且没有伦理问题和形成畸胎瘤的风险，是目前被认为用于干细胞治疗最佳的细胞类型^[10-11]。胚胎心外膜细胞是最近被发现的具有分化为心肌细胞和血管平滑肌细胞潜能的心脏干细胞^[12-13]，但其分化机制及调控因素尚不清楚。实验于体外建立小鼠胚胎心脏心外膜细胞培养模型，相差显微镜观察胚胎心外膜细胞生长特点，使用免疫荧光技术对细胞进行胚胎心外膜细胞特异性抗体Wt-1、Tbx18染色，以期为研究胚胎心脏心外膜细胞定向分化的分子机制提供新的思路。

材料方法

讨论

近年来心肌梗死患者逐渐增加，严重威胁着人类健康。心肌梗死是由于冠状动脉闭塞导致心脏血流供应中断而形成的以心肌细胞坏死为特征的疾病。心肌细胞是一种终末分化细胞，故心肌损伤后不能通过细胞再生获得修复，而是形成纤维化瘢痕。随后，梗死区心室壁延展变薄，发生心室重构，成为心肌梗死患者后期出现心功能不全和心源性死亡的主要原因。目前临床上应用的治疗方法如药物治疗(溶栓)、冠状动脉内介入治疗和冠状动脉旁路搭桥术可以使闭塞血管再通，梗死心肌血运重建，一定程度上限制和减轻心室重构的进展，改善了患者的症状，降低了病死率。但由于这些疗法无法促使梗死的心肌再生，使心肌梗死患者的远期预后不能得到显著改善。另一种治疗方法是心脏移植，因存在供体来源缺乏、免疫排斥、有创性及代价昂贵等问题而在应用上受到很大限制。因此迫切需要探寻能够修复与再生坏死心肌细胞、阻止或延缓心室重构和心功能衰竭发生的新疗法。心肌梗死患者的心肌细胞发生不可逆转的损伤，导致有效心肌能细胞数量下降，而又很难通过自身组织的再生来恢复心脏功能，因此人们尝试通过干细胞移植修复受损的心肌组织。干细胞移植治疗心肌梗死尚处于探索阶段，在选择干细胞方面还有待研究，近年来，各国科学家先后进行了骨骼肌干细胞、心肌细胞、胚胎干细胞、骨髓干细胞、外周血干细胞、诱导多能干细胞等实验，均取得了一定进展，但到底哪种细胞最适合治疗心肌梗死；何时进行干细胞移植治疗；采用哪种方法植入干细胞效果最好等问题都有待进一步研究。随着研究的深入，干细胞移植治疗将成为解决心肌梗死的有效手段。干细胞再生心肌治疗为心肌梗死提供了新的治疗方法，骨髓源成体干细胞、胚胎干细胞、诱导多能干细胞的研究虽然取得重大进展，但因其各自的局限性限制了临床应用。骨髓源成体干细胞研究虽已进展到临床阶段，但目前得到的结果尚不能在临床上推广，且其转分化为心肌细胞仍受到质疑^[19-21]；胚胎干细胞虽具有分化为三胚层所有细胞的潜能，但伦理道德问题、免疫排斥和形成畸胎瘤的风险限制了其临床应用^[22-25]；诱导多能干细胞虽解决了胚胎干细胞临床应用面临的前两个问题，但其研究还处于起步阶段，稳定性和安全性还有待于进一步观察^[26-28]；心脏干细胞优越的生物学特性逐渐吸引了研究者的兴趣。心脏干细胞移植试验性治疗缺血性心脏病作为一个新兴的研究领域，目前尚有许多问题需要解决：①对人体无损害又便于跟踪的移植细胞标记物。②移植的最佳途径和能够改善心脏功能的最佳移植细胞数量和细胞类型。③干细胞移植的临床适应证。④细胞移植后能否产生正常的心肌电-机械耦联，形成真正的功能合胞体。⑤干细胞移植后是否会在宿主体内长期存活、增殖或分化仍不清楚。 C-Kit⁺细胞是最早发现也是目前研究较多的心脏干细胞。细胞表达c-Kit一般被认为具有心脏多项分化潜能；分离心脏的c-Kit细胞，条件培养基培养后移植到大鼠急性心肌梗死心脏中，3周后治疗组左室射血分数明显改善^[29]。Sca-1⁺心脏干细胞表达Sca-1而不表达造血细胞表面因子和C-kit，Sca-1⁺细胞在体外虽不能自然分化为心脏细胞，但特定条件培养后部分细胞表达心肌细胞特异因子(Nkx2.5、TnⅠ和α-actin)；静脉注射的Sca-1⁺细胞，可以定向回到缺血再灌注心脏中并分化为心肌细胞^[30]。isl1⁺基因在心脏生长发育过程中有重要作用，且标记第二心区来源的心脏干细胞^[31-33]；在新生啮齿类和人的心脏里也能检测到这群isl1⁺细胞，且保持着未分化状态；与新生鼠的心脏细胞共培养后，这些细胞分化为可自发收缩的心肌样细胞；并与周围细胞形成电机械信号联系^[34]。胚胎心外膜细胞是最近被发现的具有分化为心肌细胞和血管平滑肌细胞潜能的心脏干细胞，但其分化机制及调控因素尚不清楚。心外膜细胞是位于心脏表面的一层间皮细胞组成的膜，在心脏发育过程中起着重要的作用，缺少心外膜的小鼠死于胚胎早期^[35-37]。低级动物斑马鱼心脏损伤试验发现，心外膜细胞在心脏损伤后可迁移入心肌层并分化冠脉血管，损伤的心肌细胞可被完整的修复^[38-41]；Wt-1及Tbx18在小鼠的心外膜前体细胞中表达，且在心外膜层细胞中持续表达；最近发现Wt-1及Tbx18标记的胚胎心外膜细胞体内外可以分化为心肌细胞、血管平滑肌细胞^[3-4]。Wt-1及Tbx18标记的胚胎心外膜细胞作为新发现的心脏干细胞为心脏损伤后的自我修复提供了崭新的思路和广阔的前景。明确心外膜细胞向心肌、血管平滑肌细胞分化的机制与调控因素，以提高心外膜源性心肌、血管平滑肌细胞的分化效能将加速心脏干细胞运用于临床。作者这次实验在体外建立了胚胎心外膜细胞的培养模型，相差显微镜观察胚胎心外膜细胞生长特点，使用免疫荧光技术对细胞进行胚胎心外膜细胞特异性抗体Wt-1、Tbx18染色。实验结果显示，细胞生长增殖迅速，几乎100%的细胞表达胚胎心外膜细胞特异因子；为进一步明确胚胎心外膜细胞定向分化分子机制提供了研究基础。