摘要
目的探索体外培养和扩增同一来源内皮细胞和平滑肌细胞的有效方法,研究联合培养的内皮细胞对平滑肌细胞形态和增殖的影响。为研究内皮细胞和平滑肌细胞相互关系和体外构建组织工程血管提供理论和实践基础。方法在体外用酶消化法和组织块贴壁法分别建立内皮细胞和平滑肌细胞的原代,并应用胰蛋白酶和乙二胺四乙酸钠传代培养。应用光镜、透射电镜、细胞计数和MTT法对内皮细胞和平滑肌细胞的形态和代谢进行研究。模拟血管壁的内层结构及内皮细胞和平滑肌细胞间的相互影响途径,建立以聚对苯二甲酸乙二醇酯(PET)膜为介质的内皮细胞与平滑肌细胞联合培养模型。分3组:内皮细胞,平滑肌细胞、平滑肌细胞/平滑肌细胞和空白/平滑肌细胞,应用3H-TdR掺入法研究联合培养的内皮细胞对平滑肌细胞增殖的影响。结果酶消化法和植块培养法可以有效地建立起内皮细胞和平滑肌细胞的原代。内皮细胞对联合培养的平滑肌细胞早期增殖有促进作用,晚期则增殖缓慢。结论以PET膜为介炙联合培养同一来源的内皮细胞和平滑肌细胞的新模型是研究两者间相互影响的良好模型。内皮细胞和平滑肌细胞作为血管构成的基本细胞,对于它们的形态学、体外培养和扩增以及相互关系的研究具有重要的意义。
Objective To develop effective method for culture and proliferation in vitro of endothelial cell （EC） and smooth muscle cell （SMC） obtained from an aorta and to study the effect of EC co-culture on SMC proliferation and morphology, this experimentation provides theoretical and practical foundation for studying mutual relation of EC and SMC and building tissue engineering blood vessel. Methods To establish primary cultures of EC and SMC by dissociating cell and explanting pieces of tissue individually, and employing 0.1% trypsin and 0.02% EDTA to subculture. Morphology and metabolism of EC and SMC were studied by use of light microscopy and election microscopy and MTT. EC and SMC had grown to 2 - 4 passages from primary euhures, and plated on either side of a porous 13μm thick polyethylene terephthalate （PET） membrane. SMC were studied in co-culture opposite from confluent EC （EC/SMC）. Controls included SMC cultured opposite SMC （SMC/SMC） or SMC alone （O/SMC）. After co-culture were established,SMC were harvested at the second day,the third day and the fifth day after release from growth arrest （n = 7 culture/group） each day. SMC proliferation was indexed by ^3H-thymidine incorporation. Results Dissociating cell by collagenase and explant pieces of tissue could validly build primary cultures of EC and SMC. EC stimulated SMC proliferation more than SMC/SMC and O/SMC cultures at early stage, and the rate of SMC proliferation was slow at late stage. Conclusion Since the co-culture model amples the effect of ECs on SMCs,it may be more useful than conditioned media to study EC-SMC interactions. It is very significant to study how to culture and proliferate endothelial cell and smooth muscle cell and mutual relation and morphology, for those are essential vascular cells.
出处
《哈尔滨医科大学学报》
CAS
北大核心
2005年第4期303-307,共5页
Journal of Harbin Medical University
基金
国家自然科学基金资助项目(30170970)国家杰出青年基金资助项目(30325042)黑龙江省自然科学基金资助项目(D00-21)黑龙江省杰出青年基金资助项目(JC-02-07)
关键词
内皮细胞
平滑肌细胞
联合培养
血管
形态学
endothelial cell
smooth muscle cell
co-culture
blood vessel
morphology
