人胚胎心脏来源间充质干细胞的获取及其生物学特征

Harvesting and biological characteristics of mesenchymal stem cells derived from human fetal hearts

背景：目前尚缺乏关于人胚胎心脏来源间充质干细胞各代在体外生物学特征的报道。目的：以不同培养方法获取人胚胎心脏来源的间充质干细胞，并观察其生物学特征，筛选适合作为心肌修复细胞源的传代次数。设计、时间及地点：细胞学体外对比观察，在南京大学医学院和国家生物医药技术重点实验室完成。材料：流产人胚胎10例，均来自南京大学医学院附属鼓楼医院，经产妇及家属知情同意，实验获得医学伦理委员会批准。方法：取心脏组织，剪为约1mm的碎块，胶原酶消化过筛。获得单细胞悬液采用细胞培养法，向细胞悬液中添加10％FBS的DMEM／F12培养基;消化后的残余组织采取组织块培养法，放于培养板中，于37℃、体积分数为0．05的CO2培养箱中放置1．5h后．添加10％FBS的DMEM/F12培养基。两种方法均采取直接贴壁方式获取贴壁细胞，常规传代。主要观察指标：倒置显微镜观察心脏组织来源各代细胞的形态特征：流式细胞仪检测各代细胞表面标志，分析细胞周期。结果：细胞培养与组织块培养至P3代后，细胞形态相似且均呈梭形。细胞培养的P3和P4代以及组织块培养的细胞群中CD29和CD44阳性比例较高．而c—Kit、CD31、CD45、GATA-4和c—Tnt均呈阴性：细胞培养的P2和P3代细胞群间充质干细胞的比例最大．组织块培养的细胞中P3代细胞群间充质干细胞的比例最大;且P2-P4代细胞状态较好．当传至P7代时细胞生长速度开始减慢。原代-P5代细胞绝大部分处于静息状态，但保留自我更生的潜能，符合干细胞特性。结论：利用细胞培养法或组织块培养法都可以成功获得人胚胎心脏来源间充质干细胞，P3代细胞更适合予心脏修复的细胞移植治疗。

BACKGROUND： There are few studies on in vitro biological characteristics of human embryo heart-derived mesenchymal stem cells at different passages. OBJECTIVE： To obtain the mesenchymal stem ceils derived from fetal heart in vitro, to study their biological features, and to explore which passage cells is the best cell source for myocardial repair. DESIGN, TIME AND SETTING： The cytology in vitro control experiment was performed at the Medical School ＆ State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University. MATERIALS： Ten samples of aborted human embryo were obtained from Gulou Hospital of Medical College of Nanjing University. The informed consent was obtained from puerperants and their family members. The protocol was approved by Medical Ethics Committee. METHODS： The heart samples were cut into 1 mm blocks, digested with collagenase and cell suspension was prepared by cell culture method. The obtained cells were incubated in DMEM/F12 including 10% fetal bovine serum. The digested heart tissues were incubated by tissue culture method at 37 ℃ in 0.05 volume fraction CO2 incubator for 1.5 hours, and then incubated in DMEM/F12 containing 10% fetal bovine serum. By these two methods, adhered cells were collected by direct adherence method for conventional passage. MAIN OUTCOME MEASURES： The morphology of heart tissues-derived cells was observed with an inverted microscope. The imrnunophenotypes of cells in different phases were artalyzed by Flow Cytometry to analyze cell cycle. RESULTS： At the third passage, cells were spindle. The third and fourth passages of cells and cell mass cultured in tissue blocks, CD29 and CD44 were positively expressed, but c-Kit, CD31, CD45, GATA-4 and c-Tnt were negatively expressed. Most of the cells of the second and third passages were mesenchymal stem cells. Most of the cells of the third passage in tissue block culture were mesenchymal stem cells. The cells in the second, third and fourth passages were better than the other passages. From the seventh passage, cell growth became slow. From the primary to fifth passage, cells were in quiescent condition, but kept self-renewal potential, which were accorded with the characteristics of stem cells. CONCLUSION： The mesenchymal stem cells can be obtained from fetal heart by both cell culture and tissue culture methods. Mesenchymal stem cells of the third passage are much better for cell transplantation therapy of acute myocardial infarction.