胎盘间充质干细胞与骨髓间充质干细胞分离培养和生物学特性研究

目的探讨兔胎盘间充质干细胞(placenta-derived mesenchymal stem cells,PMSCs)和兔骨髓间充质干细胞(bone marrow-derived MSCs,BMSCs)体外分离培养、增殖,对其生物学性状进行比较观察。方法取足月待产新西兰大耳白兔1只,采用密度梯度离心法及贴壁培养技术从兔胎盘对PMSCs进行分离、纯化和传代培养。取2周龄新西兰大耳白兔1只,采用直接贴壁法从后肢骨髓中对BMSCs进行分离、纯化和传代培养。用倒置相差显微镜观察两种细胞形态。免疫组织化学染色对第3代细胞表面标志(CD44、CD105、CD34、CD40L)进行鉴定。将BMSCs与PMSCs第2代细胞分别与生物衍生骨进行复合培养5d,每条材料接种(1.0~1.5)×106个细胞,苏木素染色观察细胞与材料复合培养情况。扫描电镜观察两种细胞分别与材料复合培养3d和8d的情况。结果在倒置相差显微镜下观察,两种细胞均为贴壁生长,形态为均一成纤维细胞样。PMSCs增殖力强,细胞的增殖能力随传代次数的增加而有所下降,细胞体外培养10代后,生长速度减慢。两种细胞均表达CD44、CD105,不表达CD34、CD40L。复合培养5d,PMSCs和BMSCs在生物衍生骨表面生长,大量黏附,细胞积聚成团,相互连接成网状,孔隙内也可见细胞生长和增殖,并分泌基质。扫描电镜观察:复合培养3d,可见较多量的细胞在生物衍生骨上黏附,呈梭形或多角形;8d两种细胞均已大量增长,呈层状排列,细胞连接紧密,分泌大量基质,细胞周围有较多的网状胶原形成。结论PMSCs与BMSCs有相似的生物学特性,可作为组织工程的另一成体干细胞来源。

EXPERIMENTAL STUDY ON ISOLATION AND CULTIVATION OF PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND BONE MARROW-DERIVED MESENCHYMAL STEM CELLS OF RABBIT AND THEIR BIOLOGICAL CHARACTERISTICS

OBJECTIVE: To explore a method to isolate, culture and multiplicate the placenta-derived mesenchymal stem cells (PMSCs) and the bone marrow-derived mesenchymal stem cells (BMSCs) of rabbit, and to compare their biological characteristics. METHODS: PMSCs were isolated from placenta of 1 fetation rabbit by Percoll density gradient centrifuge and cultured in vitro. BMSCs were isolated from hindlimb bone marrow blood of 1 new born rabbit by direct plates culture method. The 3rd passage PMSCs and BMSCs were observed by inverted phase contrast microscope. The stem cell marker (CD44, CD105, CD34 and CD40L) were examined by immunohistochemistry. The 2nd passage PMSCs and BMSCs were co-cultured with biomaterials, (1.0-1.5) x 10(6) cells in one biomaterial, and then observed by haematoxylin staining after 5 days, and by SEM after 3 days and 8 days. RESULTS: PMSCs and BMSCs were both uniformly spondle-shaped in appearance and showed active proliferative capacity. The proliferative ability of PMSCs were quite strong and declined with passages. After cultured 10 passages in vitro, its growth slowed. Both PMSCs and BMSCs expressed CD44 and CD105, but did not express CD34 and CD40L immunoreactivity. PMSCs and BMSCs poliferated and adhered to the surface of biomaterials, and cell formed clumps and network; the cells proliferation and the matrix were seen in the pore after 5 days of culture. The observation of SEM showed that many cells adhered to the biomaterials with spindle-shape and polygon after 3 days; and that PMSCs and BMSCs grew, arranged in layers and secreted many matrices; the reticular collagen formed arround cells after 8 days. CONCLUSION: PMSCs and BMSCs have similar biological characteristics and PMSCs can be served as excellent seeding-cells for tissue engineering.