人脂肪干细胞体外向成骨细胞定向诱导分化的实验研究

Experiment of osteogenic differentiation of human adipose stem cells in vitro

目的:探讨人脂肪干细胞(hASCs)的分离方法、体外增殖能力及定向诱导分化为成骨细胞的潜能。方法:从脂肪抽吸物中分离、培养具有多向分化潜能的脂肪干细胞,比较原代(P0)及传代第1、2、5代(P1、P2、P5)细胞的生长曲线。实验组取P2细胞,应用成骨诱导液向成骨细胞定向诱导分化,并以未诱导组为对照组,利用ALP染色、von Kossa染色、免疫荧光检测、RT-PCR等方法对细胞成骨潜能进行评价。结果:传代细胞较原代细胞增殖速度快。P1、P2、P5均具有一些共同特征,传代培养的潜伏期约为24h,传代培养细胞的对数增殖期约为2～3d,接种后第4～5天进入平台期;细胞诱导14d后,实验组ALP染色呈阳性反应,对照组阴性;von Kossa染色,实验组出现深棕色结节状沉积,且随诱导时间增加而加深,对照组无结节状沉积出现;免疫荧光检测,实验组和对照组均表达Ⅰ型胶原,实验组骨钙素(OCN)、骨桥蛋白(OPN)检测为阳性,对照组为阴性或弱阳性;RT-PCR检测表明,实验组诱导14d时有ALP、Osteopontin表达,对照组阴性;实验组、对照组及成骨细胞均有I型胶原阳性表达。结论:hASCs在体外培养条件下生长良好,P2细胞在诱导培养下可向成骨细胞分化,为以hASCs为种子细胞构建组织工程骨奠定了基础。

PURPOSE： To explore the feasibility of isolation, proliferation and osteogenic potential of human adipose stem cells （hASCs） in vitro. METHODS： hASCs were isolated from the adipose tissue of liposuetion aspirated by means of enzymatic digestion and proliferated in vitro. Morphology and growth kinetics by hemaeytometer of primary and continuous cells were observed. Osteogenic differentiation of hASCs cultured in conditional culture medium was evaluated by Alkaline phosphatase （ALP） staining and Von Kossa for mineralization, immunohistochemistry of osteocalcin （OCN）, ostseopotin （OPN） and collagen type I for matrix maturation, which was further eonfirmed by RT-PCR analysis for markers of osteogenie differentiation, with the non-osteogenic induced cells as controls. RESUITS： It was shown that both primary and passage cells shared similar fibroblastie-like morphology, while passage cells grew faster. In osteogenie differentiation bioassay, there was a significant difference in expression of ALP, OPN, and OCN between groups when the culture time was extended to 14 days, and collagen type Ⅰ was expressed in both groups. CONCLUSIONS： These results indicate that the hASCs grow well in vitro and could be induced to osteoblastic differentiate, hASCs can serve as a seeding cell source for bone engineering.