人髁突来源骨髓间充质干细胞的分离与鉴定

The isolation and characterization of mesenchymal stem cells derived from human condyle

目的探讨人髁突来源骨髓间充质干细胞(MSCs)分离、培养和鉴定方法以及多向分化能力,为骨组织工程提供种子细胞。方法取髁突良性肥大患者被切除的髁突,冲洗收集骨髓细胞,采用密度梯度离心和贴壁培养法进行培养和纯化MSCs,取生长良好的P3或P4代MSCs进行检测:(1)MTT法测定细胞增殖和生长曲线分析;(2)流式细胞仪检测MSCs细胞表面抗原标记和细胞增殖周期;(3)脂肪细胞、成骨细胞和神经细胞多向诱导分化及相关检测。结果(1)利用密度梯度离心和贴壁筛选的方法从人髁突分离MSCs,经传代后细胞形态呈梭形,成纤维细胞样,均匀一致;(2)MTT法测定细胞增殖和生长曲线分析结果显示MSCs在传代后经历游离期、贴壁期、潜伏期、对数生长期和平台期;(3)流式细胞仪检测结果显示所获得的MSCs纯度较高,细胞单一。CD44、CD29和CD55表面标记阳性率分别为100%、99.8%和86.6%,而CD34、CD45和CD106的阳性率仅为0.5%、2.9%和6.3%;(4)MSCs细胞周期检测结果为G0期和G1期91.9%、G2期2.4%和S期5.7%,说明大部分细胞仍处于静止期;(5)成脂诱导后细胞胞浆内可见脂滴,苏丹黑B、油红O染色均呈阳性;成骨诱导后,Vonkossa和茜素红染色均呈阳性;神经细胞诱导后,细胞由梭形变为有多个突起的神经元样细胞,甲苯胺蓝尼氏和GFAP免疫组化染色均为阳性。结论从人髁突骨髓中能分离出高度一致的MSCs,这些细胞具有多向分化能力,可作为骨组织工程的种子细胞。

Objective To investigate the methods of isolation, cultivation and characterization as well as the ability of muh-directional differentiation of mesenchymal stem cells （MSCs） derived from human condyle with hyperplasia, so as to pave the way for searching the new source of seed cells in bone tissue engineering. Methods Bone marrow were collected by irrigation from resected human condyle, mononuclear cells were isolated by density gradient centrifugation and cultured in vitro. The high purity of MSCs were obtained by keeping the adherent cells and removal of nonadherent cells repeatedly. P3 or P4 MSC populations were collected and examined as follow： （1）Drawing a growth curve of the MSCs by the methods of MTT. （2）Detecting expressed the surface marks and the growing circle of MSCs by flow cytometric analysis. （3）The effieiencies of muh-directional differentiation into adipocytes, osteoblast and neuron-like cell were assessed by related methods. Results （1）The highly uniformed MSCs were obtained by density gradient centrifugation and keeping the adherent cells derived from human condyle bone marrow and the MSCs showed long fnsiform and fibroblast-like shaped after repeated passages. （2）The growth curve by MTI＂ displayed that every passage experienced dissociation period, adherence period, incubation period, log phase and platform period. （3）The cell surface marks assay with flow cytometry indicated that these MSCs expressed CD44, CD29 and CD55 in high percentage （about 100%, 99.8% and 86.6% respectively）, and expressed CD34, CD45 and CD106 in low percentage （about 0.5%, 2.9% and 6.3% respectively）. （4）The cell circle assay displayed G0 ＋ G1 stage was in high percentage （about 91.9%）, but G2 and S stage were in low percentage （about 2.4% and 5.7% respectively）. （5） Under inductive medium in vitro, the MSCs could be induced into adipocyte which were positive when stained by Sudan black B and oil red O; into osteoblasts which were positive when stained by Von kossa and alizarin red; as well as into neuron-like cell which were positive when stained by Toluidine Blue and GFAP immunohistochemistry. Conclusions The highly uniformed MSCs derived from human condyle can be obtained. These MSCs possess the ability of multdirectional differentiation, and can be used as a new source of seed cells in bone tissue engineering.