非接触共培养脊索细胞诱导骨髓间充质干细胞向类软骨细胞分化的实验研究

目的:分离兔髓核脊索细胞(notochordal cells,NC)及骨髓间充质干细胞(mesenchymal stem cell,MSC),通过非接触共培养探讨NC对MSC细胞表型的影响。方法:4～6周龄新西兰兔8只,取胸腰段脊柱的髓核,用密度梯度离心提取NC,同时取其股骨骨髓用FICOLL液分离MSC,将NC和MSC等比例(1∶1)通过transwell培养板进行非接触共培养作为实验组,单纯MSC细胞培养作为对照组,光镜下观察细胞的生长情况。对两组的MSC行免疫组化及RT-PCR、Western-blot检测MSC细胞表型的改变情况。结果:原代NC呈圆形或椭圆形,细胞体积大,细胞增殖不明显;MSC贴壁生长,呈三角形或梭形,漩涡状排列。甲苯胺蓝染色:对照组MSC细胞核淡染,胞体染色不明显,染色阴性;实验组MSC可见从第3天开始胞体及胞外基质出现紫红色,第5天染色更加明显。Ⅱ型胶原免疫组化对照组MSC淡染,细胞形态不清楚;实验组第3天出现MSC内出现棕黄色深染,随着时间推移细胞染色加深呈阳性表现。RT-PCR检测,经过5d非接触共培养后实验组蛋白聚糖的基因表达为对照组的2.35倍(P<0.05),Ⅱ型胶原的基因表达为对照组的1.61倍(P<0.05),对照组Ⅰ型胶原的基因表达为实验组的2.56倍(P<0.05)。Western-blot检测后发现:经过5d非接触共培养,实验组蛋白聚糖的含量为对照组的1.61倍(P<0.05),Ⅱ型胶原的表达为对照组的10.04倍(P<0.05)(P<0.05)。结论:在非接触共培养条件下脊索细胞可以诱导骨髓间充质干细胞表型发生变化,向类软骨细胞方向分化,这将为组织工程化髓核的种子细胞筛选提供新选择。

Induction of mesenchymal stem cells to nucleus pulposus phenotype by indirect co-culture in vitro

Objectives: To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs) from immature nucleus pulposus(NP) of New Zealand rabbit,and to investigate the induction of notochordal cells to mesenchymal stem cells.Methods: Notochordal cells were harvested from immature NPs of 8 New Zealand rabbits(4-6 week-old) and purified by discontinuous gradient density centrifugation.MSCs were released from femur bone marrow of New Zealand rabbits and purified by discontinuous gradient density centrifugation.MSCs were cultured alone and non-contact co-cultured with NCs(1 ∶1) respectively,the expressions of collagenⅡand proteoglycan were determined by toluidine blue and immunocytochemistry staining.The expressions of collagenⅠ/Ⅱand proteoglycan in gene and protein level were detected.Results: NCs and MSCs were isolated and purified.NCs with diameter of 20-30μm had abundant intracytoplasmic vesicles and poor proliferation.MSCs showed as fusiform,arrayed with whirlpool.After indirect co-culture of NCs and MSCs for 3 days,the cells of experiment group demonstrated a robust cellular and extracellular matrix,with well-de fined cells that demonstrated intense metachromatic staining when stained with toluidine blue,which was in sharp contrast to the control and more obvious by continued co-culture.The indirect co-cultured cells also demonstrated positive immune reactivity to collagen Type II and brown particles were scattered in intracellular.After indirect co-culture for 5 days,The mRNA level of collagenⅠdecreased to a lower level in NC/MSC sample compared with the higher level in MSC sample.Collagen II and aggrecan gene expression increased significantly in NC/MSC sample,almost 1.61 and 2.35 fold than that in the control.After indirect co-cultured,MSCs from experiment group were observed expression of collagen Ⅱand proteoglycan compared with negative expression in the group of MSCs cultured alone.Conclusions: By co-culture,NCs can stimulate MSCs′ proliferation and differentiation toward nucleus pulposus cell,which may provide a new choice to cell therapy for intervertebral disc regeneration.