从腰椎弓板和棘突分离培养人骨髓间充质干细胞的研究

摘要

目的 探讨从人腰椎弓板和棘突分离培养人骨髓间充质干细胞(hMSCs)的可行性.方法 采用密度梯度离心法,从3例人腰椎弓板和棘突中分离和培养hMSCs,并以1例从髂骨内分离的hMSCs作为对照,观察hMSCs的形态和传代能力,以流式细胞仪检测其表面标记,采用不同的诱导培养基研究并比较其向成骨细胞、脂肪细胞和软骨细胞分化的能力.应用Stata 10.0统计软件进行分析.结果 在人腰椎弓板和棘突中分离的hMSCs呈现典型间充质干细胞的形态,第6代和第2代细胞之间的老化细胞数比较差异无统计学意义(6.500±1.384比6.500±1.258,t=0.000,P＞0.05),经诱导可向成骨细胞、脂肪细胞和软骨细胞分化.其向成骨细胞分化时的茜素红染色阳性面积像素值显著低于对照hMSCs(32.658±3.14比61.362±8.952,t=6.674,P＜0.01);在成脂肪细胞分化时,单位面积的油红染色阳性细胞数显著多于对照hMSCs(27.250±2.493比7.750±1.753,t=18.100,P＜0.01);在三维培养下形成的肥大软骨细胞数I-hMSCs和L-hMSCs比较差异无统计学意义(18.833±3.933比19.500±5.252,t=0.227,P＞0.05).结论 从人的腰椎弓板和棘突可以分离hMSCs,其细胞特征和髂骨来源的hMSCs相同.在hMSCs的成骨分化和成脂肪分化之间存在反向关联.%Objective To study the feasibility of the isolation and culture of human bone marrow mesenchymal stem cells (hMSCs) from human vertebral arches and spinous processes.Methods Cells were isolated and cultured from three samples of human lumbar vertebral arches and spinous processes by density gradient centrifugation.The hMSCs isolated from the iliac crest were used as control cells.We observed the morphology and passaging ability of the cells,and used cytometry to detect their surface markers.To study and compare their ability to differentiate into osteoblasts,adipocytes,and chondrocytes,the cells were cultured in osteogenic,adipogenic and chondrogenic differentiation medium respectively.Statisticalanalysis of data using the Stata10.0 software.Results The cells isolated from human lumbar vertebral arches and spinous processes showed the typical morphology of mesenchymal stem cells.There was no significant difference in the number of aged cells between cells at passage 6 and those at passage 2 (6.500 ± 1.384 vs.6.500 ± 1.258,t =0.000,P ＞ 0.05).The cells were capable of differentiating into osteoblasts,adipocytes and chondrocytes.However,the pixels value of the alizarin red-stained area when differentiated into osteoblasts was significantly less than that of the control hMSCs (32.658 ± 3.147 vs.61.362 ± 8.952,t =6.674,P ＜ 0.01),while under adipogenic differentiation conditions the number of oil red O-stained cells per unit area was significantly greater than that of the control hMSCs (27.250 ± 2.493 vs.7.750 ± 1.753,t =18.100,P ＜0.01).There was no significant difference in the number of hypertrophic chondrocytes formed in three-dimensional culture (18.833 ± 3.933 vs.19.500 ± 5.252,t =0.227,P ＞ 0.05) under chondrogenic differentiation conditions.Conclusion hMSCs can be isolated from human lumbar vertebral arches and spinous processes,and are identical in cell characteristics to hM-SCs derived from iliac crest.There is an inverse tendency between osteogenic differentiation and adipogenic differentiation of hMSCs.