同种异体肋软骨细胞与骨髓间充质干细胞共培养的研究

In vitro experimental study on co-culture of allogenic costal chondrocytes and bone marrow mesenchymal stem cells

目的探讨骨髓间充质干细胞(BMSCs)在体外与同种异体肋软骨细胞共培养的微坏境中向软骨细胞分化的可行性。方法分离、培养BMSCs和肋软骨细胞,取第二代BMSCs和肋软骨细胞2∶1混合为实验组,终质量浓度为3×108/L;另取相同浓度的单纯肋软骨细胞为对照组,1×10^8/L浓度的单纯肋软骨细胞为低浓度组,体外培养3个月,观察细胞形态变化,2周时取混合细胞进行组织学鉴定;1、2、3个月后取标本,行大体观察、组织学染色等对新生软骨进行评价。结果获取的两种细胞生物活性良好,实验组2周后BMSCs向软骨细胞转化,1个月后组织学检测显示3组在体外均形成了透明软骨,第1、2个月时实验组新生软骨的平均软骨细胞密度(72.667±1.862、98.333±1.506)均高于对照组(61.000±1.414、84.500±1.049)和低浓度组(49.833±1.941、75.000±0.894),对照组高于低浓度组,差异有统计学意义(P<0.05),3个月时3组间软骨细胞密度差异无统计学意义(98.500±1.049、98.333±0.817、97.333±0.817,P>0.05)。结论BMSCs在体外与同种异体肋软骨细胞共培养的微坏境下,被诱导分化为软骨细胞,两种细胞体外培养可形成透明软骨。

Objective To investigate the feasibility of bone marrow mesenchymal stem cells differentiating into chondrocyte in vitro under microenvironment of costal chondrocyte,co-cultured with allogenic costal chondrocyte.Methods Firstly bone marrow mesenchymal stem cells and costal chondrocytes were isolated and cultured.Then the second generation of bone marrow mesenchymal stem cells and costal chondrocytes were co-cultured at 2∶1 for the experimental group with the final concentration of 3×108/L.The costal chondrocytes with the same concentration were taken as the control group,and the chondrocytes with the concentration of 1×108/L were taken as the low concentration group.The cells were cultured in vitro for 1,2 and 3 months to observe the morphological changes.Two weeks later,the co-cultured cells were taken for histological identification.And after 1,2 and 3 months,the specimens were respectively taken for gross observation and histological staining to evaluate the new cartilage.Results The two kinds of cells had good biological activity and the bone marrow mesenchymal stem cells had transformed into chondrocytes 2 weeks later in the experimental group.And histological examinationshowed that hyaline cartilages were formed in all three groups in vitro one month later.The average density of chondrocyte in the experimental group was higher than that in the other two groups in the first and second months(72.667±1.862,98.333±1.506),and that in the control group(61.000±1.414,84.500±1.049)was higher than that in the low concentration group(49.833±1.941,75.000±0.894,P<0.05).and there was no significant difference among the three groups at three months(98.500±1.049,98.333±0.817,97.333±0.817,P>0.05).Conclusion BMSCs can be induced to differentiate into chondrocyte under the condition of co-culture with allogenic costal chondrocyte in vitro.The co-cultured cells can form a thin layer of hyaline cartilage in vitro.