Optimization of the protocols for induction and differentiation of bone marrow mesenchymal stem cells into chondrocytes: a synergistic action between transforming growth factor beta1 and insulin-like growth factor 1

doi:10.3969/j.issn.2095-4344.0512

Abstract

Abstract:

BACKGROUND: Inducing factors are currently used as a main method for the differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes.
OBJECTIVE: To investigate the collaborative stimulation of transforming growth factor beta1 (TGF-β1) and insulin-like growth factor 1 (IGF-1) to induce the directed differentiation of BMSCs to chondrocytes, and to explore the best inductive effect.
METHODS: Rat BMSCs were isolated, cultured and purified using adherent culture. Then, different inducing factors were added in the induction medium: TGF-β1+IGF-1 group, TGF-β1 group, IGF-1 group, and control group without growth factors. Immunofluorescence was carried out at 21 days of induction. The expression of collagen type II was evaluated by immuncytochemical staining at 7, 14 and 21 days of induction.
RESULTS AND CONCLUSION: (1) Immunofluorescence detection of the TGF-β1+IGF-1 and TGF-β1 groups showed highly expressed collagen type II (brown red-stained cytoplasm), while negatively expressed collagen type II in the other two groups. (2) Findings from the immuncytochemical staining showed that the expression of collagen type II was stronger in the TGF-β1+IGF-1 group than the TGF-β1 group (P < 0.01), and the expression was gradually enhanced with time. Meanwhile, there was also no expression of collagen type II in the IGF-1 and control groups. To conclude, the combination of TGF-β1 and IGF-1 can achieve the better inductive effect on the chondrogenic differentiation of BMSCs in vitro.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Chondrocytes, Cell Differentiation, Insulin-Like Growth Factor I, Transforming Growth Factor beta1, Tissue Engineering

CLC Number:

R394.2

Tan Rong-bang, Chen Hong-ming, Luo Shi-guan, Li Ri-zhu, Zeng De-chuang. Optimization of the protocols for induction and differentiation of bone marrow mesenchymal stem cells into chondrocytes: a synergistic action between transforming growth factor beta1 and insulin-like growth factor 1[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2631-2636.

Figures/Tables 1

2.1 骨髓间充质干细胞诱导前后形态学变化原代培养24 h后首次换液，去除未贴壁细胞，倒置显微镜下见贴壁细胞呈分散、克隆集落样生长，大部分细胞形态呈梭形、三角形，小部分细胞形态呈圆形，细胞增殖迅速。3-5 d后大部分细胞呈纺锤形成纤维样细胞形态，可见明显细胞集落形成，细胞呈放射状排列，有突起。随着细胞增殖，细胞集落间互相融合。7-10 d后细胞长满瓶底80%-90%进行传代。传代细胞开始呈圆形，贴壁后伸展为梭形，细胞核居中，有1个或2个核仁。第3代后细胞纯化，杂质细胞极少见，细胞形态均一，极个别细胞呈宽大多边形，见图1A。诱导7 d后细胞形态开始发生改变，逐渐转变成三角形、星形、多边形，随着诱导时间延长，三角形、星形、多边形细胞逐渐增多，21 d后绝大多数细胞呈三角形、星形、多边形细胞形态，可见多个突起，见图1B。 2.2 骨髓间充质干细胞鉴定结果流式细胞仪检测结果显示实验所培养的细胞广泛高度表达CD90，CD29，阳性率分别为99.6%，99.7%，而极少表达CD34，CD45，阳性率分别为0.1%，0.7%，见图2。免疫荧光显示骨髓间充质干细胞表达CD90，CD29在99%以上，而不表达CD34，CD45，见图3。说明实验中分离培养的细胞是非造血干细胞的骨髓间充质干细胞。 2.3 骨髓间充质干细胞诱导分化为软骨细胞的鉴定 2.3.1 Ⅱ型胶原免疫荧光染色诱导21 d后，TGF-β1+ IGF-1组、TGF-β1组免疫荧光显示胞浆呈棕红色，高度表达Ⅱ型胶原，而IGF-1组和对照组结果呈阴性，见图4。 2.3.2 Ⅱ型胶原免疫细胞化学染色 TGF-β1+ IGF-1组、TGF-β1组Ⅱ型胶原染色呈阳性，胞浆见棕黄色、棕褐色颗粒，近胞核处明显。而IGF-1组和对照组结果呈阴性，见图5。以JEDR8010D形态图像分析软件VESION1.0扫描图片，结果见表1。TGF-β1+ IGF-1组Ⅱ型胶原表达量明显高于TGF-β1组(P < 0.01)，且随着时间的延长，表达量逐渐增加，表明TGF-β1与IGF-1共同诱导骨髓间充质干细胞定向分化为软骨细胞时两者具有协同刺激作用。"

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