Intra-articular injection of Sox9-transfected bone marrow mesenchymal stem cells for treatment of knee osteoarthritis

doi:10.3969/j.issn.2095-4344.2017.05.014

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells are considered to have good proliferation and differentiation potentials. Sox9 is a transcription factor that is essential for chondrogenesis and has been termed as a “master regulator” of the chondrocyte phenotype.
OBJECTIVE: To study the therapeutic effects of Sox9-transfected bone marrow mesenchymal stem cells on knee osteoarthritis.
METHODS: The bone marrow mesenchymal stem cells were transfected with Lenti-Sox9-EGFP in vitro. The model of murine knee osteoarthritis was established by cutting off the anterior cruciate ligament. Thirty model mice were randomly divided into three groups, as normal saline group, bone mesenchymal stem cell group and Sox9-transfected bone mesenchymal stem cell group. 0.1 mL of normal saline, 0.1 mL of normal saline containing non-transfected bone marrow mesenchymal cells (non-transfected group), or 0.1 mL of normal saline containing Sox9-transfected bone marrow mesenchymal cells (Sox9-transfected group) was injected into the knee joint cavity of mice in the corresponding group, respectively. After 4, 8, 12 weeks, the repair of articular cartilage lesions was evaluated by toluidine blue and immunohistochemical staining.
RESULTS AND CONCLUSION: The lesions of articular cartilage were more serious in the normal saline group, compared with the other two groups, and the difference became more obvious over time. Damaged articular cartilage was improved in the non-transfected group, but the improvement was less than that in the Sox9-transfected group. Immunohistochemistry staining revealed that in the Sox9-transfected group, the positive type II collagen expression was stronger than that in the other two groups, but this positive expression was decreased over time in all the three groups. These results suggest that Sox9-transfected bone marrow mesenchymal stem cells promote the repair of damaged cartilage in mice with knee osteoarthritis.

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: Tissue Engineering, Osteoarthritis, Mesenchymal Stem Cells, Cartilage

CLC Number:

R394.2

Zhuo Qun-hao, Zhang Wei-na, Li Jian, Wang Hong-wei, Zheng Feng, Ying Bin-bin . Intra-articular injection of Sox9-transfected bone marrow mesenchymal stem cells for treatment of knee osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 736-741.

Figures/Tables 2

2.1 Sox9 基因转导的骨髓间充质干细胞鉴定经流式细胞仪鉴定显示，检测的标记物CD34(-)，CD45(-)，CD44(+)，CD105(+)，证明所培养的细胞为较纯的骨髓间充质干细胞。 2.2 基因转染效率在荧光显微镜下，骨髓间充质干细胞转导3 d后，可观察到绿色荧光蛋白阳性细胞最多，流式细胞仪检测其转染效率为65%, 免疫组织化学检测提示Sox9表达阳性。RT-PCR结果提示：Lenti-Sox9-EGFP转染骨髓间充质干细胞在转染后1周时Sox9表达率最高，并在其后3周内维持在较高水平。 2.3 关节软骨组织学变化甲苯胺蓝染色：注射后4周，生理盐水组甲苯胺蓝染色稍变浅，软骨表层有裂缝；单纯骨髓间充质干细胞组软骨细胞增多，软骨层增厚；Sox9转染骨髓间充质干细胞组与正常关节软骨比较无显著差异(图1)。注射后8周，生理盐水组软骨细胞减少，可见软骨细胞簇，排列紊乱，甲苯胺蓝染色变浅；单纯骨髓间充质干细胞组软骨细胞成簇增殖明显，甲苯胺蓝染色中度减退；Sox-9转染骨髓间充质干细胞组软骨细胞轻度减少，软骨层变薄，甲苯胺蓝色染色稍变浅(图2)。注射后12周，生理盐水组软骨细胞明显减少，软骨层剥脱，软骨下骨暴露，甲苯胺蓝色染色几乎消失；单纯骨髓间充质干细胞组软骨表层裂隙达软骨全层，甲苯胺蓝染色重度减退；Sox9转染骨髓间充质干细胞组软骨细胞减少，软骨表层有浅的裂隙，甲苯胺蓝染色中度减退(图3)。各组各时间点标本改良Mankin评分结果如表2所示。生理盐水组软骨损伤程度较单纯骨髓间充质干细胞组、Sox9转染骨髓间充质干细胞组明显严重，且时间越长差别越明显，单纯骨髓间充质干细胞组软骨损伤有改善，但改善情况差于Sox9转染骨髓间充质干细胞组，差异有显著性意义(P < 0.05)。"

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