Lentiviral-mediated Runx2 transfection of bone marrow mesenchymal stem cells in the repair of joint injury due to osteoarthritis

doi:10.3969/j.issn.2095-4344.0996

Abstract

Abstract:

BACKGROUND: Bone marrow mesenchymal stem cells as seed cells can differentiate into chondrocytes and osteoblasts. Runx2 can induce bone marrow mesenchymal stem cells to differentiate and mature into chondrocytes and osteoblasts, thus promoting bone healing.
OBJECTIVE: To investigate the effect of lentiviral-mediated Runx2 transferred into bone marrow mesenchymal stem cells in the repair of joint injury due to osteoarthritis.
METHODS: (1) Bone marrow mesenchymal stem cells were isolated and cultured from C57BL/6 mice. Lenti-Runx2-EGFP lentivirus (Runx2 group) was transfected into bone marrow mesenchymal stem cells, lentivirus without Runx2 gene (control group) was transfected, and a blank control group was setup. After 48 hours of transfection, the expression of green fluorescent protein was observed under the fluorescence microscope, and the transfection efficiency was calculated. The stably transfected cell line was screened by purinomycin. (2) A model of osteoarthritis was established in C57BL/6 mice by cutting off the anterior cruciate ligament of the knee joint. All of the model mice were randomly divided into normal saline group, bone mesenchymal stem cell group and Runx2-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 (1×10⁷), or 0.1 mL of normal saline containing Runx2-transfected bone marrow mesenchymal cells (1×10⁷) was injected into the knee joint cavity of mice in the corresponding group, respectively.
RESULTS AND CONCLUSION: (1) CD90 and CD105 were highly expressed in bone marrow mesenchymal stem cells. The transfection efficiency of Lenti-Runx2-EGFP lentivirus was (88.57±3.07)%, and the stably transfected cell line was screened by puromycin (4 mg/L). (2) The knee articular cartilage of mice was intact with smooth knee articular surface in the Runx2-transfected bone mesenchymal stem cell group, while knee articular cartilage degeneration and uneven knee articular surfaces were observed in the other two groups. (3) The chondrocytes decreased and the cartilaginous layer thinned in bone mesenchymal stem cell group, while a lot of cracks on the surface of the cartilage in the saline group. Runx2-transfected bone mesenchymal stem cell group had many chondrocytes and thickened cartilaginous layer, and the Mankin’s score was significantly lower than the other two groups. (4) The positive rate of type II collagen in Runx2-transfected bone mesenchymal stem cell group was significantly higher than that in the other two groups, while the positive rate of type X collagen was lower than the other two groups. (5) The mRNA and protein expressions of Runx2, bone morphogenetic protein-2, alkaline phosphatase, osteocalcin and osteopontin in Runx2-transfected bone mesenchymal stem cell group were significantly higher than those in the other two groups. To conclude, Runx2-transfected bone marrow mesenchymal stem cells can promote the repair of damaged cartilage, thus providing a new direction for the clinical treatment of 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: Osteoarthritis, Lentivirus Infections, Tissue Engineering

CLC Number:

R394.2

Duan Zhi-bin, Qiu Zhi-qiang, Peng Kun, Xu Ze-min, Cheng Ke, Chen Xiang, Shi Qing-ming. Lentiviral-mediated Runx2 transfection of bone marrow mesenchymal stem cells in the repair of joint injury due to osteoarthritis[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(29): 4607-4613.

Figures/Tables 2

2.1 骨髓间充质干细胞形态观察和鉴定第3代骨髓间充质干细胞，形态基本一致，呈长梭形，“鱼群”样朝向同一样方向排列，见图1。流式细胞仪检测显示，骨髓间充质干细胞CD90阳性率99.6%，CD105阳性率99.4%，见图2。 2.2 Runx2慢病毒转染骨髓间充质干细胞及稳定转染细胞株筛选转染48h后，Runx2组和阴性对照组转染效率分别为(88.57±3.07)%和(89.43±2.09)%，并经最佳浓度的嘌呤霉素(4 mg/L)筛选得到了稳定转染细胞株，见图3。 2.3 实验动物数量分析各组10只小鼠在实验期间均无脱失，均结果结果分析。 2.4 膝关节X射线结果植入2周后，生理盐水组小鼠膝关节软骨退变明显，有缺损迹象，关节面不平整(图4A)；单纯骨髓间充质干细胞组软骨退变程度较生理盐水组轻微，尚未发现缺损迹象，关节面较平整(图4B)；Runx2转染骨髓间充质干细胞组软骨完整，未见明显退化，关节面平整(图4C)。 2.5 膝关节软骨组织形态及Mankin’s评分植入2周后，生理盐水组软骨表层存在大量裂隙(图5A)；单纯骨髓间充质干细胞组软骨细胞稍减少，软骨层稍变薄(图5B)；Runx2转染骨髓间充质干细胞组软骨细胞多，软骨层厚(图5C)。 Runx2转染骨髓间充质干细胞组Mankin's评分为(2.47±0.56)分，显著低于单纯骨髓间充质干细胞组和生理盐水组[分别为(4.17±0.48)，(6.82±0.63)分]，差异有显著性意义(F=153.231，t=6.788、17.369，P < 0.001)。 2.6 膝关节软骨组织Ⅱ型及Ⅹ型胶原蛋白表达植入2周后，免疫组化染色结果显示，Runx2转染骨髓间充质干细胞组Ⅱ型胶原蛋白阳性率显著大于单纯骨髓间充质干细胞组和生理盐水组(t=14.347，23.484，均P < 0.001)；而Ⅹ型胶原蛋白阳性率显著小于单纯骨髓间充质干细胞组和生理盐水组(t=10.823，22.461，均P < 0.001)，见表1，图6。 2.7 膝关节滑膜组织Runx2、骨形成蛋白2、碱性磷酸酶、骨钙素和骨桥蛋白mRNA表达水平植入2周后，反转录PCR检测结果显示，Runx2转染骨髓间充质干细胞组Runx2、骨形成蛋白2、碱性磷酸酶、骨钙素和骨桥蛋白mRNA表达显著高于单纯骨髓间充质干细胞组和生理盐水组(P < 0.001)，见表2。 2.8 膝关节滑膜组织Runx2、骨形成蛋白2、碱性磷酸酶、骨钙素和骨桥蛋白表达水平植入2周后，蛋白印迹法检测结果显示，Runx2转染骨髓间充质干细胞组Runx2、骨形成蛋白2、碱性磷酸酶、骨钙素和骨桥蛋白表达显著高于单纯骨髓间充质干细胞组和生理盐水组(P < 0.001)，见表3，图7。"

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