Objective: To observe the effect of superparamagnetic iron oxide (SPIO) on the differentiation of rat bone marrow stem cells (BMSCs) into chondrocytes in vitro and explore the possible mechanism.
Methods: CCK8 assay was performed to examine the cytotoxicity of SPIO (1 and 5 µg/mL) on cultured SD rat BMSCs. Prussian blue staining and fluorescence excitation assay were used to assess the binding of the SPIO to BMSCs after the cells had been cultured in chondrocytes-induced medium in the presence of SPIO (1 and 5 µg/mL) for 9 days. The mRNA levels of COL2 α2, aggrecan and MMP13 in the cell culture were examined using Q-PCR, and the chondrogenic differentiation of the BMSCs was analyzed using alcian blue staining and immunofluorescence staining for COL2 α2. The protein levels of COL2 α2, aggrecan, MMP13, Ihh and PTHrP in the cells were examined using Western blotting.
Results: CCK8 assay showed no significant toxicity of SPIO on BMSCs. Compared with the control cells, the cells cultured in the presence of SPIO showed increased expressions of COL2 α2 and aggrecan and decreased expression of MMP13 at both mRNA and protein levels with also significantly increased expressions of Ihh and PTHrP proteins.
Conclusion: SPIO can promote the differentiation of rat BMSCs into chondrocytes and up-regulate the Ihh/PTHrP signal pathway, suggesting the potential of SPIO as a new therapeutic agent for chondrocyte-related diseases.
目的: 探讨超顺磁氧化铁（SPIO）纳米微粒对SD大鼠骨髓间充质干细胞（BMSCs）成软骨分化的影响并探讨背后可能的机制。
方法: 体外培养SD大鼠BMSCs，Caspase-8比色测定工具包（CCK8法）检测SPIO（1和5 μg/mL）对BMSCs细胞毒性；SPIO（1和5 μg/mL）与成软骨培养基混合培养9 d，普鲁士蓝染色和自发荧光检测验证SPIO与BMSCs结合情况；荧光定量聚合酶链反应检测成软骨相关因子mRNA的表达量，如：二型胶原α2、聚集蛋白聚糖、基质金属蛋白酶13；体外行阿利新蓝染色及二型胶原α2免疫荧光染色检测BMSCs成软骨量；Western blot检测软骨标记蛋白二型胶原α2，聚集蛋白聚糖和基质金属蛋白酶13表达量以及Ihh/PTHrP信号通路蛋白Ihh、PTHrP表达量明确机制。
结果: 体外CCK8法检测提示SPIO（1和5 μg/mL）对BMSCs细胞活性无影响，差异无统计学意义（P＞0.05）；荧光定量聚合酶链反应检测二型胶原α2和聚集蛋白聚糖的mRNA表达提示，和对照组（单纯加入溶剂）相比，1 μg/mL SPIO的mRNA量明显增加，5 μg/mL SPIO的mRNA量大于1 μg/mL SPIO，基质金属蛋白酶13的mRNA表达出现浓度依赖抑制，和对照组相比1 μg/mL SPIO表达减少，5 μg/mL SPIO的mRNA量小于1 μg/mL SPIO；体外阿利新蓝染色和二型胶原α2免疫荧光染色提示阳性细胞随着SPIO浓度增加而增加，且均大于对照组（P＜0.05）；蛋白免疫印迹提示二型胶原α2和聚集蛋白聚糖蛋白表达随SPIO升高而升高，且均大于对照组，但基质金属蛋白酶13表达量随SPIO升高而降低，且均小于对照组，Ihh和PTHrP蛋白表达量随SPIO浓度增加而升高，且均大于对照组（P＜0.05）。SPIO促进Ihh/PTHrP信号通路，除CCK8法检测结果外均出现浓度依赖性。
结论: 超顺磁氧化铁纳米微粒在体外实验促进BMSCs向软骨细胞分化，并上调Ihh/PTHrP信号通路，为治疗软骨退变相关疾病提供可能的治疗手段。