Li Yan, Liu Zhitao, Tong Zhaochen, Huang Heqing, Liu Zhendong
Department of Orthopedics, Third Xiangya Hospital, Central South University, Changsha 410013, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2018 Sep 28;43(9):971-981. doi: 10.11817/j.issn.1672-7347.2018.09.007.
To examine the growth activity and osteogenic differentiation of bone mesenchymal stem cells (BMSCs) in rats with Type 2 diabetes mellitus (T2DM) as well as the expression level of Dickkopf-1 (DKK-1) in bone marrow, and to explore the relationship between the osteogenic activity of BMSCs and the expression of DKK-1. Methods: The BMSCs were isolated from T2DM rats and were cultured in vitro. The BMSCs were divided into a T2DM group and a control group. The proliferation of BMSCs was detected by cell counting kit-8 (CCK8). Apoptosis rate was detected by annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining. In the osteogenic induction phase, the expression level of alkaline phosphatase (ALP) in BMSCs was detected by ALP staining and ALP activity assay kit. The osteogenic differentiation of BMSCs was analyzed by alizarin red staining and mineralized nodule quantification. In addition, the expression of Runx2 and DKK-1 in BMSCs was detected by qRT-PCR. Results: Compared with the control group, the proliferation of BMSCs was decreased and the apoptosis was increased in the T2DM group (both P<0.01). In the osteogenic induction process of BMSCs, the expression of ALP significantly decreased, the formation of calcium nodules reduced, and the expression of osteoblast transcription factor Runx2 was down-regulated in the T2DM group compared with those in the control group (all P<0.01). The levels of DKK-1 protein and mRNA were up-regulated in the T2DM group, which were higher than those in the control group (both P<0.01). The levels of DKK-1 protein and mRNA were related to the increase of Runx2 (both P<0.01). Conclusion: The growth activity of BMSCs and the potential of osteogenic differentiation are attenuated in the T2DM rats, which may be related to the increase of DKK-1 expression in BMSCs.
观察2型糖尿病（T2DM）大鼠骨髓间充质干细胞（BMSCs）的生长活性和成骨分化情况以及骨髓中Dickkopf-1（DKK-1）的表达水平，探讨BMSCs成骨活性与DKK-1表达之间的关系。方法：从T2DM大鼠中分离BMSCs并进行体外培养。将BMSCs分为T2DM组和对照组。采用细胞计数试剂盒-8（CCK8）检测BMSCs的增殖情况。通过膜联蛋白V-异硫氰酸荧光素（FITC）/碘化丙啶（PI）双染法检测凋亡率。在成骨诱导阶段，采用碱性磷酸酶（ALP）染色和ALP活性检测试剂盒检测BMSCs中ALP的表达水平。通过茜素红染色和矿化结节定量分析BMSCs的成骨分化情况。此外，采用qRT-PCR检测BMSCs中Runx2和DKK-1的表达。结果：与对照组相比，T2DM组BMSCs的增殖降低，凋亡增加（均P<0.01）。在BMSCs成骨诱导过程中，与对照组相比，T2DM组ALP表达显著降低，钙结节形成减少，成骨细胞转录因子Runx2表达下调（均P<0.01）。T2DM组DKK-1蛋白和mRNA水平上调，高于对照组（均P<0.01）。DKK-1蛋白和mRNA水平与Runx2的增加相关（均P<0.01）。结论：T2DM大鼠BMSCs的生长活性和成骨分化潜能减弱，这可能与BMSCs中DKK-1表达增加有关。
