Shao Bing-Yi, Wang Lan, Yu Yang, Chen Liang, Gan Ning, Huang Wen-Ming
Department of Operative Dentistry and Endodontics, Stomatological Hospital of Chongqing Medical University, Chongqing 400047, P.R. China.
Department of Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Stomatological Hospital of Chongqing Medical University, Chongqing 400047, P.R. China.
Exp Ther Med. 2020 Nov;20(5):84. doi: 10.3892/etm.2020.9212. Epub 2020 Sep 11.
The present study was designed to investigate the effects of T cells on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs were co-cultured with CD4 T cells that had been pretreated with anti-TNF-α or controls and were derived from ovariectomized (OVX) mice or sham control mice. MTT was used to assess the proliferative ability of BMMSCs and flow cytometry was used to analyze the BMMSC cell cycle. Following the induction of osteogenic differentiation in BMMSCs, calcium nodules were observed using alizarin red staining and alkaline phosphatase (ALP) staining. The expression levels of the osteogenesis-associated genes, runt related transcription factor 2 (Runx2) and osteocalcin (OCN) in BMMSCs were quantified using reverse transcription-quantitative PCR and western blotting. Osteogenesis-related signaling pathways, including ERK, JNK and p38 MAPK were also examined by western blotting. BMMSCs co-cultured with CD4 T cells from OVX mice exhibited reduced proliferative ability compared with sham mice and the cell cycle was arrested at the G2/M phase. Additionally, BMMSCs co-cultured with CD4 T cells from OVX mice presented with reduced levels of osteogenic differentiation and lower ALP activity, less calcium deposition and reduced expression of Runx2 and OCN compared with sham mice. The reduced levels of proliferation and osteogenic differentiation of BMMSCs induced by CD4 T cells were not seen when the T cells were had been pretreated with anti-TNF-α. The results indicated that CD4 T cells from OVX mice inhibited the proliferation and osteogenic differentiation of BMMSCs by producing high levels of TNF-α and may provide a novel insight into the dysfunction of BMMSCs caused by estrogen deficiency.
本研究旨在探讨T细胞对骨髓间充质干细胞（BMMSCs）增殖和成骨分化的影响。将BMMSCs与经抗TNF-α预处理的CD4 T细胞或对照组共培养，这些CD4 T细胞来源于去卵巢（OVX）小鼠或假手术对照小鼠。采用MTT法评估BMMSCs的增殖能力，流式细胞术分析BMMSC细胞周期。在诱导BMMSCs成骨分化后，用茜素红染色和碱性磷酸酶（ALP）染色观察钙结节。采用逆转录定量PCR和蛋白质印迹法检测BMMSCs中成骨相关基因 runt相关转录因子2（Runx2）和骨钙素（OCN）的表达水平。还通过蛋白质印迹法检测了包括ERK、JNK和p38 MAPK在内的成骨相关信号通路。与假手术小鼠相比，与OVX小鼠的CD4 T细胞共培养的BMMSCs增殖能力降低，细胞周期停滞在G2/M期。此外，与假手术小鼠相比，与OVX小鼠的CD4 T细胞共培养的BMMSCs成骨分化水平降低，ALP活性降低，钙沉积减少，Runx2和OCN表达降低。当T细胞用抗TNF-α预处理时，未观察到CD4 T细胞诱导的BMMSCs增殖和成骨分化水平降低。结果表明，OVX小鼠的CD4 T细胞通过产生高水平的TNF-α抑制BMMSCs的增殖和成骨分化，这可能为雌激素缺乏导致的BMMSCs功能障碍提供新的见解。
