Lu Ruinan, Wang Qian, Li Jianyong, Miao Dengshun
Department of Hematology, The First Affiliated Hospital of Nanjing Medical University Nanjing, China.
Research Center for Bone and Stem Cells, Key Laboratory for Aging & Disease, Nanjing Medical University Nanjing 211166, China.
Am J Transl Res. 2020 Mar 15;12(3):787-799. eCollection 2020.
Previous studies have reported that p27 deletion stimulates the proliferation of bone marrow mesenchymal stem cells (BM-MSCs) and their differentiation into osteoblasts, it also increases bone marrow hematopoietic progenitor cells (HPCs). However, it is unknown whether the enhanced hematopoiesis induced by p27 deficiency was associated with releasing hematopoietic stem cell (HSC) and HPC supporting factors by BM-MSCs. To answer this question, we cultured the BM-MSCs from wild-type (WT) or p27 knockout (KO) mice, analyzed their proliferation, apoptosis and osteogenesis and harvested their conditioned medium (CM); We also cultured the bone marrow cells (BMCs) with normal medium or CM from WT or KO BM-MSCs and analyzed changes of HSCs and HPCs and colony forming cells (CFCs). Our results showed that the proliferation and osteogenic differentiation of BM-MSCs were increased significantly and their apoptosis was reduced significantly in p27 deficient mice. Simultaneously, we demonstrated that the CM from p27 deficient BM-MSCs stimulated the expansion of HSCs/HPCs more dramatically than that from WT BM-MSCs. Five 2-fold up-regulated proteins were identified in the CM from p27 deficient BM-MSCs by protein chip assays, including interleukin-22 (IL-22), transforming growth factor-β type I receptor, tumor necrosis factor-related Apoptosis-inducing ligands, VE-cadherin and vascular endothelial growth factor B. We confirmed that expression of IL22 at both mRNA and protein levels were up-regulated significantly in p27 deficient BM-MSCs. The treatment of IL22 increased the percentages of HSCs and HPCs in BMC cultures and the number of CFCs in the colony formation assay, whereas the increased HSC/HPC expansion induced by the CM derived from p27 deficient BM-MSCs was blocked by the addition of anti-IL22 antibody in a dose dependent manner. We also found that the percentages of IL22R1, Stat3 and p-Stat3-S727 positive HSCs and HPCs were increased significantly in p27 deficient BMCs. Our findings in this study indicate that p27 deficiency stimulates HSC/HPC expansion by increasing secretion of IL22 by BM-MSCs and activating IL22-Stat3 signaling in HSCs and HPCs.
先前的研究报道，p27缺失可刺激骨髓间充质干细胞（BM-MSCs）增殖并分化为成骨细胞，还可增加骨髓造血祖细胞（HPCs）。然而，p27缺乏诱导的造血增强是否与BM-MSCs释放造血干细胞（HSC）和HPC支持因子有关尚不清楚。为了回答这个问题，我们培养了野生型（WT）或p27基因敲除（KO）小鼠的BM-MSCs，分析了它们的增殖、凋亡和成骨情况，并收集了它们的条件培养基（CM）；我们还用正常培养基或WT或KO BM-MSCs的CM培养骨髓细胞（BMCs），并分析了HSCs、HPCs和集落形成细胞（CFCs）的变化。我们的结果表明，p27缺陷小鼠中BM-MSCs的增殖和成骨分化显著增加，其凋亡显著减少。同时，我们证明，p27缺陷BM-MSCs的CM比WT BM-MSCs的CM更显著地刺激HSCs/HPCs的扩增。通过蛋白质芯片分析，在p27缺陷BM-MSCs的CM中鉴定出5种上调2倍的蛋白质，包括白细胞介素-22（IL-22）、转化生长因子-β I型受体、肿瘤坏死因子相关凋亡诱导配体、VE-钙黏蛋白和血管内皮生长因子B。我们证实，p27缺陷BM-MSCs中IL22的mRNA和蛋白水平均显著上调。IL22处理增加了BMC培养物中HSCs和HPCs的百分比以及集落形成试验中CFCs的数量，而添加抗IL22抗体以剂量依赖的方式阻断了p27缺陷BM-MSCs来源的CM诱导的HSC/HPC扩增增加。我们还发现，p27缺陷BMCs中IL22R1、Stat3和p-Stat3-S727阳性的HSCs和HPCs百分比显著增加。我们在本研究中的发现表明，p27缺乏通过增加BM-MSCs分泌IL22并激活HSCs和HPCs中的IL22-Stat3信号来刺激HSC/HPC扩增。
