hMSCs成骨诱导前后分泌免疫相关细胞因子的实验研究

目的:研究hMSCs、DOC(osteogenic cells differentiated from mesenchymal stem cells,成骨诱导后骨髓间充质干细胞)分泌免疫相关细胞因子的变化,从细胞因子角度探讨hMSCs、DOC形成免疫抑制微环境的可能机理。方法:hMSCs、DOC(成骨诱导18天),采用ELISA技术,分别检测IL-2、IL-4、IL-10和TGF-β1分泌水平的变化。结果:在MSCs、DOC中IL-2、IL-4h低表达或忽略表达,IL-10中度表达,TGF-β1高表达。对IL-2、IL-4、IL-10,hMSCs、DOC的表达水平有统计学差异(P<0.05)。结论:在体外实验中,hMSCs、DOC可能形成免疫抑制微环境并通过其维持免疫调节功能。     

The effects of cyclin-dependent kinase inhibitors on adipogenic differentiation of human mesenchymal stem cells

The molecular mechanisms that couple growth arrest and cell differentiation were examined during adipogenesis. Here, to understand the cyclin-dependent kinase inhibitor (CKI) genes involved in the progression of adipogenic differentiation, we examined changes in the protein and mRNA expression levels of CKI genes in vitro. During the onset of growth arrest associated with adipogenic differentiation, two independent families of CKI genes, p27Kip1 and p18INK4c, were significantly increased. The expressions of p27Kip1 and p18INK4c, regulated at the level of protein and mRNA accumulation, were directly coupled to adipogenic differentiation. This finding was supported by the inhibition of adipogenic differentiation caused by short interfering RNA (siRNA). In this study, we investigated the regulatory effects of transforming growth factor beta-1 (TGFβ-1) on CKI genes involved in adipogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Only the up-regulation of p18INK4c during adipogenic differentiation, and not that of the p27Kip1 gene was prevented by treatment with TGFβ-1, one of the factors that inhibit adipogenesis in vitro. This finding indicates a close correlation between adipogenic differentiation and p18INK4c induction in hMSCs. Thus, these data demonstrate a role for the differentiation-dependent cascade expression of cyclin-dependent kinase inhibitors in regulating adipogenic differentiation, thereby providing a molecular mechanism that couples growth arrest and differentiation.     

Accelerated adipogenic differentiation of hMSCs in a microfluidic shear stimulation platform

The use of transplanted adipose tissue to repair crucial defects is clinically interesting for surgical reconstruction. Terminally differentiated adipocytes are utilized to promote the healthy regeneration of defective tissue. Use of differentiated mesenchymal stem cells, capable of differentiation into adipocytes, is advantageous because of their regenerative properties. Conventionally, the differentiation of hMSCs toward adipocytes occurs through chemical stimulation. We designed a microfluidic system, consisting of plastic tubing and a syringe pump, to create an environment of shear to accelerate this differentiation process. This system employed a flow rate equivalent to the accelerated flow rates found within the arterial system in order to promote and activate intracellular and extracellular proteins associated with the adipogenic lineage. Confirmation of sustained viability following shear exposure was obtained using a fluorescent live‐dead assay. Visualization of intracellular lipid accumulation was achieved via Oil Red O staining. When placed into culture, shear stimulated hMSCs were further induced toward brown adipose tissue, as evidenced by a greater quantity of lipid triglycerides, relative to unstimulated hMSCs. qRT‐PCR analysis validated the phenotypic changes observed when the hMSCs were later cultured in adipogenic differentiation media. Additionally, increased fold change for adipogenic markers such as LPL1, CFL1, and SSP1 were observed as a result of shear stimulation. The significance of this work lies in the demonstration that transient fluid shear exposure of hMSCs in suspension can influence differentiation into adipocytes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:440–446, 2016