Viability of bone marrow mesenchymal stem cells in inflammation-induced ischemia-reperfusion environment

doi:10.3969/j.issn.2095-4344.0659

Abstract

Abstract:

BACKGROUND: Local tissue ischemia-hypoxia and inflammatory microenvironment mainly contribute to the low survival rate of bone marrow mesenchymal stem cells.
OBJECTIVE: To study the effect of ischemia-hypoxia on viability of bone marrow mesenchymal stem cells in inflammatory environment and to explore the relationship between cell viability and inflammatory factors.
METHODS: The whole bone marrow culture method was used to separate and culture bone marrow mesenchymal stem cells and flow cytometry was used for cell identification. The third passage cells were treated by oxygen-glucose deprivation (OGD) preconditioning for different periods (6, 12 and 24 hours) as well as inflammatory induction (OGD, OGD+20 μg/L tumor necrosis factor alpha (TNF-α), OGD+100 μg/L lipopolysaccharide (LPS)). The treated cells were observed under inverted microscope. Cell viability was tested by MTT assay and flow cytometry. The levels of interleukin-6, interleukin-10, and TNF-α in the cell supernatant were measured by ELISA and the mRNA expression levels of interleukin-6, interleukin-10, TNF-α and nuclear factor-κB in the cells were detected by RT-PCR.
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells with a purity of 95% or more were successfully isolated and cultured. MTT and flow cytometry results showed that the cell viability was significantly increased after 6 and 12 hours of OGD, but was significantly decreased after 24 hours of OGD. The viability of bone marrow mesenchymal stem cells decreased after 6, 12, and 24 hours of OGD under the induction of TNF-α and lipopolysaccharide. The results of ELISA and RT-PCR showed that the levels of interleukin-6 and interleukin-10 in the cell supernatant were lowered after 6 and 12 hours of OGD and the gene expression of interleukin-6, interleukin-10, TNF-α, and nuclear factor-κB was lower compared with normal conditions. The levels of interleukin-6 and TNF-α in the cell supernatant increased significantly and the expression of interleukin-6, interleukin-10, TNF-α and nuclear factor-κB genes increased significantly after 6, 12 hours of OGD under TNF-α and LPS induction compared with simple OGD. Therefore, under the induction of TNF-α and lipopolysaccharide, the viability of bone marrow mesenchymal stem cells is affected by the expression and secretion of inflammatory factors. With the increase of the expression and secretion of inflammatory factors, the viability of bone marrow mesenchymal stem cells shows a decreasing trend.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Ischemia, Anoxia, Tumor Necrosis Factor-alpha, Lipopolysaccharides, Tissue Engineering

CLC Number:

R394.2

Zhang Ni, Chen Lan-ying, Li Xue-liang, Guan Zi-yi, Fang Cong, Zhou Meng-jing, Luo Ying-ying, Liu Rong-hua. Viability of bone marrow mesenchymal stem cells in inflammation-induced ischemia-reperfusion environment[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(33): 5259-5267.

Figures/Tables 4

2.1 原代BM-MSCs的培养、传代及鉴定结果接种24 h观察有大量悬浮细胞，见瓶底有少量细胞附着，细胞形态多为圆形，48 h观察细胞明显贴壁，贴壁细胞呈梭形，72 h观察有明显的集落形成，多为长梭形，5 d集落明显扩大，集落中间细胞密度增加，细胞开始呈螺旋或者放射性生长，约8 d细胞融合达到70%-80%，即可传代。传代细胞生长形态与原代相似，细胞生长状态良好，形态上多呈现长梭形或纺锤形，形态单一均匀，紧密贴于瓶底，呈现放射状或螺旋状生长(图1A，1B)。流式细胞仪对大鼠BM-MSCs表面抗原标志CD29、CD90、CD34、CD45表达分析(图1C)，结果显示CD29阳性表达率为99.8%，CD90阳性表达率为99.5%，而CD34、CD45阴性表达，其表达率分别为0.2%，4.7%。 2.2 显微镜下观察各组细胞损伤情况在6 h时，氧糖剥夺组、氧糖剥夺+肿瘤坏死因子α组、氧糖剥夺+脂多糖组BM-MSCs与正常组相比形态上无明显差异，贴壁良好，见图2A-D；在12 h时，与正常组相比，氧糖剥夺组、氧糖剥夺+肿瘤坏死因子α组、氧糖剥夺+脂多糖组BM-MSCs形态有皱缩趋势，且氧糖剥夺+肿瘤坏死因子α组、氧糖剥夺+脂多糖组细胞比氧糖剥夺组稀疏，细胞贴壁良好，见图2E-H；在24 h时，与正常组相比，氧糖剥夺组、氧糖剥夺+肿瘤坏死因子α组、氧糖剥夺+脂多糖组BM-MSCs明显皱缩变小，长梭形形态消失，细胞贴壁减弱，细胞数量明显减少，细胞损伤明显，见图2I-L。 2.3 各组BM-MSCs的生存率 MTT结果显示，与正常组比较，氧糖剥夺6 h和12 h后BM-MSCs存活率升高，分别为115.13%和106.97%，其中6 h差异有显著性意义(P=0.012)，氧糖剥夺24 h后BM-MSCs存活率降低，为85.95%，见图3。由结果可得，氧糖剥夺6，12 h使细胞活力增强，氧糖剥夺24 h对细胞造成损伤。与正常组比较，氧糖剥夺+肿瘤坏死因子α处理6，12，24 h后BM-MSCs存活率下降，分别为98.85%，89.70%和51.40%，氧糖剥夺+脂多糖处理6，12，24 h后BM-MSCs存活率下降，分别为97.92%，81.30%和40.27%，其中处理24 h细胞生存率与正常组相比差异有显著性意义(P < 0.01)，见图4。相较于单纯进行氧糖剥夺处理，加入肿瘤坏死因子α和脂多糖诱导，导致BM-MSCs活力降低。 2.4 各组BM-MSCs的凋亡情况与正常组比较，氧糖剥夺6 h后死亡细胞显著性减少(P < 0.01，n=3)，氧糖剥夺24 h后死亡细胞增加，见图5。由结果可得，氧糖剥夺6 h使细胞活力增强，氧糖剥夺24 h细胞活力降低。与正常组比较，氧糖剥夺+肿瘤坏死因子α处理6，12，24 h死亡细胞明显增多，氧糖剥夺+脂多糖处理6，12，24 h死亡细胞也明显增多，其中处理24 h细胞死亡比例与正常组相比差异有显著性意义(P < 0.01，n=3)，随着处理时间延长，死亡细胞增多，见图6。由结果可得，加入肿瘤坏死因子α和脂多糖诱导，导致BM-MSCs活力降低。 2.5 各组BM-MSCs的炎症因子表达在6 h时，氧糖剥夺组白细胞介素6、白细胞介素10和肿瘤坏死因子α基因表达量显著低于正常组(P < 0.01，n=3)；在12 h时，炎症因子表达量与6 h时一致，均显著低于正常组表达水平(P < 0.01，n=3)，见图7。结果显示，氧糖剥夺处理6 h和12 h使BM-MSCs炎症因子表达降低。氧糖剥夺+肿瘤坏死因子α组、氧糖剥夺+脂多糖组处理6 h和12 h时白细胞介素6、白细胞介素10和肿瘤坏死因子α基因表达量显著高于氧糖剥夺组(P < 0.01)，见图8。RT-PCR结果显示，加入肿瘤坏死因子α和脂多糖诱导，使BM-MSCs炎症因子基因表达增加。 2.6 各组BM-MSCs的炎症因子分泌在6 h时，氧糖剥夺组上清液中白细胞介素6、白细胞介素10分泌量显著低于正常组(P < 0.01，n=3)，肿瘤坏死因子α分泌量高于正常组(P=0.067，n=3)，其中白细胞介素6含量低于检测限；在12 h时，上清液中的炎症因子含量与6 h趋势一致，白细胞介素6含量仍低于检测限，见图9。结果显示，氧糖剥夺处理6 h和12 h减少BM-MSCs分泌白细胞介素6和白细胞介素10。氧糖剥夺+肿瘤坏死因子α组、氧糖剥夺+脂多糖组处理6 h和12 h上清液中白细胞介素6、肿瘤坏死因子α分泌量显著高于氧糖剥夺组，白细胞介素10分泌量差异无显著性意义，见图10。结果显示，加入肿瘤坏死因子α、脂多糖使BM-MSCs炎症因子分泌增加。 2.7 各组BM-MSCs中NF-κB的表达氧糖剥夺组NF-κB表达均显著低于正常组；氧糖剥夺+肿瘤坏死因子α组NF-κB表达均显著高于正常组和氧糖剥夺组；氧糖剥夺+脂多糖组NF-κB表达均显著高于正常组和氧糖剥夺组。由结果得到加入肿瘤坏死因子α、脂多糖激活BM-MSCs中NF-κB表达，各组NF-κB表达水平高低顺序为：氧糖剥夺+脂多糖组>氧糖剥夺+肿瘤坏死因子α组>正常组>氧糖剥夺组，见图11。"

References

[1] Shi Y, Hu G, Su J, et al. Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair.Cell Res. 2010;20(5):510-518.

[2] El Agha E, Kramann R, Schneider RK, et al. Mesenchymal Stem Cells in Fibrotic Disease.Cell Stem Cell. 2017;21(2): 166-177.

[3] Prabhu SD, Frangogiannis NG.The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis.Circ Res. 2016;119(1):91-112.

[4] HaiderHKh, Ashraf M.Bone marrow cell transplantation in clinical perspective.J Mol Cell Cardiol. 2005;38(2):225-235.

[5] Ali EHA, Ahmed-Farid OA, Osman AAE.Bone marrow-derived mesenchymal stem cells ameliorate sodium nitrite-induced hypoxic brain injury in a rat model.Neural Regen Res. 2017; 12(12):1990-1999.

[6] Teng X, Chen L, Chen W, et al. Mesenchymal Stem Cell-Derived Exosomes Improve the Microenvironment of Infarcted Myocardium Contributing to Angiogenesis and Anti-Inflammation.Cell PhysiolBiochem. 2015;37(6): 2415-2424.

[7] Han D, Huang W, Li X, et al. Melatonin facilitates adipose-derived mesenchymal stem cells to repair the murine infarcted heart via the SIRT1 signaling pathway.J Pineal Res. 2016;60(2):178-192.

[8] Huang W, Fan W, Wang Y, et al. Mesenchymal stem cells in alleviating sepsis-induced mice cardiac dysfunction via inhibition of mTORC1-p70S6K signal pathway.Cell Death Discov. 2017;3:16097.

[9] Joggerst SJ, Hatzopoulos AK.Stem cell therapy for cardiac repair: benefits and barriers.Expert Rev Mol Med. 2009;11: e20.

[10] Gnecchi M, He H, Liang OD, et al. Paracrine action accounts for marked protection of ischemic heart by Akt-modified mesenchymal stem cells.Nat Med. 2005;11(4):367-368.

[11] Pers YM, Ruiz M, Noël D, et al. Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives.Osteoarthritis Cartilage. 2015;23(11): 2027-2035.

[12] Enomoto M.The future of bone marrow stromal cell transplantation for the treatment of spinal cord injury.Neural Regen Res. 2015;10(3):383-384.

[13] HaiderHKh, Ashraf M.Implantation of genetically manipulated BM cells for cardiac repair.Cytotherapy. 2005;7(1):74-75.

[14] Kim SW, Houge M, Brown M, et al. Cultured human bone marrow-derived CD31(+) cells are effective for cardiac and vascular repair through enhanced angiogenic, adhesion, and anti-inflammatory effects.J Am CollCardiol. 2014;64(16): 1681-1694.

[15] Chen L, Zhang Y, Tao L, et al. Mesenchymal Stem Cells with eNOS Over-Expression Enhance Cardiac Repair in Rats with Myocardial Infarction.Cardiovasc Drugs Ther. 2017;31(1): 9-18.

[16] Toma C, Pittenger MF, Cahill KS, et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation. 2002;105(1):93-98.

[17] Westman PC, Lipinski MJ, Luger D, et al. Inflammation as a Driver of Adverse Left Ventricular Remodeling After Acute Myocardial Infarction.J Am CollCardiol. 2016;67(17): 2050-2060.

[18] Prabhu SD, Frangogiannis NG.The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis.Circ Res. 2016;119(1):91-112.

[19] Reesink HL, Sutton RM, Shurer CR, et al. Galectin-1 and galectin-3 expression in equine mesenchymal stromal cells (MSCs), synovial fibroblasts and chondrocytes, and the effect of inflammation on MSC motility.Stem Cell Res Ther. 2017; 8(1):243.

[20] Chen Z, Eadie AL, Hall SR, et al. Assessment of hypoxia and TNF-alpha response by a vector with HRE and NF-kappaB response elements.Front Biosci (Schol Ed). 2017;9:46-54.