Lü Cui, Liu Qian, Zeng Xianwei
Electrocardiographic Room, Affiliated Hospital of Weifang Medical University, Weifang Shandong, 261031, P.R.China.
Electrocardiographic Room, Affiliated Hospital of Weifang Medical University, Weifang Shandong, 261031,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 Feb 15;31(2):240-245. doi: 10.7507/1002-1892.201605095.
To explore the effects of interleukin 10 (IL-10) gene modified bone marrow mesenchymal stem cells (BMSCs) on the expression of inflammatory cytokines and neuronal apoptosis in rats after cerebral ischemia reperfusion injury.
BMSCs were cultured by whole bone marrow adherence screening method. The properties of BMSCs were identified by immunocytochemical methods. BMSCs at passage 3 were transfected with recombinant adenovirus IL-10 gene (AdIL-10-BMSCs). The model of middle cerebral artery occlusion was made in 40 adult male Sprague Dawley rats by thread embolism method. The rats were randomly divided into 4 groups ( =10). At 3 hours after modelling, the rats of groups A, B, C, and D received tail intravenous injection of 1 mL L-DMEM medium containing 10% FBS, 61.78 ng IL-10, 1 mL BMSCs suspension (2×10 cells/mL), and 1 mL AdIL-10-BMSCs cell suspension (2×10 cells/mL), respectively. The cells were labelled with BrdU before cell transplantation in groups C and D. At 7 days after reperfusion, the brain tissue was harvested to detect the expression of OX42 by immunohistochemical assay, to determine the concentration of tumor necrosis factor α (TNF-α) and IL-1β by ELISA, and to detect the apoptosis by TUNEL assay. BrdU labelled cells were observed by immunofluorescence staining in groups C and D.
BrdU labelled positive cells with green fluorescence were observed in the brain tissue of groups C and D, which mainly distributed in the striatum, cerebral cortex, and subcortex around the infarction area. The number of OX42 positive cells was significantly less in groups B, C, and D than group A ( <0.05), and in group D than groups B and C ( <0.05). Compared with the other 3 groups, the contents of TNF-α and IL-1β significantly decreased in group D ( <0.05). TUNEL assay showed that the apoptotic cells (TUNEL positive cells) were mainly seen in the striatum and fronto parietal subcortical tissues (equivalent to ischemic penumbra). The number of TUNEL positive cells in group D was significantly less than that in groups A, B, and C ( <0.05).
AdIL-10-BMSCs can inhibit secretion of TNF-α and IL-1β from microglial cells and inhibit the nerve cell apoptosis around infarct brain tissue, which might contribute to its protective role upon cerebral ischemia reperfusion injury.
探讨白细胞介素10（IL-10）基因修饰的骨髓间充质干细胞（BMSCs）对大鼠脑缺血再灌注损伤后炎性细胞因子表达及神经元凋亡的影响。
采用全骨髓贴壁筛选法培养BMSCs。通过免疫细胞化学方法鉴定BMSCs的特性。用重组腺病毒IL-10基因转染第3代BMSCs（AdIL-10-BMSCs）。采用线栓法对40只成年雄性Sprague Dawley大鼠制作大脑中动脉闭塞模型。将大鼠随机分为4组（每组n = 10）。造模后3小时，A、B、C、D组大鼠分别尾静脉注射1 mL含10%胎牛血清的L-DMEM培养基、61.78 ng IL-10、1 mL BMSCs悬液（2×10⁶细胞/mL）和1 mL AdIL-10-BMSCs细胞悬液（2×10⁶细胞/mL）。C组和D组在细胞移植前用BrdU标记细胞。再灌注7天后，取脑组织通过免疫组织化学法检测OX42的表达，用酶联免疫吸附测定法（ELISA）测定肿瘤坏死因子α（TNF-α）和IL-1β的浓度，并用末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）检测细胞凋亡。C组和D组通过免疫荧光染色观察BrdU标记的细胞。
C组和D组脑组织中观察到带有绿色荧光的BrdU标记阳性细胞，主要分布在梗死灶周围的纹状体、大脑皮质及皮质下。B、C、D组OX42阳性细胞数均明显少于A组（P < 0.05），且D组少于B组和C组（P < 0.05）。与其他3组相比，D组TNF-α和IL-1β含量明显降低（P < 0.05）。TUNEL检测显示凋亡细胞（TUNEL阳性细胞）主要见于纹状体及额顶叶皮质下组织（相当于缺血半暗带）。D组TUNEL阳性细胞数明显少于A、B、C组（P < 0.05）。
AdIL-10-BMSCs可抑制小胶质细胞分泌TNF-α和IL-1β，抑制梗死脑组织周围神经细胞凋亡，这可能是其对脑缺血再灌注损伤发挥保护作用的机制。
