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目的探讨低氧环境下视网膜神经胶质细胞介导的低氧反应分子通路对血管内皮前体细胞(EPCs)的作用及其机制。方法人外周血循环EPCs(CD34+、CD33+双标阳性细胞)经荧光免疫激活流式细胞仪分选收集后培养;分别使用低氧(5%O2,实验组)和正常氧(20%O2,对照组)的人视网膜神经胶质细胞生长培养液作为条件培养液,多次处理EPCs,检测和分析EPCs的增殖活性(WST-1比色法)和分化能力(免疫荧光染色法);采用酶联免疫吸附试验检测实验组和对照组视网膜神经胶质细胞血管内皮生长因子(VEGF)的释放量,采用免疫印迹法检测细胞核内低氧反应因子(HIF)-1α的表达。结果实验组EPCs的增殖活性(吸光度值为1·53±0·10)和分化能力[细胞数目为(29±5)个]均较对照组增殖活性(吸光度值为0·88±0·25)和分化能力[细胞数目为(18±8)个]明显增强(P<0·05)。实验组培养6、24h后神经胶质细胞核内均可检测到HIF-1α表达,而对照组表达缺失。实验组视网膜神经胶质细胞VEGF的释放量较对照组明显增加,且具有时相性,培养24h实验组VEGF的释放量[(319·16±34·12)pg/ml]较对照组[(220·28±24·33)pg/ml]增加44·88%(P<0·01)。结论视网膜神经胶质细胞在低氧培养状态下可通过细胞因子的分泌作用直接促进培养的EPCs增殖和分化,这可能在EPCs参与的新生血管形成中发挥重要的介导作用,该作用与神经胶质细胞HIF-1α和VEGF的信号通道激活有关。本研究对神经胶质细胞介入血管前体细胞的病理生理作用进行了初步探讨。
Objective To investigate the effect and mechanism of retinal glial cell-mediated hypoxia-responsive molecular pathway on vascular endothelial progenitor cells (EPCs) under hypoxia. Methods Human peripheral blood circulating EPCs (CD34 +, CD33 + double-labeled positive cells) were collected by flow cytometry after fluorescence immunofluorescence. The cells were incubated with hypoxia (5% O2, experimental group) and normal oxygen ) Of human retinal glial cell growth medium as a conditioned medium, EPCs were treated multiple times, and the proliferation activity (WST-1 colorimetric method) and differentiation ability (immunofluorescence staining) of EPCs were detected and analyzed. Adsorption assay was used to detect the release of vascular endothelial growth factor (VEGF) in retinal glial cells in experimental group and control group. The expression of hypoxia-response factor (HIF-1α) in the nucleus was detected by Western blotting. Results Proliferation activity (absorbance value was 1.53 ± 0.10) and differentiation ability (29 ± 5) of EPCs in experimental group were higher than those in control group (absorbance value was 0.88 ± 0.25 ) And differentiation ability [(18 ± 8) cells] were significantly increased (P <0.05). The expression of HIF-1α was detected in glial cell nucleus 6 h and 24 h after the experimental group was cultured, while the expression of HIF-1α was absent in the control group. Compared with the control group, the release of VEGF in the experimental group was significantly increased compared with the control group, and the release of VEGF in the experimental group was significantly higher than that in the control group [(319 · 16 ± 34 · 12) pg / ml] · 28 ± 24 · 33) pg / ml] increased by 44 · 88% (P <0.01). CONCLUSIONS: Under hypoxic conditions, retinal glia can directly promote the proliferation and differentiation of cultured EPCs through the secretion of cytokines, which may play an important mediating role in the angiogenesis of EPCs. HIF-1alpha cells and VEGF signaling pathway activation. In this study, the pathophysiological effects of glial cells intervening in vascular precursor cells were explored.