内皮祖细胞培养上清对高氧暴露新生大鼠肺结构的改善作用

发布时间：2018-08-08 22:07

【摘要】：目的:研究内皮祖细胞培养上清(endothelial progenitor cell-conditioned medium,EPC-CM)对高氧暴露新生大鼠肺损伤时肺泡结构的改善作用及其机制。方法:从新生SD大鼠骨髓中获取内皮祖细胞并鉴定,收集第3代细胞的培养上清备用。另取新生SD大鼠40只随机分为4组,即空气组:仔鼠在空气(21%O_2)中喂养21天;高氧组:仔鼠在85% O_2中喂养21天;内皮细胞基础培养基(endothelial cell basal medium,EBM)干预组:仔鼠在85%O_2中喂养至第14天时,经气道给予100μL EBM,然后喂养至第21天;EPC-CM干预组:仔鼠在85% O_2中喂养至第14天,经气道给予100μL EPC-CM,喂养至第21天。第21天处死小鼠,左肺用4%多聚甲醛固定,留作石蜡切片,随后HE染色进行肺组织病理形态学观察,并做辐射状肺泡计数(radical alveolar count,RAC)及肺泡平均线性截距(mean linear intercept,MLI)测量;免疫组织化学方法对血管内皮细胞FVIII染色,计数肺组织微血管密度;右肺留作实时荧光定量PCR检测肺组织KGF、VEGF、SP-A和SP-C的mRNA表达。结果:培养所得细胞具有典型的EPCs形态改变,能结合异硫氰酸荧光素标记的荆豆凝集素1并摄取Di I荧光标记的乙酰化低密度脂蛋白。高氧组及EBM干预组的仔鼠体重、RAC、MLI和微血管密度较空气组显著降低(P0.05),EPC-CM干预组的RAC和微血管密度较高氧组和EBM干预组明显增加(P0.05),而体重和MLI的变化无明显差异,但有增高的趋势。高氧组和EBM干预组肺组织KGF、VEGF、SP-A和SP-C的mRNA表达较空气组显著降低(P0.05),EPC-CM干预组的表达显著高于高氧组和EBM干预组(P0.05)。结论:EPC-CM可改善高氧暴露新生大鼠的肺泡化和肺血管发育,可能与促进肺内KGF和VEGF mRNA的表达相关。
[Abstract]:Objective: To study the effect and mechanism of endothelial progenitor cell-conditioned medium (EPC-CM) on pulmonary alveolar structure in neonatal rats with hyperoxia exposure. Methods: to obtain endothelial progenitor cells from the bone marrow of newborn SD rats and identify the third generation cell culture supernatant. Another newborn SD rat was taken. 40 rats were randomly divided into 4 groups, that is, the air group: the young rats were fed in the air (21%O_2) for 21 days; the high oxygen group was fed for 21 days in 85% O_ 2; the endothelial cell basal medium (endothelial cell basal medium, EBM) intervention group: when the offspring were fed in 85%O_ 2 for fourteenth days, the channel was given 100 u L EBM, and then fed to twenty-first days; EPC-CM intervention group: Offspring Rats For fourteenth days in 85% O_ 2, 100 mu L EPC-CM was given through the airway and twenty-first days were fed for twenty-first days. The left lung was fixed with 4% polyformaldehyde and left for paraffin section. Then, the lung tissue was observed by HE staining, and the radiated alveolar count (radical alveolar count, RAC) and the mean linear intercept of pulmonary alveolus (mean linear inter) Cept, MLI) measurement; immunohistochemical method for FVIII staining of vascular endothelial cells, counting the microvascular density of lung tissue, and right lung for real-time fluorescence quantitative PCR to detect the mRNA expression of KGF, VEGF, SP-A and SP-C in lung tissue. Results: the cultured cells have typical EPCs morphologic changes and can be combined with fluorescein isothiocyanate labeled agglutinin 1. The body weight, RAC, MLI and microvascular density in the high oxygen group and the EBM intervention group were significantly lower than those in the air group (P0.05). The RAC and microvascular density in the EPC-CM intervention group increased significantly (P0.05) in the EPC-CM intervention group (P0.05), but there was no significant difference in weight and MLI, but there was an increased trend in the EPC-CM intervention group. The expression of KGF, VEGF, SP-A and SP-C in the lung tissue of the hyperoxic group and the EBM intervention group was significantly lower than that in the air group (P0.05). The expression of the EPC-CM intervention group was significantly higher than that of the hyperoxia group and the EBM intervention group (P0.05). Conclusion: EPC-CM can improve the alveolation and the development of pulmonary blood tube in the newborn rats with hyperoxia exposure, which may be related to the promotion of the expression of lung KGF and the expression of the lungs.
【作者单位】：复旦大学附属儿科医院;
【基金】：国家自然科学基金资助项目(No.81270727)
【分类号】：R722.6

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