骨髓间充质干细胞外泌体在高氧诱导的新生大鼠支气管肺发育不良模型中的修复作用

Repair effect of bone morrow mesenchymal stem cell-derived exosomes on neonatal rats model with bronchopulmonary dysplasia induced by hyperoxia

目的 明确骨髓间充质干细胞外泌体(BMSCs-Exo)对高氧诱导的大鼠支气管肺发育不良(BPD)模型的修复作用。方法 将新生4 d的Sprague Dawley(SD)大鼠吸入85%高氧建立高氧肺损伤模型。分为空气对照组、高氧组、高氧+PBS组和高氧+Exo组4组。使用BMSCs-Exo腹腔注射1周后处死SD大鼠,取肺组织进行石蜡包埋,分别进行HE染色,辐射状肺泡计数(radial alveolar counts, RAC),Masson染色、免疫组化染色和TUNEL染色。ELISA和Real time-PCR(qPCR)检测血清和肺组织蛋白白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、组织金属蛋白酶-9(MMP-9)及血管内皮生长因子-A(VEGF-A)的表达。结果 动物模型中,HE染色后对各组肺泡进行统计学分析显示,高氧+Exo组的肺泡数量显著高于高氧组及高氧+PBS组(P<0.001)。Masson染色后对蓝色胶原纤维所占面积百分比进行定量分析显示,高氧+Exo组胶原纤维所占面积百分比显著低于高氧组和高氧+PBS组(P<0.001)。免疫组化染色示高氧+Exo组的Ki-67和Bcl-2阳性细胞数量较高氧组和高氧+PBS组显著增高(P<0.05),而Bax阳性细胞数量较以上两组显著降低(P<0.001)。TUNEL染色检测凋亡的肺泡上皮细胞,高氧+Exo组显著低于高氧组和高氧+PBS组(P<0.001)。ELISA和qPCR检测显示,高氧+Exo组相较于高氧组和高氧+PBS组的IL-1β、TNF-α、MMP-9显著降低(P<0.001),而VEGF-A表达显著增高(P<0.001)。结论 BMSCs-Exos在肺发育不良模型干预中能抗高氧诱导的细胞凋亡,促进肺上皮细胞发育和组织修复。

Objective To study the repair effect of bone morrow mesenchymal stem cell-derived exosomes(BMSCs-Exo)on neonatal rats model with huperoxia-induced pulmonary dysplasia.Methods Sprague Dawley(SD)rats of 4 days old were inhaled with 85%oxygen to establish a hyperoxic lung injury model,and then were divided into 4 groups:air control group,hyperoxia group,hyperoxia+PBS group and hyperoxia+Exo group.The SD rats were sacrificed after intraperitoneal injection of BMSCs-Exos for 1 week,and lung tissues were taken for paraffin embedding.HE staining,radial alveolar counts(RAC),Masson staining and immunohistochemical staining were performed.ELISA assay and real time-PCR(qPCR)were carried out to assess the expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),matrix metalloproteinase-9(MMP-9)and vascular endothelial growth factor-A(VEGF-A).Results RAC in the hyperoxia+Exo group was significantly higher than those in the hyperoxia group and hyperoxia+PBS group(P<0.001).The area percentage of blue collagen fiber with the Masson staining method in hyperoxia+Exo group was significantly lower than those in hyperoxia group and hyperoxia+PBS group(P<0.001).Immunohistochemical staining was observed,the number of Ki-67 and Bcl-2 positive cells in the hyperoxia+Exo group was significantly higher,while the Bax positive cells was significantly lower,than those in hyperoxia group and hyperoxia+PBS group(P<0.05 or P<0.001).TUNEL staining showed that the rate of apoptotic alveolar epithelial cells in hyperoxia+Exo group was significantly lower than those in hyperoxia group and hyperoxia+PBS group.Compared with hyperoxia group and hyperoxia+PBS group,the transcriptional expression levels and serum concentrations of IL-1β,TNF-α,MMP-9 in the hyperoxia+Exo group were significantly reduced and that of VEGF-A was significantly increased(P<0.001).Conclusion BMSCs-Exo can protect from hyperoxia-induced apoptosis and promote alveolar epithelial cells development and tissue repair in lung dysplasia model.