胎牛肺间充质干细胞对脂多糖介导小鼠急性肺损伤治疗的研究

Research on Fetal Bovine Lung Mesenchymal Stem Cells on Lipopolysaccharide-induced Acute Lung Injury in Mice

目的探讨胎牛肺间充质干细胞的生物学特性及其对脂多糖诱导的小鼠急性肺损伤的治疗修复作用。方法采用脂多糖(LPS)对小鼠进行直接腹腔灌注,造小鼠急性肺损伤模型,采用胎牛肺间充质干细胞(LMSCs)对小鼠进行注射治疗,通过观察组织学切片及血清学检测对小鼠肺损伤程度及预后进行评估。结果成功分离出胎牛LMSCs,细胞形态多呈长梭形或纺锤形,核质比大,边缘不规则。RT-PCR检测显示,胎牛LMSCs阳性表达CD29、CD44、CD166和CD73,不表达CD31。免疫荧光检测显示,胎牛LMSCs阳性表达CD29、CD73、CD44和CD166。病理学切片显示,对照组小鼠肺泡壁结构完整,肺泡间质无渗出大小相近,存在正常的肺泡结构,无炎症细胞出现在管壁及周围,无胶原蛋白沉淀;损伤组小鼠肺组织出现大面积肺泡结构受损,大多数正常肺泡结构消失,肺泡腔改变可见变形闭塞,大量炎症细胞浸润,结构变性和异常的胶原蛋白沉淀,损伤作用时间越长,损伤程度加重;治疗组小鼠在LMSCs移植治疗后肺损伤病变面积和损伤程度较损伤组下降,可见炎症细胞浸润减少、间质有所增厚及胶原少量沉积,肺切片更接近于正常肺组织肺泡形态。ELISA血清检测显示,损伤组6、24、48 h血清TNF-α、IL-4、IL-6水平高于治疗组和对照组,且治疗组高于对照组,差异有统计学意义(P<0.05)。结论胎牛LMSCs在体外培养体系中可以稳定的保持其生物学特性,低代次LMSCs具有良好的增殖活性及分泌细胞因子的能力,可以有效降低LPS诱导的小鼠急性肺损伤血清中TNF-α水平,参与调控IL-4、IL-6的分泌及免疫应答作用,对急性炎性反应有一定治疗作用。

Objective To investigate the biological characteristics of fetal bovine lung mesenchymal stem cells and their therapeutic and repair effects on lipopolysaccharide-induced acute lung injury in mice.Methods Lipopolysaccharide(LPS)was used to infuse mice directly into the abdominal cavity to create a mouse model of acute lung injury.The mice were injected with fetal bovine lung mesenchymal stem cells(LMSCs).The histological sections and serological examinations were carried out.The degree of lung injury and prognosis of mice were evaluated.Results The fetal bovine LMSCs were successfully isolated,and the cell morphology was mostly long fusiform or spindle-shaped,with large nucleus to cytoplasmic ratio and irregular edges.RT-PCR detection showed that fetal bovine LMSCs positively expressed CD29,CD44,CD166 and CD73,but did not express CD31.Immunofluorescence detection showed that fetal bovine LMSCs positively expressed CD29,CD73,CD44 and CD166.The pathological section showed that the structure of the alveolar wall of the mice in the control group was intact,the alveolar interstitium had no exudation of similar size,there was a normal alveolar structure,no inflammatory cells appeared on and around the tube wall,and there was no collagen precipitation;The lung tissues of the mice in the injury group showed large areas of alveolar structure damage,most of the normal alveolar structure disappeared,the alveolar cavity changes showed deformation and occlusion,a large number of inflammatory cell infiltration,structural degeneration and abnormal collagen precipitation.The longer the damage action time,the more severe the damage;After LMSCs transplantation,the area and extent of lung injury of the mice in the treatment group were lower than those in the LPS group.The infiltration of inflammatory cells was reduced,the interstitium was thickened,and the collagen was slightly deposited.The lung slices were closer to the alveolar morphology of normal lung tissue.ELISA serum test showed that the serum levels of TNF-α,IL-4,and IL-6 in the injury group were higher than the treatment group and the control group at 6,24,and 48 h,and the treatment group was higher than the control group,the difference was statistically significant(P<0.05).Conclusion Fetal bovine LMSCs can maintain their biological characteristics stably in an in vitro culture system.Low-passage LMSCs have good proliferation activity and cytokine secretion ability,which can effectively reduce TNF-αin serum of mice with acute lung injury induced by LPS It participates in the regulation of IL-4 and IL-6 secretion and immune response,and has a certain therapeutic effect on acute inflammatory reactions.