干细胞动员对急性肺损伤幼鼠肺泡表面活性蛋白的影响_黄国日.pdf

干细胞动员对急性肺损伤幼鼠肺泡表面活性蛋白的影响基金项目:广西壮族自治区卫生厅计划课题(Z2014457);南宁市科技攻关计划课题(20153336)作者简介:黄国日，男，副主任医师，医学学士，研究方向:儿童危重症及心血管疾病。E-mail:380112810 qqcom通信作者:潘革。E-mail:pange0506 163com 本文引用格式 黄国日，潘革，莫锦丽，等干细胞动员对急性肺损伤幼鼠肺泡表面活性蛋白的影响 J 右江医学，2023，51(1):15-23黄国日1，潘革2a，莫锦丽2a，杨兰2b，黄忠向2c(1广西医科大学第二附属医院儿科，广西南宁 530007;2广西医科大学第三附属医院 a儿科，b检验科，c病理科，广西南宁 530031)【摘要】目的分析干细胞动员对急性肺损伤(ALI)幼鼠肺泡表面活性蛋白表达的影响。方法选择 SPF 级BALB/c 品系健康幼鼠 120 只，随机分为动员组、对照组各 60 只，均经尾静脉注射精制大肠杆菌内毒素脂多糖(LPS)5 mg/kg 复制内毒素性肺损伤幼鼠模型，动员组采用皮下注射重组人粒细胞刺激因子(rhG-CSF)30 g/(kgd)进行干细胞动员干预，对照组注射同等剂量生理盐水，连续 5 d，在干预后 5 d、10 d、14 d 对两组肺组织进行半定量肺损伤评分法及辐射状肺泡计数(AC)评估，对两组肺表面活性物质蛋白 SP-A、SP-D、水通道蛋白 5(AQP5)，肺组织匀浆SP-A、SP-D、AQP5 mNA 含量的表达水平进行检测。结果对照组 5 d、10 d、14 d 半定量肺损伤评分逐渐增加(P005);动员组干预后 10 d 与 14 d 半定量肺损伤评分均高于干预后 5 d(P005);干预后 5 d、10 d、14 d，动员组半定量肺损伤评分均低于对照组;干预后 10 d 半定量肺损伤评分与 14 d 比较差异有统计学意义(P005);干预后 5 d、10 d、14 d 动员组 AC 值高于对照组(P005);动员组干预后 5 d、10 d、14 d 肺组织中 SP-A、SP-D、AQP5 蛋白表达高于对照组(P005)，SP-A、SP-D、AQP5 mNA 含量高于对照组(P005)。结论干细胞动员可减轻 LPS 诱导的幼鼠急性肺损伤，促进其肺泡表面活性物质再生，促进肺恢复。【关键词】干细胞动员;急性肺损伤;幼鼠;肺泡表面活性蛋白中图分类号:3633文献标志码:ADOI:103969/jissn1003-1383202301003Effect of stem cell mobilization on pulmonary surfactantprotein in juvenile rats with acute lung injuryHUANG Guori1，PAN Ge2a，MO Jinli2a，YANG Lan2b，HUANG Zhongxiang2c(1 Department of Pediatrics，the Second Affiliated Hospital of Guangxi Medical University，Nanning 530007，Guangxi，China;a Department of Pediatrics，b Department of Laboratory Medicine，c Department ofPathology，2 the Third Affiliated Hospital of Guangxi Medical University，Nanning 530031，Guangxi，China)【Abstract】ObjectiveTo analyze the effect of stem cell mobilization on the expression of pulmonary surfactant protein injuvenile rats with acute lung injury(ALI)Methods120 healthy BALB/c SPF juvenile rats were selected and randomly di-vided into mobilization group and control group，with 60 rats in each group Juvenile rat models of endotoxin-induced ALIwere reproduced by injecting refined escherichia coli endotoxin lipopolysaccharide(5 mg/kg)through tail vein The mobili-zation group were given subcutaneous injection of recombinant human granulocyte stimulating factor(rhG-CSF)30 g/(kgd)for stem cell mobilization，and rats in the control group were injected with the equivalent dose of normal saline for 5 con-secutive days At the 5th，10th，and 14thday after intervention，the lung tissues of both groups were assessed through semi-quantitative lung injury score and radial alveolar count(AC)，and the pulmonary surfactant protein SP-A，SP-D，aquapor-in 5(AQP5)and the expression levels of SP-A，SP-D，and AQP5 mNA in lung tissue homogenate were detected esultsThe semi-quantitative lung injury scores of the control group gradually increased on the 5th，10th，and 14thday after inter-51右江医学 2023 年第 51 卷第 1 期Chinese Youjiang Medical Journal 2023，Vol51 No1vention(P005)The semi-quantitative lung injury scores of the mobilization group on the 10thand 14thday after interven-tion were all higher than that on the 5thday after intervention(P005)The semi-quantitative lung injury scores of the mo-bilization group on the 5th，10th，and 14thafter intervention were all lower than those of the control group There was statisti-cally significant difference in the semi-quantitative lung injury scores between the 10thand 14thday after intervention(P005)The AC values of the mobilization group on the 5th，10th，and 14thday after intervention were all higher than thoseof the control group(P005)The protein expression levels of SP-A，SP-D and AQP5 in lung tissues of the mobilizationgroup on the 5th，10th，and 14thday after intervention were all higher than those in the control group(P005)The mNAlevels of SP-A，SP-D and AQP5 in the mobilization group on the 5th，10th，and 14thday after intervention were all higherthan those in the control group(P005)ConclusionStem cell mobilization can alleviate LPS-induced ALI in juvenilerats，promote the regeneration of pulmonary surfactant，and promote lung recovery【Key words】stem cell mobilization;acute lung injury(ALI);juvenile rat;pulmonary surfactant protein急性肺损伤(acute lung injury，ALI)以呼吸困难为主要表现，患者可在各个年龄段发病，是目前临床危重医学研究重点1。ALI 的病理改变在于过度炎症反应使肺血管内皮细胞、肺泡上皮细胞遭受广泛性破坏，如肺泡表面活性物质(SP-A、SP-D、AQP5等)合成减少，因此改善肺泡表面活性物质水平逐渐成为 ALI 患者治疗靶点2。骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMMSCs)为一类非造血细胞的多能干细胞，具有向多种组织细胞分化的能力3。近年来随着对干细胞多向分化潜能研究深入，发现干细胞可再生修复 ALI 的肺泡上皮细胞和内皮细胞，重建损伤的肺组织结构及功能，成为 ALI 治疗新靶点，并可能是未来治疗 ALI 患者的关键所在4。本实验通过对幼鼠自体骨髓干细胞进行动员，扩增入血，分析动员后肺泡表面活性蛋白水平变化，进而找到治疗 ALI 的新途径，为下一步临床开展儿童 ALI 的救治提供理论依据。1材料与方法1111实实验验动物动物选取无特定病原体(specific patho-gen free，SPF)级 BALB/c 品系健康幼鼠120 只，体重174230 g，平均(201322056)g，由广州四和生物科技有 限 公 司 提 供，实 验 动 物 生 产 许 可 证 号:44007200101674，使用许可证号:SCXK(粤)2016-0041，采用随机数字表将实验动物随机分为对照组和动员组，每组 60 只。对照组雄性 20 只，雌性 40只，平均体重(201182121)g;动员组雄性 27 只，雌性 33 只，平均体重(202762205)g。两组性别、体重比较差异无统计学意义(P005)，具有可比性。实验开展前，幼鼠均饲养 1 周(饲养温度设置为 21)，自由进食和饮水。饲养观察 1 周无异常后进行实验，实验期间无幼鼠死亡。本实验获得广西医科大学动物实验中心伦理委员会批准。1212实实验验主要主要仪器仪器及及试剂试剂苏州安泰洁净工作台(SW-CJ-IFD)，低速离心机(中佳，SC3614)，倒置光学显微镜(OLYMPUS CKX41，U-CT30-2)，倒置荧光显微镜(生产商:Leica，型号:DMI6000B)、台式高速冷冻离心机(湖南湘仪实验室仪器开发有限公司，TG