PD-1/PD-L1参与骨髓间充质干细胞归巢修复慢阻肺大鼠的肺损伤

PD-1/PD-L1 participates in the homing of bone marrow mesenchymal stem cells to repair lung injury in COPD rats

目的 研究程序性死亡蛋白1(programmed death-1, PD-1)/程序性死亡蛋白配体1(programmed death ligand-1,PD-L1)在间充质干细胞(mesenchymal stem cells, MSCs)归巢修复慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)中的影响。方法 分离纯化骨髓来源MSCs,流式细胞术检测细胞表面分子表达,油红O染色、碱性磷酸酶染色分别观察MSCs成脂及成骨分化情况。采用烟气暴露法构建COPD大鼠模型,造模完成后将绿色荧光蛋白(GFP)标记的MSCs经尾静脉分别注射入正常及COPD大鼠模型中,通过观察肺部荧光评价MSCs的归巢效果;经尾静脉向COPD模型大鼠注射MSCs作为MSCs治疗组,模型组和正常对照组注射等体积生理盐水,HE染色观察各组大鼠肺脏组织病理改变,qRT-PCR比较各组大鼠肺脏组织PD-1的表达情况;体外qRT-PCR检测PD-L1在MSCs的表达,Transwell实验法观察PD-1及PD-L1抗体对MSCs定向迁移(归巢)的影响。结果 大鼠骨髓来源MSCs提取分离后,细胞贴壁生长,呈长梭形,流式细胞术结果显示MSCs上CD29、CD44呈阳性表达,CD34、CD45阴性表达,油红O染色可见呈深橙红色的脂滴,碱性磷酸酶染色可见典型的成骨表现;GFP标记的MSCs能够有效到达正常及COPD损伤肺组织中,荧光信号在COPD肺组织中明显强于正常肺组织,且PD-1在COPD肺组织中表达强于正常组织;与正常对照组相比,COPD模型组HE染色可见明显COPD肺损伤改变,移植MSCs细胞治疗后,肺损伤得到明显改善;分离培养的MSCs表达PD-L1;体外Transwell迁移实验表明,与对照组相比,加入PD-1可增加MSCs定向迁移,PD-L1抗体组相较于PD-1组,MSCs迁移数量明显减低。结论 PD-1/PDL1参与MSCs定向归巢到COPD大鼠肺部并达到较好的损伤修复作用。

Objective To explore the impact of programmed death-1(PD-1)/programmed death ligand-1(PD-L1)on the homing and repair of mesenchymal stem cells(MSCs)in chronic obstructive pulmonary disease(COPD).Methods The mesenchymal stem cells(MSCs)were isolated and purified from bone marrow,and flow cytometry was used to detect the expression of cell surface molecules.The adipogenic and osteogenic differentiation of MSCs were observed through Oil Red O staining and alkaline phosphatase staining,respectively.A COPD rat model was established using a smoke exposure method.After the model was established,MSCs labeled with green fluorescent protein(GFP)were injected into the tail vein of both normal and COPD rat models to evaluate the homing effect of MSCs by observing lung fluorescence.MSCs were injected into the tail vein of COPD model rats as an MSC treatment group,while the model group and the normal control group were injected with an equal volume of saline.HE staining was used to observe the pathological changes in the lung tissues of the rats in each group. The expression of PD-1 in the lung tissues of the rats in each group was compared using qRT-PCR. In vitro, qRT-PCR was used to detect the expression of PD-L1 in MSCs, and a Transwell assay was used to observe the effect of PD-1 and PD-L1 antibodies on the directed migration (homing) of MSCs. Results After the extraction and isolation of rat bone marrow-derived MSCs, the cells adhered and grew in a spindle shape. Flow cytometry results showed positive expression of CD29 and CD44, and negative expression of CD34 and CD45 on MSCs. Oil Red O staining revealed deep orange-red lipid droplets, and alkaline phosphatase staining showed typical osteogenic characteristics. GFP-labeled MSCs were effectively delivered to both normal and COPD-damaged lung tissues, with fluorescence signals being significantly stronger in COPD lung tissues than in normal tissues, and PD-1 expression was higher in COPD lung tissues than in normal tissues. Compared to the normal control group, HE staining of the COPD model group revealed obvious COPD lung damage changes, which were significantly improved after transplantation of MSCs. The isolated and cultured MSCs expressed PD-L1. In vitro Transwell migration experiments showed that compared to the control group, adding PD-1 increased the directed migration of MSCs, and the number of MSCs migrated in the PD-L1 antibody group was significantly lower than in the PD-1 group. Conclusion PD-1/PD-L1 are involved in the directed homing of MSCs to the lungs of COPD rats, achieving effective damage repair.