超声引导注射骨髓间充质干细胞治疗胎兔支气管肺发育不良

The Study of Ultrasound Guidance Injection of Bone Marrow Mesenchymal Stem Cells to Treat Fetal Rabbit Bronchopulmonary Dysplasia

目的探讨超声引导下注射骨髓间充质干细胞(BM-MSCs)治疗胎兔支气管肺发育不良(BPD)的作用及可能机制。方法检测BM-MSCs细胞表面标志物、三系分化能力、慢病毒包装与感染能力。将新西兰孕兔随机分为A组(正常组)、B组(LPS组)和C组(LPS+BM-MSCs组),每组选取3只新西兰孕兔、10只胎兔。于孕23 d在超声引导下B、C组经右肺组织注射脂多糖(LPS),A组注射等量生理盐水。孕27 d超声引导下A、B组经右肺组织注射生理盐水,C组注射BM-MSCs。孕29 d后行剖宫产分离胎兔肺组织。采用苏木精-伊红(H-E)染色观察肺组织病理改变,RT-PCR检测肺组织中白细胞介素1β(IL-1β)、IL-6、IL-10、肿瘤坏死因子-α(TNF-α)、血管内皮生长因子(VEGF)、肺泡表面活性蛋白A(SPA)、肺泡表面活性蛋白B(SPB)和肺泡表面活性蛋白C(SPC)mRNA的表达。结果BM-MSCs细胞表面标记物CD29和CD44阳性,CD11b/c和CD45阴性,具有三系分化能力。慢质-增强型绿色荧光蛋白(EGFP)感染所有rBM-MSCs需24 h。不同组别中,B组肺组织辐射状肺泡计数(RAC)数量较A组明显减少;C组肺组织RAC数量较A组稍减少,较B组稍增加,差别有统计学意义(P<0.05)。与A组比较,B组胎兔肺组织肺泡壁增厚,炎性细胞浸润,肺泡结构简化,数量减少,部分表现为充气不均匀;与A组比较,C组胎兔肺组织肺泡壁稍增厚,肺泡少量炎性细胞浸润;与B组比较,C组肺组织肺泡壁变薄,肺泡数量增多,炎性细胞浸润减少。与A组比较,B组胎兔肺组织中IL-1β、IL-6、IL-10、TNF-α和VEGF mRNA的表达上升,C组较B组表达下降;与A组比较,B组胎兔肺组织中SPA、SPB和SPC mRNA的表达下降,C组较B组表达上升,差别有统计学意义(P<0.05)。结论超声引导下注射LPS可引起支气管肺发育不良,BM\|MSCs胎肺注射治疗可减轻肺组织病理变化,推测可能通过分泌各种因子减轻炎症反应,促进肺泡发育、损伤修复和血管重建。

Objective To investigate the role and possible mechanism of ultrasound-guided injection of bone marrow mesenchymal stem cells(BM-MSCs)for treatment of fetal rabbit bronchopulmonary dysplasia.Methods Detection of BM-MSCs cell surface markers,three-line differentiation ability,lentivirus packaging and infection ability.Pregnant New Zealand rabbits were randomly divided into group A(normal),group B(LPS)and group C(LPS+BM-MSCs),with 3 pregnant New Zealand rabbits in each group and 10 fetal rabbits in each group.On the 23rd day of gestation in New Zealand pregnant rabbits,lipopolysaccharide(LPS)was injected into the right lung tissue of fetal rabbits in groups B and C under the guidance of ultrasound,and the same amount of normal saline was injected into group A.Under ultrasound guidance on the 27th day of pregnancy,each fetal rabbit in group A and group B was injected with normal saline through right lung tissue,and each fetal rabbit in group C was injected with BM-MSCs through right lung tissue.After 29 days of pregnancy,through a cesarean section separated lung tissue from fetal rabbit.The pathological changes of the lung tissue were evaluated by hematoxylin-eosin(H-E)staining,and mRNA expression of IL-1β,IL-6,IL-10,TNF-α,VEGF,SPA,SPB,and SPC was detected by RT-PCR.Results The surface markers of BM-MSCs were positive for CD29 and CD44,and negative for CD11b/c and CD45.BM-MSCs had the ability of three-line differentiation.It took about 24 hours for all rBM-MSCs to be infected by lentiviruses expressing enhanced green fluorescent protein(EGFP).In different groups,the number of RAC in group B was significantly reduced compared with group A;the number of RACs in group C was slightly reduced compared with group A.The number of RAC in group C was slightly increased compared to group B.The difference was statistically significant(P<0.05).Compared with group A,the fetal rabbit lung tissue in group B alveolar wall was thickened,inflammatory cells infiltrated,the alveolar structure simplified,the number reduced,and some manifestations were uneven inflatable.Compared with group A,the fetal rabbit lung tissue in group C alveolar wall was slightly thickened,and inflammatory cells infiltrated.Compared with group B,the fetal rabbit lung tissue in group C alveolar wall became thinner,the number of alveolar increased,and the inflammatory cell infiltration reduced.Compared with group A,the expression of IL-1β,IL-6,IL-10,TNF-α,and VEGF mRNA in the lung tissue of fetal rabbits in group B increased,while the expression decreased in group C compared with group B.Compared with group A,the expression of SPA,SPB,and SPC mRNA in the lung tissue of fetal rabbits in group B decreased,and the expression in group C increased compared with group B.The difference was statistically significant(P<0.05).Conclusion Ultrasound guided injection of lipopolysaccharide can cause bronchopulmonary dysplasia,and BM\|MSCs fetal lung injection therapy can alleviate pathological changes in lung tissue.It is speculated that various factors may be secreted to alleviate inflammation,promote alveolar development,injury repair,and vascular reconstruction.