骨髓间充质干细胞对烟雾吸入伤大鼠Th17/Treg免疫平衡的影响

Effect of bone mesenchymal stem cell on T helper cell 17/regulatory T cells immune homeostasis in rats with smoke inhalation injury

目的观察骨髓间充质干细胞(BMSC)对烟雾吸入伤(SII)大鼠辅助性T细胞17/调节性T细胞(Th17/Treg)免疫平衡的影响。方法将30只雄性Wistar大鼠按随机数字表法分为正常对照组(6只)、SII模型组(12只)、SII+BMSC组(12只)。采用烟雾一对一口鼻吸入法复制SII模型。制模成功12 h后,正常对照组和SII模型组经尾静脉注射磷酸盐缓冲液(PBS),SII+BMSC组经尾静脉注射BMSC细胞悬液。正常对照组于给药后1 d、SII模型组和SII+BMSC组于给药后1 d、3 d取材。光镜下观察肺组织损伤程度,并进行肺损伤评分;比较各组大鼠不同时间点动脉血氧分压(PaO_(2))、氧合指数(PaO_(2)/FiO_(2))、Th17、Treg、Th17/Treg、白细胞介素-17(IL-17)、转化生长因子-β(TGF-β)、转录因子RAR相关孤儿受体-C(RORC)、叉头翼状转录因子-3(Foxp-3)的mRNA表达水平及血清IL-17、TGF-β蛋白含量的差异。结果光镜下可见:烟雾暴露后大鼠肺组织出现明显肺泡间质增厚,肺间质断裂融合,大量炎症细胞浸润等病变;BMSC移植后可显著减轻肺泡损伤,抑制炎症细胞浸润。与正常对照组比较,制模后1 d起,SII模型组肺损伤评分、Th17、Th17/Treg、血清IL-17蛋白含量和mRNA表达及RORC mRNA表达水平均升高,PaO_(2)、PaO_(2)/FiO_(2)、Treg、TGF-βmRNA和血清蛋白含量均明显降低(均P<0.05),持续到制模后3 d(P<0.05)。与SII模型组比较,SII+BMSC组制模后3 d肺损伤评分、Th17和制模后1 d Th17/Treg、IL-17 mRNA表达与血清蛋白含量及RORC mRNA表达水平均明显降低〔肺损伤评分(分):2.00±0.63比3.33±0.82,Th17:1.47±0.13比3.00±0.54,Th17/Treg:0.29±0.13比1.02±0.32,IL-17 mRNA(2^(-ΔΔCt)):0.64±0.09比1.40±0.41,IL-17(ng/L):142.43±22.86比309.83±13.45,RORC mRNA(2^(-ΔΔCt)):1.42±0.81比3.50±0.42,均P<0.05〕,制模后1 d起PaO_(2)、PaO_(2)/FiO_(2)、Treg、TGF-βmRNA表达与蛋白含量均明显升高〔PaO_(2)(mmHg,1 mmHg≈0.133 kPa):120.60±13.38比84.25±18.32,PaO_(2)/FiO_(2)(mmHg):596.43±51.22比401.19±87.24,Treg:4.00±0.56比2.46±0.60,TGF-βmRNA(2^(-ΔΔCt)):1.91±0.37比0.25±0.16,TGF-β(ng/L):2901.40±28.58比1855.96±345.72,均P<0.05〕。3组不同时间点Foxp-3 mRNA表达水平比较差异均无统计学意义。结论SII大鼠体内存在Th17/Treg细胞比例失衡,BMSC通过调节Th17/Treg免疫平衡抑制炎症反应,促进肺损伤修复,对SII发挥治疗作用。

Objective To investigate the effect of bone mesenchymal stem cell(BMSC)on T helper cell 17/regulatory T cell(Th17/Treg)immune homeostasis in rats with smoke inhalation injury(SII).Methods Thirty male Wistar rats were randomly divided into three groups:normal control group(6 rats),SII model group(12 rats)and SII+BMSC group(12 rats).The SII model was replicated using the smoke pair one-bite nasal inhalation method.Twelve hours after successful modeling,the normal control and SII model groups were injected with phosphate buffer solution(PBS)via the tail vein,and the SII+BMSC group was injected with BMSC cell suspension via the tail vein.Samples were taken on the 1st day after administration in the normal control group and on the 1st day and 3rd day after administration in the SII model and SII+BMSC groups.The degree of lung tissue damage was observed under light microscope and the lung injury score was made;The differences of arterial partial pressure of oxygen(PaO_(2)),oxygenation index(PaO_(2)/FiO_(2)),Th17,Treg,and Th17/Treg,the mRNA expression levels of interleukin-17(IL-17),transforming growth factor-β(TGF-β),transcription factors RAR-related orphan receptor C(RORC),forkhead box protein P-3(Foxp-3),and in serum IL-17,TGF-βprotein levels at different time points in each group of rats were compared.Results Under light microscope,the lung tissue of rats showed significant alveolar interstitial thickening,interstitial fracture and fusion,and massive inflammatory cell infiltration after smoke exposure;BMSC transplantation significantly reduced alveolar injury and inhibited inflammatory cell infiltration.Compared with normal control group,the lung injury score,Th17,Th17/Treg,serum IL-17 protein levels and mRNA expression,RORC mRNA expression levels were higher in the SII model group from the 1st day after modeling,PaO_(2),PaO_(2)/FiO_(2),Treg,TGF-βmRNA and serum protein levels were significantly lower(all P<0.05),lasting until 3 days after molding(P<0.05);Compared with the SII model group,lung injury score,Th17 on the 3rd day post modeling and Th17/Treg,IL-17 mRNA expression and serum protein levels and RORC mRNA expression levels on the 1st day post modeling were significantly lower in the SII+BMSC group[lung injury score:2.00±0.63 vs.3.33±0.82,Th17:1.47±0.13 vs.3.00±0.54,Th17/Treg:0.29±0.13 vs.1.02±0.32,IL-17 mRNA(2^(-ΔΔCt)):0.64±0.09 vs.1.40±0.41,IL-17(ng/L):142.43±22.86 vs.309.83±13.45,RORC mRNA(2^(-ΔΔCt)):1.42±0.81 vs.3.50±0.42,all P<0.05],PaO_(2),PaO_(2)/FiO_(2),Treg,TGF-βmRNA expression and protein content were significantly increased from the 1st day after modeling[PaO_(2)(mmHg,1 mmHg≈0.133 kPa):120.60±13.38 vs.84.25±18.32,PaO_(2)/FiO_(2)(mmHg):596.43±51.22 vs.401.19±87.24,Treg:4.00±0.56 vs.2.46±0.60,TGF-βmRNA(2^(-ΔΔCt)):1.91±0.37 vs.0.25±0.16,TGF-β(ng/L):2901.40±28.58 vs.1855.96±345.72,all P<0.05].The differences in Foxp-3 mRNA expression levels among the three groups at different time points were not statistically significant.Conclusion The Th17/Treg cell ratio is imbalanced in SII rats,and BMSC play a therapeutic role in SII by regulating the Th17/Treg immune balance to suppress the inflammatory response and promote the repair of lung injury.