【作者】 王薇；

【导师】 李志远；

【作者基本信息】 中南大学 ， 神经生物学， 2022， 硕士

【摘要】 目的:PF是一种不可逆转的疾病,严重影响患者的生活质量。目前尚无针对PF的特异性治疗方法。而hucMSCs具有抗炎、促进组织修复的功能。PF的病程进展首先是机体发生炎症反应,炎症因子集聚,进一步发展成纤维化。本研究旨在探求Ang Ⅱ介导hucMSCs免疫调节肺纤维化及通过脑肺轴调节对BDNF的影响。方法:流式细胞术对hucMSCs的标志性表面抗原进行鉴定。用成骨、成脂的诱导培养基培养hucMSCs,检测其分化能力。不同浓度的Ang Ⅱ预处理hucMSCs筛选出最适的实验浓度。气管注射BLM建立动物模型,即用1 ml的注射针吸取BLM（5 U/Kg）沿气管环之间快速注入SD大鼠肺部,注射完以后迅速将大鼠直立旋转5 min。SD大鼠分成四组:假手术组（Sham组）、造模组注射PBS（BLM+PBS组）、造模组注射hucMSCs（BLM+hucMSCs组）以及造模组注射Ang Ⅱ预处理的hucMSCs（BLM+hucMSCs-Ang Ⅱ组）。造模后第15天分别尾静脉注射相同量的PBS、hucMSCs和hucMSCs-Ang Ⅱ,于造模后第23天取材。H&E染色观察肺组织炎症浸润情况;Massion染色和天狼星红染色观察肺组织中胶原纤维的情况;免疫组化检测cr-1、α-SMA、5-HT和MMP-9在肺组织中的分布情况,BDNF在海马中的表达;蛋白免疫印迹技术（WB）检测α-SMA、MMP-9和AT1R表达。结果:（1）流式细胞术结果显示实验用的hucMSCs表面分子CD29、CD44、CD90和CD105呈阳性,CD31、CD34、CD45、CD47和HLA-DR呈阴性。成脂和成骨分化实验说明实验用的hucMSCs可分化为成骨细胞和脂肪细胞;这说明实验用的细胞是hucMSCs;（2）合适浓度(10^-7M)下Ang Ⅱ通过和受体AT2R结合促进hucMSCs的迁移,但对hucMSCs增殖几乎无影响;（3）小动物活体成像结果显示Ang Ⅱ预处理后,会有更多的hucMSCs到达损伤的肺组织;（4）经过hucMSCs和hucMSCs-Ang Ⅱ治疗后,SD大鼠肺部炎症降低,中性粒细胞减少,抗炎因子IL-10表达上调;（5）hucMSCs和hucMSCs-Ang Ⅱ治疗后,SD大鼠肺组织中HYP的量减少,α-SMA和AT1R在BLM+PBS组中上调,治疗后BLM+hucMSCs-Ang Ⅱ组中的α-SMA和AT1R的表达比给予单一hucMSCs治疗组中更低;（6）hucMSCs治疗后促进SD大鼠肺组织中胶原蛋白和胶原纤维的降解;（7）hucMSCs治疗能下调肺内5-HT的表达,促进肺损伤修复,还可上调海马区BDNF的表达,起到神经保护的作用。结论:hucMSCs治疗可逆转SD大鼠肺纤维化,促进炎症浸润、胶原蛋白及胶原纤维降解减少,显著改善肺纤维化症状。此外,Ang Ⅱ预处理可以提高hucMSCs的归巢能力,使更多的hucMSCs能够到达肺损伤部位并发挥作用。hucMSCs通过促进PNECs分泌修复肺损伤,还可通过脑肺轴的影响BDNF的表达。图9幅,表7个,参考文献187篇更多还原

【Abstract】 Aim:PF is an irreversible disease that seriously affects the quality of life of patients.Currently,there is no specific treatment for PF.HucMSCs have strong differentiation function and can differentiate into many kinds of cells.Moreover,hucMSCs had anti-inflammatory and tissue repair functions.The progression of PF begins with an inflammatory response in the body,the accumulation of inflammatory factors,and finally the development of fibrosis.The purpose of this study was to explore the effects of Ang Ⅱ mediated hucMSCs immune regulation on pulmonary fibrosis and BDNF regulation through brain-lung axis.Methods:The signature surface antigens of hucMSCs were identified by flow cytometry.hucMSCs were cultured in osteogenic and lipid-induced medium and their differentiation ability was detected.The optimal concentration of Ang Ⅱ pretreated hucMSCs brush was selected.Animal models were modeled by tracheal injection of BLM,in which BLM（5 U/Kg）was absorbed with a 1m L injection needle and rapidly injected into the lungs of SD rats between the tracheal rings.After the injection,the rats were quickly rotated vertically for 5 min.SD rats were divided into four groups:Sham operation group（Sham group）,model group injected with PBS（BLM+PBS group）,model group injected with hucMSCs（BLM+hucMSCs group）,and model group injected with Ang Ⅱ pretreated hucMSCs（BLM+hucMSCs-Ang Ⅱ group）.On day 15 after modeling,PBS,hucMSCs and hucMSCs-Ang Ⅱ with the same dose were injected into tail vein,respectively.The samples were made on the 23^rdday after modeling.Hematoxylin-eosin staining was used to observe the inflammatory infiltration of lung tissue.The collagen fibers in lung tissues were observed by Massion staining and Sirius red staining.Immunohistochemistry was used to detect the distribution of Cr-1,α-SMA,5-HT and MMP-9 in lung tissues and the expression of BDNF in hippocampus.The expression ofα-SMA,MMP-9 and AT1R were detected by western blot（WB）.Result:（1）Flow cytometry results showed that the surface molecules of hucMSCs were positive for CD29,CD44,CD90 and CD105,and negative for CD31,CD34,CD45,CD47 and HLA-DR.The results showed that hucMSCs could differentiate into osteoblasts and adipocytes.These two experimental results indicated that the cells used in the experiment were hucMSCs;（2）At an appropriate concentration(10^-7M),Ang Ⅱ can bind to the receptor AT2R to promote the migration of hucMSCs,but has almost no effect on the proliferation of hucMSCs;（3）In vivo imaging results of small animals showed that after pretreatment with Ang Ⅱ,more hucMSCs could reach the injured lung tissues;（4）After treatment with hucMSCs and huc MSCS-Ang Ⅱ,lung inflammation was reduced,neutrophils decreased,and the expression of anti-inflammatory factor IL-10 was up-regulated;（5）After treatment with hucMSCs and hucMSCs-Ang Ⅱ,the amount of HYP in lung tissue of SD rats was decreased,andα-SMA and AT2R were up-regulated in BLM+PBS group.After treatment,the expression ofα-SMA and AT1R in BLM+hucMSCs-Ang Ⅱ group were lower than that in BLM+hucMSCs group;（6）hucMSCs treatment promoted the degradation of collagen protein and collagen fiber in lung tissue of SD rats;（7）hucMSCs treatment can down-regulate the expression of 5-HT in lung,promote the repair of lung injury,and up-regulate the expression of BDNF in hippocampus,playing a neuroprotective role.Conclusion:hucMSCs treatment could reverse pulmonary fibrosis,promote the reduction of inflammatory infiltration,collagen and collagen fiber degradation,and significantly improved pulmonary fibrosis symptoms in SD rats.Moreover,Ang Ⅱ pretreatment could improve the homing ability of hucMSCs,so that more hucMSCs could reach the site of lung injury and play a role.HucMSCs can repair lung injury by promoting the secretion of PNECs,and also affect the expression of BDNF by the brain-lung axis.更多还原