肺巨噬细胞移植治疗

肺巨噬细胞移植治疗(PMT)Pulmonary Macrophage Transplantation Therapy专业：细胞生物学姓名：李倩倩导师：毕秀丽hPAP遗传性肺泡蛋白质沉积症简介 基因治疗 摘要 实验方法 实验过程和结果阐述 相关基因治疗案例 hPAPHereditary pulmonary alveolar proteinosis:即 hPAP,遗传性肺泡蛋白质沉积症 纤维支气管镜活检病理学检查示肺泡腔内充满 PAS阳性的粗颗粒状物质 ,肺泡灌洗液可见大量无定形的碎片 ,常伴 PAS染色阳性的巨噬细胞。在 hPAP中，肺泡充满了 表面活性物质，肺生成这种物质来降低表面张力，保持肺泡开放状态。 hPAP患儿具有 CSFR2RA或 CSFR2RB突变。这些突变降低了肺泡巨噬细胞清除这些孩子肺部表面活性物质的能力。 表面活性物质累积于肺部，填满肺泡会造成呼吸困难或呼吸衰竭。对于这些孩子当前唯一的治疗方法就是在全身麻醉情况下完成侵袭性的洗肺程序。但其效应是暂时的，必须频繁重复操作，给受累患儿造成生活质量问题。GM-CSF：粒细胞集落刺激生物因子，能够刺激骨髓细胞对肺泡细胞的胞饮以及对面面活性物质的降解。 GM-CSF受体即 CSFR的缺陷或者 GM-CSF抗体均可导致 hPAP。GM-CSF的受体为 CSFR,CSFR由 和 链组成， 编码 和 的基因分别为 csf2ra和 csf2rb。csf2ra和 csf2rb突变CSFRB表达缺陷阻断 GM-CSF信号转导 巨噬细胞对肺泡表面蛋白清除能力下降hPAP也就是说， hPAP患者肺泡血清中 GM-CSF升高，而 GM-CSF抗体为阴性基因治疗简介基因治疗（ gene therapy）是指将外源正常基因导入靶细胞，以纠正或补偿因基因缺陷和异常引起的疾病，以达到治疗目的。也就是将外源基因通过基因转移技术将其插入病人的适当的受体细胞中，使外源基因制造的产物能治疗某种疾病。基因治疗的特异性强，其药物本质为 DNA，在体内表达为蛋白质，且基因治疗作用有持续性。基因治疗的策略有基因替代，基因修正，基因抑制，基因增强和免疫调节。 摘要：以往用骨髓移植 (BMT)来治疗 hPAP小鼠模型，会破坏现有骨髓。最近发现定居巨噬细胞可以自我维持，不需来自骨髓的细胞直接再生。于是作者想到测试新型巨噬细胞移植疗法。结果表明，自然的健康巨噬细胞和基因矫正巨噬细胞同样能够很好地发挥作用纠正小鼠的 hPAP。在小鼠 基因 Csf2rb丧失表达、模拟 hPAP的小鼠中，研究人员利用一种 病毒载体 将矫正 Csf2rb基因传递到取自动物的异常肺泡巨噬细胞中。随后将基因矫正细胞直接灌注到小鼠的肺部。这种方法使得疾病相关生物标记物正常化，至少在一年内降低了疾病死亡率。实验方法 支气管肺泡灌洗液和细胞收集、处理ELISA ,Quantitative RT-PCRBone marrow derived macrophages (BMDMs)Colony forming cell (CFC) assay集落形成实验Surfactant Clearance AssayPulmonary macrophage transplantation (PMT)Flow cytometry 流式细胞术STAT5 phosphorylation index assayWestern blottingMicroarray analysis微阵列分析Localization of PMT-derived cells after transplantationLentiviral vectors（ 慢病毒载体） , and differentiation and expansion of macrophages实验过程简介三种小鼠模型 1） WT mice(wind type)2)KO mice(Csf2rb-/-)3)KO+PMT mice(pulmonary macrophage transplantation)WT HSPCs isolated expanded differentiated into macrophagestransplantationhPAP markers： 1） PAS染色阳性 ORO染色阳性2） BAL turbiditySP-Dconcentration3） BAL中， GM-CSF M-CSF CSF1 MCR1 mRNA for PU1, PPAR ABCG1 结果 KO小鼠 可以作为人类 hPAP患者的模拟生物(a) Typical lung pathology and identical pulmonary histopathology in aKO mouse. PAS stain.(b) Photographs of ilkyappearing BAL from a 14 month-old KO mouseand normal-appearing BAL from an age-matched WT mouse(d) BAL fluid biomarkers of hPAP(GM-CSF, M-CSF, and MCP-1) in 4 month-old KO mice(e) Alveolar macrophage biomarkers (PU.1, Pparg, Abcg1 mRNA) arereduced in 4 month-old KO(f)(g) Progressive increase in BAL turbidityBAL fluid,GM-CSF level in KO mice (h) GM-CSF bioactivity in BAL fluid from 10 month-old KO or WT mice Result: We validated KO mice as a model of human hPAP by demonstrating they had the same clinical,physiological, histopathological, and biochemical abnormalities, disease biomarkers, natural historyas children with hPAP3. Macrophage characterization after PMT(a-b) Photomicrographs of WT BMDMs prior to transplantation phase-contrast (a) or DiffQuick staining (b) (c) Flow cytometry evaluation of cell-surface phenotypic markerson WT BMDMs before PMT.(d)Photographs ofmethylcellulose cultures of Lincells from bone marrow and BMDMs and typical colonies(e) Colony counts of BFU-E, CFU-GEMM andCFU-GM showing BMDMs contained 0.005% CFU-GM and no BFU-E or CFU-GEMMprogenitors, corresponding to 93 CFU-GM per dose of BMDMs administered (n=3determinations per condition). (f-g) Evaluation of surfactant clearance capacity.These results demonstrated the cells used for PMT were highly purified, mature macrophages capable of surfactant clearance.Efficacy of PMT of WT macrophagesTo determine the therapeutic potential of PMT, KO mice received WT (Csf2rb+/+) BMDMs by PMT once (Fig. 1a). One year later, PMT-derived CD131 + BAL cells were present (Fig.1b), alveolar macrophages expressed Csf2rb(c) Appearance of BAL fluid(left) or sediment (right). (b)Representative cytology of BAL obtained one year afterPMT after staining with PAS or oil-red-O (ORO)PMT nearly completely resolved theabnormal pulmonary histopathology (c)Representative photomicrographs of PASstained whole-mount lung sections one year after PMTPMT nearly completely resolved theabnormal pulmonary histopathology (d) Lung histology after staining with H&E, PAS,Masson trichrome (MT), or surfactant protein B (SP-B)(e) BAL turbidity and SP-D concentration. (f) BAL biomarkers. ResultThese results demonstrate PMT had a highly efficacious and durable therapeutic effect on the primary pulmonary and secondary systemic manifestations of hPAP in KO mice.Macrophage engraftment efficiency is significantly different compared to untreated WT mice (Mann-Whitney Rank Sum Test, P0.001). is significantly different compared to untreated KO mice not significantly significantly different compared to untreated KO mice P=0.133.Neither significantly affected efficacy in the range evaluated and one dose of2 million cells was used for PMT in the remaining studies.To determine if WT macrophages had a survival advantage over KO macrophage we measured GM-CSF bioactivity in BAL fluid and found it was detectable in KO but not WT mice WT macrophages had increased survival/proliferationcompared to KO macrophages in vitro (Fig. 2a) and accumulated to greater numbers after PMT in KO mice than in WT mice (Fig. 2b and Extended Data Fig. 3d)(b) Quantification of GFP+ BAL cells 2 months after PMT of Lys-MGFP BMDMs into WT (n=3) or KO (n=6) mice. (d) GFP+ cells in BAL cells from WT or KO mice 2 monthsafter PMT of Lys-MGFP BMDMs.PMT of WT LysMGFP knock-in mouse25 BMDMs into KO mice followed by Ki67 immunostaining revealed PMT-derived cells replicated in vivo (Extended Data Fig. 3e-g). Ki67是与增殖相关的核抗原， Ki67标记 G0期 (非增殖期 )以外的细胞， Ki67阳性细胞比例越高，处于增殖时期的细胞比例越高，肿瘤恶性程度越高。GFPKi67GFP+Ki67(e) Macrophage replication after PMT. KO mice received Lys-MGFP BMDMs by PMT and paraffin-embedded lung was immunostained for Ki67 one month or one year later(c)The percentage of Ki67+PMT-derived alveolar macrophages was 32.2 _+ 6.05% one month after PMT and declined to 11.29 _+ 2.2% by one year (Fig. 2c)(f) Ki67 staining of BAL cells froman untreated WT mice To further define survival advantage, we evaluated the engraftment kinetics after one PMT of WT BMDMs in KO mice. CD131 + cells increased steadily from zero to 69.0?2.5% of BAL cells(Fig. 2d) (e) a smooth decline in pulmonary GM-CSF to near normal (f) BAL turbidity declined with the increase inCD131+ alveolarmacrophages.One year after PMT, CD131 + cells were present , Csf2rb protein was detectable in alveolar macrophages, and Csf2rb mRNA in BAL cells from PMT-treated KO mice was only slightly less than in WT and undetectable in untreated KO BAL cells (Fig. 2g). numbers of CD131 + alveolar macrophages in PMT-treated KO and untreated WT mice were similar one year after PMT (Fig. 2h).These results 1) WT macrophages had a selective survival advantage over KO macrophages, 2)WT macrophages after PMT into KO mice They proliferated in vivo at a rate that slowed over time synchronous with reduction in pulmonary GM-CSF, replaced dysfunctional KO alveolar macrophages3)The numbers of CD131+,GM-CSF responsive alveolar macrophages similar to WT mice.Macrophage characterization after PMT(h)Localization of macrophages after PMT of Lys-MGFP BMDMs into KO mice and after CD68 immunostaining,DAPI staining,and fluorescence microscopy to detect CD68+/GFP+ cells PMT derived macrophages or CD68+/GFPcells (i.e., non-PMT-derived macrophages).CD68+/GFP+ revealed 88.9 +_ 0.87% were intra-alveolar and 11.1 _+0.87% were interstitial GFPimmunohistochemical staining was done to eliminate potential interference from autofluorescence and confirmed these results; 90.5_+1.1% PMT-derived macrophages were intra-alveolar and 9.4_+ 1.1% were interstitial (Fig. 3a-b )GFP+ cells Localization of GFP+ macrophages to intra-alveolarLocalization was done in WT Lys-MGFP or KO mice and PMT-treated KO mice by flow cytometry to detect the percentage of GFP+ cells one year after PMT (Extended Data Fig. 4a-b) and by PCR amplification of Lys-MGFP transgene-specific DNA (Extended Data Fig. 4c),PMT-derived cells were present in the lungs but not detected in blood, bone marrow, or spleen. One year after PMT of CD45.1 + WT BMDMs into CD45.2+ KO mice,flow cytometric detection of CD45.1 + cells confirmed thease findings (Extended Data Fig.4e-g). the percentage of CD45.1 +cells in the gated regions are shown Results show that the transplanted macrophages remained in the lungs, primarily within the intra-alveolar space.The effects of the lung milieu on the phenotype of transplanted macrophages were evaluated by measuring cell-surface markers. One year after PMT of WT Lys-MGFP BMDMs into KO mice,similar to the phenotype of WT alveolar macrophages and different from recipient KO mice微阵列分析 Microarray analysisUnsupervised hierarchicalclustering dendrogram 层析聚类分析heat-map of selected GM-CSF-regulated genesExpression of genes regulated by GM-CSF was reduced in KOmice and restored by PMT (Fig. 4, Extended Data Fig. 5a).(a) Expression of Spi1 (PU.1) and Pparg (PPAR were confirmed by qRT-PCR using independent samples(b)Venn diagrams showing numbers of genes whoseexpression was altered in alveolar macrophages from KO compared to WT mice (WTKO)or PMT-treated compared to untreated KO mice (KOKO+PMT).Only genes with statistically significant changes of at least two-fold were marked as increased (up arrows) or decreased (down arrows). Of 776 genes for which expression was disrupted in KO mice, PMT normalized expression of 600 including 80% of genes up-regulated and 76% of genes down-regulated in KO compared to WT mice(Extended Data Fig. 5b).(d) Heat maps showing differentially expressed genes including PPARregulated genes, glycophospholipid metabolism, peroxisome function apoptosis, cell cycle control, Genes with increased or decreased transcript levels are shown by red and blue colors, respectively.Efficacy of gene therapy by PMTSince PMT in humans would likely employ aut