1、肺巨噬細(xì)胞移植治療肺巨噬細(xì)胞移植治療(PMT)Pulmonary MacrophageTransplantation Therapy專業(yè)：細(xì)胞生物學(xué)姓名：李倩倩導(dǎo)師：畢秀麗 hPAP遺傳性肺泡蛋白質(zhì)沉積癥簡介 基因治療 摘要 實驗方法 實驗過程和結(jié)果闡述 相關(guān)基因治療案例 hPAPHereditary pulmonary alveolar proteinosis:即hPAP,遺傳性肺泡蛋白質(zhì)沉積癥 纖維支氣管鏡活檢病理學(xué)檢查示肺泡腔內(nèi)充滿PAS陽性的粗顆粒狀物質(zhì),肺泡灌洗液可見大量無定形的碎片,常伴PAS染色陽性的巨噬細(xì)胞。 在hPAP中，肺泡充滿了表面活性物質(zhì)，肺生成這種物質(zhì)來降低表面張力，

2、保持肺泡開放狀態(tài)。 hPAP患兒具有CSFR2RA或CSFR2RB突變。這些突變降低了肺泡巨噬細(xì)胞清除這些孩子肺部表面活性物質(zhì)的能力。 表面活性物質(zhì)累積于肺部，填滿肺泡會造成呼吸困難或呼吸衰竭。對于這些孩子當(dāng)前唯一的治療方法就是在全身麻醉情況下完成侵襲性的洗肺程序。但其效應(yīng)是暫時的，必須頻繁重復(fù)操作，給受累患兒造成生活質(zhì)量問題。GM-CSF：粒細(xì)胞集落刺激生物因子，能夠刺激骨髓細(xì)胞對肺泡細(xì)胞的胞飲以及對面面活性物質(zhì)的降解。GM-CSF受體即CSFR的缺陷或者GM-CSF抗體均可導(dǎo)致hPAP。 GM-CSF的受體為CSFR,CSFR由和和鏈鏈組成，組成， 編碼編碼和和的基因分別為的基因分別為cs

3、f2racsf2ra和和csf2rbcsf2rb。csf2racsf2ra和和csf2rbcsf2rb突變突變 CSFRBCSFRB表達(dá)缺陷表達(dá)缺陷 阻斷阻斷GM-CSFGM-CSF信號轉(zhuǎn)導(dǎo)信號轉(zhuǎn)導(dǎo) 巨噬細(xì)胞對肺泡表面蛋白清除能力下降巨噬細(xì)胞對肺泡表面蛋白清除能力下降 hPAP hPAP也就是說，也就是說，hPAPhPAP患者肺泡血清中患者肺泡血清中GM-CSFGM-CSF升升高，而高，而GM-CSFGM-CSF抗體為陰性抗體為陰性 基因治療簡介基因治療（gene therapy）是指將外源正?；?qū)氚屑?xì)胞，以糾正或補償因基因缺陷和異常引起的疾病，以達(dá)到治療目的。也就是將外源基因通過基因轉(zhuǎn)移

4、技術(shù)將其插入病人的適當(dāng)?shù)氖荏w細(xì)胞中，使外源基因制造的產(chǎn)物能治療某種疾病?；蛑委煹奶禺愋詮?qiáng)，其藥物本質(zhì)為DNA，在體內(nèi)表達(dá)為蛋白質(zhì)，且基因治療作用有持續(xù)性?；蛑委煹牟呗杂谢蛱娲蛐拚?，基因抑制，基因增強(qiáng)和免疫調(diào)節(jié)。 摘要：以往用骨髓移植(BMT)來治療hPAP小鼠模型，會破壞現(xiàn)有骨髓。最近發(fā)現(xiàn)定居巨噬細(xì)胞可以自我維持，不需來自骨髓的細(xì)胞直接再生。于是作者想到測試新型巨噬細(xì)胞移植療法。結(jié)果表明，自然的健康巨噬細(xì)胞和基因矯正巨噬細(xì)胞同樣能夠很好地發(fā)揮作用糾正小鼠的hPAP。在小鼠基因Csf2rb喪失表達(dá)、模擬hPAP的小鼠中，研究人員利用一種病毒載體將矯正Csf2rb基因傳遞到取自動物的異

5、常肺泡巨噬細(xì)胞中。隨后將基因矯正細(xì)胞直接灌注到小鼠的肺部。這種方法使得疾病相關(guān)生物標(biāo)記物正常化，至少在一年內(nèi)降低了疾病死亡率。實驗方法實驗方法 支氣管肺泡灌洗液和細(xì)胞收集、處理支氣管肺泡灌洗液和細(xì)胞收集、處理 ELISA ,Quantitative RT-PCRELISA ,Quantitative RT-PCR Bone marrow derived macrophages Bone marrow derived macrophages (BMDMs)(BMDMs) Colony forming cell (CFC) assayColony forming cell (CFC) assay集

6、落形成集落形成實驗實驗 Surfactant Clearance AssaySurfactant Clearance Assay Pulmonary macrophage transplantation Pulmonary macrophage transplantation (PMT)(PMT) Flow cytometryFlow cytometry流式細(xì)胞術(shù)流式細(xì)胞術(shù) STAT5 phosphorylation index assaySTAT5 phosphorylation index assay Western blottingWestern blotting Microarray

7、analysisMicroarray analysis微陣列分析微陣列分析 Localization of PMT-derived cells Localization of PMT-derived cells after transplantationafter transplantation Lentiviral vectorsLentiviral vectors（慢病毒載體）慢病毒載體）, and , and differentiation and expansion of differentiation and expansion of macrophagesmacrophages實驗

8、過程簡介三種小鼠模型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染色陽性 ORO染色陽性2）BAL turbiditySP-Dconcentration3）BAL中，GM-CSF M-CSF CSF1 MCR1 mRNA for PU1, PPAR ABCG1 結(jié)果結(jié)果 K

9、O小鼠可以作為人類hPAP患者的模擬生物(a) Typical lung pathology and identical pulmonary histopathology in aKO mouse. PAS stain.(b) Photographs of ilkyappearing BAL from a 14 month-old KO mouse and 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-o

10、ld 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 b

11、y 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 stai

12、ning (b) (c) Flow cytometry evaluation of cell-surface phenotypic markerson WT BMDMs before PMT.(d)(d)Photographs ofPhotographs of methylcellulose cultures of methylcellulose cultures of Lincells from bone marrow Lincells from bone marrow and BMDMs and typical and BMDMs and typical coloniescolonies

13、(e) Colony counts of BFU-E, (e) Colony counts of BFU-E, CFU-GEMM andCFU-GEMM andCFU-GM showing BMDMs CFU-GM showing BMDMs contained 0.005% CFU-GM contained 0.005% CFU-GM and no BFU-E or CFU-GEMMand no BFU-E or CFU-GEMMprogenitors, corresponding to progenitors, corresponding to 93 CFU-GM per dose of

14、93 CFU-GM per dose of BMDMs administered (n=3BMDMs administered (n=3d e t e r m i n a t i o n s p e r d e t e r m i n a t i o n s p e r condition). condition). (f-g) Evaluation of surfactant clearance capacity.(f-g) Evaluation of surfactant clearance capacity.These results demonstrated the cells use

15、d for PMT were highly purified, mature macrophages capable of surfactant clearance.Efficacy of PMT of WT macrophagesEfficacy 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 cel

16、ls were present (Fig.1b), alveolar macrophages expressed Csf2rb(c) Appearance of BAL fluid(c) Appearance of BAL fluid (left) or sediment (right). (left) or sediment (right). (b)Representative cytology of (b)Representative cytology of BAL obtained one year afterBAL obtained one year after PMT after s

17、taining with PAS PMT after staining with PAS or oil-red-O (ORO)or oil-red-O (ORO)PMT nearly completely resolved theabnormal pulmonary histopathology (c)Representative photomicrog r aphs of (c)Representative photomicrog r aphs of PASstained whole-mount lung sections one year PASstained whole-mount lu

18、ng sections one year after PMTafter PMTPMT nearly completely resolved theabnormal pulmonary histopathology (d) Lung histology after (d) Lung histology after staining with H&E, PAS,staining with H&E, PAS,Masson trichrome (MT), Masson trichrome (MT), or surfactant protein B or surfactant protein B (SP

19、-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 diff

20、erent compared to untreated WT m i c e ( M a n n -Whitney Rank Sum Test, P0.001). is significantly different compared to untreated KO mice not significantly s i g n i f i c a n t l y different compared to untreated KO mice P=0.133.Neither significantly affected efficacy in the range evaluated and on

21、e 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 WT macrophages had increased surviv

22、al/proliferationsurvival/proliferationcompared to KO macrophages compared to KO macrophages in vitro in vitro (Fig. 2a) and (Fig. 2a) and accumulated to greater numbers after PMT in KO mice accumulated to greater numbers after PMT in KO mice than in WT mice (Fig. 2b and Extended Data Fig. 3d)than in

23、 WT mice (Fig. 2b and Extended Data Fig. 3d)(b) Quantification of GFP+ BAL cells 2 months after PMT (b) Quantification of GFP+ BAL cells 2 months after PMT of Lys-MGFP BMDMs into WT (n=3) or KO (n=6) mice. of Lys-MGFP BMDMs into WT (n=3) or KO (n=6) mice. (d) GFP+ cells in BAL cells from WT or KO mi

24、ce 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是與增殖相關(guān)的核抗原，Ki67標(biāo)記G0期(非增殖期)以外的細(xì)胞，Ki67陽性細(xì)胞比例越高，處于增殖時期的細(xì)胞比例越高，腫瘤惡性程度越高。GFPKi67GFP+Ki67(e) Macrophage

25、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

26、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) B

27、AL turbidity declined with the increase inCD131+ alveolarmacrophages.One year after PMT, CD131 + cells were present , One year after PMT, CD131 + cells were present , Csf2rb Csf2rb protein was detectable in alveolar macrophages, and protein was detectable in alveolar macrophages, and Csf2rb Csf2rb m

28、RNAmRNA in BAL cells from PMT-treated KO mice was in BAL cells from PMT-treated KO mice was only slightly less than in WT and undetectable in only slightly less than in WT and undetectable in untreated KO BAL cells (Fig. 2g). numbers ofuntreated KO BAL cells (Fig. 2g). numbers of CD131 + CD131 + alv

29、eolar macrophagesalveolar macrophages in PMT-treated KO and untreated in PMT-treated KO and untreated WT mice wereWT mice were similar similar one year after PMT (Fig. 2h). one year after PMT (Fig. 2h).These results 1) WT macrophages had a selective survival advantage over KO macrophages, 2)WT macro

30、phages 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 K O a l v e o l a r m a c r o p h a g e s3 ) T h e n u m b e r s o f C D 1 3 1 + , G M - C S F responsive alveolar macrophages similar to WT

31、mice.Macrophage characterization after PMTMacrophage characterization after PMT( h )( h ) L o c a l i z a t i o nL o c a l i z a t i o n o f o f macrophages after PMT macrophages after PMT of Lys-MGFP BMDMs into of Lys-MGFP BMDMs into KO mice and after CD68 KO mice and after CD68 immunostaining,DAPI

32、 immunostaining,DAPI s t a i n i n g , a n d s t a i n i n g , a n d fluorescence microscopy fluorescence microscopy to detect CD68+/GFP+ to detect CD68+/GFP+ c e l l s P M T d e r i v e d c e l l s P M T d e r i v e d m a c r o p h a g e s o r m a c r o p h a g e s o r CD68+/GFPcells (i.e., CD68+/G

33、FPcells (i.e., n o n - P M T - d e r i v e d n o n - P M T - d e r i v e d macrophages).macrophages). CD68+/GFP+ revealed 88.9 +_ 0.87% were intra-alveolar CD68+/GFP+ revealed 88.9 +_ 0.87% were intra-alveolar and 11.1 _+0.87% were interstitial and 11.1 _+0.87% were interstitial GFPimmunohistochemic

34、al staining was done to eliminate GFPimmunohistochemical staining was done to eliminate potential interference from autofluorescence and potential interference from autofluorescence and confirmed these results; 90.5_+1.1% PMT-derived confirmed these results; 90.5_+1.1% PMT-derived macrophages were i

35、ntra-alveolar and 9.4_+ 1.1% were macrophages were intra-alveolar and 9.4_+ 1.1% were interstitial (Fig. 3a-b )interstitial (Fig. 3a-b )GFP+ cellsGFP+ cellsL o c a l i z a t i o n o f G F P + L o c a l i z a t i o n o f G F P + macrophages to intra-alveolarmacrophages to intra-alveolarLocalization w

36、as done in WT Lys-MGFP or KO mice and Localization was done in WT Lys-MGFP or KO mice and PMT-treated KO mice by flow cytometry to detect the PMT-treated KO mice by flow cytometry to detect the percentage of GFP+ cells one year after PMT (Extended percentage of GFP+ cells one year after PMT (Extende

37、d Data Fig. 4a-b) and by PCR amplification of Lys-MGFP Data Fig. 4a-b) and by PCR amplification of Lys-MGFP transgene-specific DNA (Extended Data Fig. 4c),transgene-specific DNA (Extended Data Fig. 4c),PMT-derived cells were PMT-derived cells were present in the lungs but present in the lungs but no

38、t detected in blood, not detected in blood, bone marrow, or spleen. bone marrow, or spleen. One year after PMT of CD45.1 + WT BMDMs into CD45.2+ One year after PMT of CD45.1 + WT BMDMs into CD45.2+ KO mice,flow cytometric detection of CD45.1 + cells KO mice,flow cytometric detection of CD45.1 + cell

39、s confirmed thease findings (Extended Data Fig.4e-g). confirmed thease findings (Extended Data Fig.4e-g). the percentage of CD45.1 +cells in the gated regions the percentage of CD45.1 +cells in the gated regions are shown are shown Results show that the transplanted Results show that the transplante

40、d macrophages remained in the lungs, macrophages remained in the lungs, primarily within the intra-alveolar space.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

41、 of WT Lys-MGFP BMDMs into KO mice,similar to the phenotype of WT alveolar macrophages and different from recipient KO mice微陣列分析Microarray analysisMicroarray analysis Unsupervised hierarchicalUnsupervised hierarchicalclustering dendrogramclustering dendrogram層析聚層析聚類分析類分析heat-map of selected GM-CSF-h

42、eat-map of selected GM-CSF-regulated genesregulated genesExpression of genes regulated by GM-CSF was reduced in Expression of genes regulated by GM-CSF was reduced in KOmice and restored by PMT (Fig. 4, Extended Data Fig. KOmice and restored by PMT (Fig. 4, Extended Data Fig. 5a).5a).(a) Expression

43、of (a) Expression of Spi1 Spi1 (PU.1) and (PU.1) and Pparg Pparg (PPAR were (PPAR were confirmed by qRT-PCR using independent samplesconfirmed by qRT-PCR using independent samples(b)Venn diagrams showing (b)Venn diagrams showing numbers of genes whosenumbers of genes whoseexpression was altered in e

44、xpression was altered in alveolar macrophages from KO alveolar macrophages from KO c o m p a r e d t o W T m i c e c o m p a r e d t o W T m i c e (WTKO)or PMT-tr eated (WTKO)or PMT-tr eated compared to untreated KO compared to untreated KO mice (KOKO+PMT).Only mice (KOKO+PMT).Only genes with statis

45、tically genes with statistically significant changes of at least significant changes of at least two-fold were marked as two-fold were marked as increased (up arrows) or increased (up arrows) or decreased (down arrows). decreased (down arrows). Of 776 genes for which Of 776 genes for which expressio

46、n was disrupted in expression was disrupted in KO mice, PMT normalized KO mice, PMT normalized expression of 600 including expression of 600 including 80% of genes up-regulated80% of genes up-regulated and and 76% of genes down-regulated76% of genes down-regulated in KO compared to WT micein KO comp

47、ared to WT mice(Extended Data Fig. 5b).(Extended Data Fig. 5b).(d) Heat maps showing differentially expressed genes (d) Heat maps showing differentially expressed genes including PPARregulated genes, glycophospholipid including PPARregulated genes, glycophospholipid metabolism, peroxisome function a

48、poptosis, cell cycle metabolism, peroxisome function apoptosis, cell cycle control, Genes with increased or decreased transcript control, Genes with increased or decreased transcript levels are shown by red and blue colors, respectively.levels are shown by red and blue colors, respectively.Efficacy

49、of gene therapy by PMTEfficacy of gene therapy by PMTSince PMT in humans would likely employ autologous, Since PMT in humans would likely employ autologous, gene-corrected HSPC-derived macrophages, we evaluated gene-corrected HSPC-derived macrophages, we evaluated PMT of KO macrophages derived from LSK cells after PMT of KO macrophages derived from LSK cells after lentiviral vector (LV)-mediated lentiviral vector (LV)-mediated Csf2rb Csf2rb cDNA expression cDNA expression (Fig. 5a).(Fig. 5a).只有WT,KO+GM-R-GFP中的CD131+ 表達(dá)CD68+都表達(dá)(c) Western blotting to detect GM-CSF r