骨髓祖细胞对肝脏纤维化细胞没有贡献。

Bone marrow progenitor cells do not contribute to liver fibrogenic cells.

作者信息

Paredes Bruno Diaz, Faccioli Lanuza Alaby Pinheiro, Quintanilha Luiz Fernando, Asensi Karina Dutra, do Valle Camila Zaverucha, Canary Paulo César, Takiya Christina Maeda, de Carvalho Antonio Carlos Campos, Goldenberg Regina Coeli Dos Santos

机构信息

Bruno Diaz Paredes, Lanuza Alaby Pinheiro Faccioli, Luiz Fernando Quintanilha, Karina Dutra Asensi, Camila Zaverucha do Valle, Christina Maeda Takiya, Antonio Carlos Campos de Carvalho, Regina Coeli dos Santos Goldenberg, Carlos Chagas Filho Biophysics Institute, Rio de Janeiro 21941-902, Brazil.

出版信息

World J Hepatol. 2012 Oct 27;4(10):274-83. doi: 10.4254/wjh.v4.i10.274.

DOI:10.4254/wjh.v4.i10.274

PMID:23293712

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3537161/

Abstract

AIM

To investigate the contribution of bone marrow (BM) cells to hepatic fibrosis.

METHODS

To establish a model of chimerism, C57Bl/6 female mice were subjected to full-body irradiation (7 Gy) resulting in BM myeloablation. BM mononuclear cells obtained from male transgenic mice expressing enhanced green fluorescent protein (GFP) were used for reconstitution. Engraftment was confirmed by flow cytometry. To induce liver injury, chimeric animals received carbon tetrachloride (CCl(4)) 0.5 mL/kg intraperitoneally twice a week for 30 d (CCl(4) 30 d) and age-matched controls received saline (Saline 30 d). At the end of this period, animals were sacrificed for post mortem analysis. Liver samples were stained with hematoxylin and eosin to observe liver architectural changes and with Sirius red for collagen quantification by morphometric analysis. α-smooth muscle actin (α-SMA) was analyzed by confocal microscopy to identify GFP+ cells with myofibroblast (MF) characteristics. Liver tissue, BM and peripheral blood were collected and prepared for flow cytometric analysis using specific markers for detection of hepatic stellate cells (HSCs) and precursors from the BM.

RESULTS

Injury to the liver induced changes in the hepatic parenchymal architecture, as reflected by the presence of inflammatory infiltrate and an increase in collagen deposition (Saline 30 d = 11.10% ± 1.12% vs CCl(4) 30 d = 12.60% ± 0.73%, P = 0.0329). Confocal microscopy revealed increased reactivity against α-SMA in CCl(4) 30 d compared to Saline 30 d, but there was no co-localization with GFP+ cells, suggesting that cells from BM do not differentiate to MFs. Liver flow cytometric analysis showed a significant increase of CD45+/GFP+ cells in liver tissue (Saline 30 d = 3.2% ± 2.2% vs CCl(4) 30 d = 5.8% ± 1.3%, P = 0.0458), suggesting that this increase was due to inflammatory cell infiltration (neutrophils and monocytes). There was also a significant increase of common myeloid progenitor cells (CD117+/CD45+) in the livers of CCl(4)-treated animals (Saline 30 d = 2.16% ± 1.80% vs CCl(4) 30 d = 5.60% ± 1.30%, P = 0.0142). In addition the GFP-/CD38+/CD45- subpopulation was significantly increased in the CCl(4) 30 d group compared to the Saline 30 d group (17.5% ± 3.9% vs 9.3% ± 2.4%, P = 0.004), indicating that the increase in the activated HSC subpopulation was not of BM origin.

CONCLUSION

BM progenitor cells do not contribute to fibrosis, but there is a high recruitment of inflammatory cells that stimulates HSCs and MFs of liver origin.

摘要

目的

研究骨髓（BM）细胞对肝纤维化的作用。

方法

为建立嵌合体模型，对C57Bl/6雌性小鼠进行全身照射（7 Gy）以造成BM骨髓消融。使用从表达增强型绿色荧光蛋白（GFP）的雄性转基因小鼠获得的BM单个核细胞进行重建。通过流式细胞术确认植入情况。为诱导肝损伤，嵌合动物每周两次腹腔注射0.5 mL/kg四氯化碳（CCl₄），持续30天（CCl₄ 30天组），年龄匹配的对照组注射生理盐水（生理盐水30天组）。在此期间结束时，处死动物进行尸检分析。肝组织样本用苏木精和伊红染色以观察肝脏结构变化，用天狼星红染色通过形态计量分析进行胶原定量。通过共聚焦显微镜分析α平滑肌肌动蛋白（α-SMA）以鉴定具有肌成纤维细胞（MF）特征的GFP⁺细胞。收集肝组织、BM和外周血并使用用于检测肝星状细胞（HSCs）和BM来源前体细胞的特异性标记物进行流式细胞术分析。

结果

肝损伤导致肝实质结构发生变化，表现为炎症浸润和胶原沉积增加（生理盐水30天组 = 11.10% ± 1.12% vs CCl₄ 30天组 = 12.60% ± 0.73%，P = 0.0329）。共聚焦显微镜显示，与生理盐水30天组相比，CCl₄ 30天组中α-SMA的反应性增加，但未与GFP⁺细胞共定位，表明BM细胞未分化为MF。肝脏流式细胞术分析显示肝组织中CD45⁺/GFP⁺细胞显著增加（生理盐水30天组 = 3.2% ± 2.2% vs CCl₄ 30天组 = 5.8% ± 1.3%，P = 0.0458），表明这种增加是由于炎症细胞浸润（中性粒细胞和单核细胞）。在CCl₄处理动物的肝脏中，常见髓系祖细胞（CD117⁺/CD45⁺）也显著增加（生理盐水30天组 = 2.16% ± 1.80% vs CCl₄ 30天组 = 5.60% ± 1.30%，P = 0.0142）。此外，与生理盐水30天组相比，CCl₄ 30天组中GFP⁻/CD38⁺/CD45⁻亚群显著增加（17.5% ± 3.9% vs 9.3% ± 2.4%，P = 0.004），表明活化的HSC亚群增加并非源于BM。