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Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression
Bal液体调控成纤维细胞α-SMA表达的评估实验   

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参见作者原研究论文

本实验方案简略版
Immunity
Mar 2016

Abstract

Because transforming growth factor-β (TGF-β1) induces differentiation of fibroblasts to myofibroblasts, we developed a protocol to evaluate alveolar macrophage-derived TGF-β1 regulation of lung fibroblast differentiation (Larson-Casey et al., 2016). The protocol evaluates the ability of mouse bronchoalveolar lavage (BAL) fluid to alter fibroblast differentiation. Fibroblast differentiation was measured by the expression of α-smooth muscle actin (α-SMA).

Background

Alveolar macrophages play an integral role in pulmonary fibrosis development by increasing the expression of TGF-β1 (He et al., 2011). Our prior data demonstrate that alveolar macrophages are a critical source of TGF-β1 as mice harboring a conditional deletion of TGF-β1 in macrophages were protected from pulmonary fibrosis (Larson-Casey et al., 2016). The expression of α-SMA is a defining feature of myofibroblasts, and TGF-β1 is a well-characterized pro-fibrotic mediator that induces transformation of fibroblasts to myofibroblasts both in vitro (Desmoulière et al., 1993) and in vivo (Sime et al., 1997). Prior studies exposed fibroblasts to recombinant TGF-β1 to show its effect on differentiation and function (Horowitz et al., 2007). Here we have developed a protocol for determining the ability of mouse BAL fluid to alter the differentiation of human lung fibroblasts to myofibroblasts, the cells that produce extracellular matrix proteins.

Materials and Reagents

  1. 6-well cell culture plates (Corning, Costar®, catalog number: 3516 )
  2. Normal human fibroblasts (IMR-90) (ATCC, catalog number: CCL-186 )
  3. DMEM
  4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 26140095 )
  5. Penicillin-streptomycin (10,000 U/ml, 10,000 µg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  6. Amphotericin B (Thermo Fisher Scientific, GibcoTM, catalog number: 15290018 )
  7. RPMI 1640 medium, no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 11835030 )
  8. DPBS (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  9. NP-40
  10. Sodium chloride (NaCl)
  11. Protease inhibitor tablets (Sigma-Aldrich, catalog number: 11836170001 )
  12. Phosphatase inhibitor (EMD Millipore, catalog number: 524625 )
  13. α-SMA antibody (American Research Products, catalog number: 03-61001 )
  14. β-actin antibody (Sigma-Aldrich, catalog number: A5441 )
  15. Tween 20
  16. Fibroblast culture media (see Recipes)
  17. Lysis buffer (see Recipes)

Equipment

  1. Cell culture incubator, 37 °C, 5% CO2: 95% air atmosphere (Thermo Fisher Scientific, FormaTM, model: Direct Heat CO2 Incubator )

Procedure

  1. Seed fibroblasts at a density of 1 x 106 per well of a 6-well cell culture plate in a total volume of 1 ml. Allow cells to adhere to cell culture plate (2-6 h).
  2. Harvest bronchoalveolar lavage (BAL) fluid from mice (Han and Ziegler, 2013). Mice can be treated with bleomycin (1.75 U/kg) or saline as a negative control. Determine the protein concentration of bronchoalveolar lavage (BAL) fluid. Using RPMI 1640 media, normalize all BAL samples to the same protein concentration in a total volume of 1 ml.
    Notes:
    1. Typically, 0.5-1 ml of BAL fluid is used and RPMI is used to normalize the protein concentration to a final total volume of 1 ml.
    2. Alternatively, this assay can be performed in vitro using conditioned media from macrophage cell lines or bone marrow derived macrophages. Harvest the conditioned media from macrophages post-transfection or post-treatment, using 1 million cells per 1 ml of media.
  3. Remove media from fibroblasts and rinse with 1.5 ml room temperature 1x PBS, add normalized BAL fluid to fibroblasts.
    1. Incubate fibroblasts with BAL fluid for 24 h in a cell culture incubator, 37 °C, 5% CO2: 95% air atmosphere.
    2. Harvest fibroblasts by lysing cells in lysis buffer (see Recipes).
  4. Determine α-SMA expression by immunoblot analysis. The expression of α-SMA protein was determined by Western blotting using 20 μg of total cellular protein. After blocking in 5% milk, the membranes were probed with mouse α-SMA primary antibody using a 1:3,000 dilution in Tris buffered saline containing 0.1% Tween 20, the membranes were incubated with the anti-mouse secondary antibody (1:2,000) in antibody dilution buffer for 1 h.

Data analysis

Results can be shown as a representative immunoblot (Figure 1); also see Larson-Casey et al. (2016).


Figure 1. Immunoblot analysis of α-SMA in IMR-90 fibroblasts cultured in BAL fluid (BALF) from saline or bleomycin (Bleo) exposed WT mice

Recipes

  1. Fibroblast culture media
    DMEM
    10% heat-inactivated FBS
    100 U/ml, 100 μg/ml penicillin-streptomycin
    1.25 μg/ml amphotericin B (fungizone)
  2. Lysis buffer
    1% NP-40
    0.15 M NaCl
    0.05 M Tris pH 7.4
    1 protease tablet
    Phosphatase inhibitor diluted 1:100

Acknowledgments

This work was supported by 2R01ES015981 & VA merit review BX001135.

References

  1. Desmoulière, A., Geinoz, A., Gabbiani, F. and Gabbiani, G. (1993). Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122(1): 103-111.
  2. Han, H. and Ziegler, S. F. (2013). Bronchoalveolar lavage and lung tissue digestion. Bio-protocol 3(16): e859.
  3. He, C., Murthy, S., McCormick, M. L., Spitz, D. R., Ryan, A. J. and Carter, A. B. (2011). Mitochondrial Cu,Zn-superoxide dismutase mediates pulmonary fibrosis by augmenting H2O2 generation. J Biol Chem 286(17): 15597-15607.
  4. Horowitz, J. C., Rogers, D. S., Sharma, V., Vittal, R., White, E. S., Cui, Z. and Thannickal, V. J. (2007). Combinatorial activation of FAK and AKT by transforming growth factor-beta1 confers an anoikis-resistant phenotype to myofibroblasts. Cell Signal 19(4): 761-771.
  5. Larson-Casey, J. L., Deshane, J. S., Ryan, A. J., Thannickal, V. J. and Carter, A. B. (2016). Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity 44(3): 582-596.
  6. Sime, P. J., Xing, Z., Graham, F. L., Csaky, K. G. and Gauldie, J. (1997). Adenovector-mediated gene transfer of active transforming growth factor-beta1 induces prolonged severe fibrosis in rat lung. J Clin Invest 100(4): 768-776.

简介

因为转化生长因子-β(TGF-β1)诱导成纤维细胞分化成肌成纤维细胞,我们开发了评价肺泡巨噬细胞衍生的TGF-β1调节肺成纤维细胞分化的方案(Larson-Casey等人)。 ,2016)。该方案评价小鼠支气管肺泡灌洗液(BAL)流体改变成纤维细胞分化的能力。通过α平滑肌肌动蛋白(α-SMA)的表达测量成纤维细胞分化。

[背景] 肺泡巨噬细胞通过增加肺纤维化发展起到不可或缺的作用TGF-β1的表达(He等人,2011)。我们的先前数据表明,肺泡巨噬细胞是TGF-β1的关键来源,因为在巨噬细胞中具有条件缺失的TGF-β1的小鼠被保护免于肺纤维化(Larson-Casey等人,2016)。 α-SMA的表达是肌成纤维细胞的定义特征,并且TGF-β1是良好表征的促纤维化介质,其在体外诱导成纤维细胞向肌成纤维细胞的转化(Desmoulière等人。,1993)和 in vivo (Sime等人,1997)。先前的研究将成纤维细胞暴露于重组TGF-β1以显示其对分化和功能的作用(Horowitz等人,2007)。在这里,我们开发了一个协议,确定小鼠BAL液的能力改变人类肺成纤维细胞分化成肌纤维母细胞,细胞外基质蛋白的细胞。

材料和试剂

  1. 6孔细胞培养板(Corning,Costar ,目录号:3516)
  2. 正常人成纤维细胞(IMR-90)(ATCC,目录号:CCL-186)
  3. DMEM
  4. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM ,目录号:26140095)
  5. 青霉素 - 链霉素(10,000U/ml,10,000μg/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  6. 两性霉素B(Thermo Fisher Scientific,Gibco TM ,目录号:15290018)
  7. RPMI 1640培养基,无酚红(Thermo Fisher Scientific,Gibco TM ,目录号:11835030)
  8. DPBS(Thermo Fisher Scientific,Gibco TM ,目录号:14190144)
  9. NP-40
  10. 氯化钠(NaCl)
  11. 蛋白酶抑制剂片剂(Sigma-Aldrich,目录号:11836170001)
  12. 磷酸酶抑制剂(EMD Millipore,目录号:524625)
  13. α-SMA抗体(American Research Products,目录号:03-61001)
  14. β-肌动蛋白抗体(Sigma-Aldrich,目录号:A5441)
  15. 吐温20
  16. 成纤维细胞培养基(参见配方)
  17. 裂解缓冲液(见配方)

设备

  1. 细胞培养温箱,37℃,5%CO 2:95%空气气氛(Thermo Fisher Scientific,Forma TM,型号:Direct Heat CO 2, sub>孵化器)

程序

  1. 种子成纤维细胞以1×10 6个/孔的密度接种在6孔细胞培养板中,总体积为1ml。允许细胞粘附到细胞培养板(2-6小时)。
  2. 收获小鼠的支气管肺泡灌洗液(BAL)(Han和Ziegler,2013)。可以用博莱霉素(1.75U/kg)或盐水作为阴性对照处理小鼠。确定支气管肺泡灌洗液(BAL)的蛋白质浓度。使用RPMI 1640培养基,将所有BAL样品标准化至相同蛋白质浓度,总体积为1ml。
    注意:
    1. 通常,使用0.5-1ml的BAL液,并使用RPMI将蛋白质浓度标准化至最终总体积为1ml。
    2. 或者,可以使用来自巨噬细胞细胞系或骨髓来源的巨噬细胞的条件培养基在体外进行该测定。从转染后或后处理的巨噬细胞收获条件培养基,每1ml培养基使用1百万个细胞。
  3. 从成纤维细胞中取出介质,用1.5 ml室温1 x PBS冲洗,加入标准化的BAL液到成纤维细胞。
    1. 在37℃,5%CO 2:95%空气气氛的细胞培养箱中将成纤维细胞与BAL液孵育24小时。
    2. 通过在裂解缓冲液中裂解细胞来收获成纤维细胞(参见Recipes)
  4. 通过免疫印迹分析确定α-SMA表达。通过使用20μg总细胞蛋白的Western印迹测定α-SMA蛋白的表达。在5%牛奶中封闭后,使用在含有0.1%Tween 20的Tris缓冲盐水中的1:3,000稀释液,用小鼠α-SMA一抗探测膜,将膜与抗小鼠二抗(1:2000)在抗体稀释缓冲液中1小时。

数据分析

结果可以显示为代表性的免疫印迹(图1);也见Larson-Casey等人。 (2016)。


图1.在来自盐水或博来霉素(Bleo)暴露的WT小鼠的BAL液(BALF)中培养的IMR-90成纤维细胞中α-SMA的免疫印迹分析

食谱

  1. 成纤维细胞培养基
    DMEM
    10%热灭活的FBS 100U/ml,100μg/ml青霉素 - 链霉素 1.25μg/ml两性霉素B(二甲双胍)
  2. 裂解缓冲液
    1%NP-40
    0.15 M NaCl
    0.05 M Tris pH 7.4 1蛋白酶片剂
    磷酸酶抑制剂稀释1:100

致谢

这项工作由2R01ES015981& VA优点评论BX001135。

参考文献

  1. Desmoulière,A.,Geinoz,A.,Gabbiani,F.和Gabbiani,G。(1993)。  转化生长因子-β1在肉芽组织肌成纤维细胞中以及在静止和生长培养的成纤维细胞中诱导α-平滑肌肌动蛋白表达。 122(1):103-111。
  2. Han,H.和Ziegler,SF(2013)。  支气管肺泡灌洗和肺组织消化。 生物协议 3(16):e859
  3. He,C.,Murthy,S.,McCormick,ML,Spitz,DR,Ryan,AJ and Carter,AB(2011)。  线粒体Cu,Zn-超氧化物歧化酶通过增加H 2 O 2亚型来介导肺纤维化。通过增加H 2 O 2 -/a> J Biol Chem 286(17):15597-15607
  4. Horolditz,JC,Rogers,DS,Sharma,V.,Vittal,R.,White,ES,Cui,Z.and Thannickal,VJ(2007)。  通过转化生长因子-β1组合激活FAK和AKT赋予肌成纤维细胞一种无遗传表型抗性表型。 Cell Signal 19(4):761-771。
  5. 线粒体Cu,Zn-超氧化物歧化酶通过增加H 2 O 2亚型来介导肺纤维化。通过增加H 2 O 2 -/a> J Biol Chem 286(17):15597-15607
  6. Horolditz,JC,Rogers,DS,Sharma,V.,Vittal,R.,White,ES,Cui,Z.and Thannickal,VJ(2007)。  Larson-Casey,JL,Deshane,JS,Ryan,AJ,Thannickal,VJ和Carter,AB(2016)。  巨噬细胞Akt1激酶介导的线粒体调节凋亡抵抗和肺纤维化。 44(3):582-596。 >
  7. Sime,PJ,Xing,Z.,Graham,FL,Csaky,KG和Gauldie,J。(1997)。 
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Larson-Casey, J. L. and Carter, A. (2016). Assay to Evaluate BAL Fluid Regulation of Fibroblast α-SMA Expression. Bio-protocol 6(22): e2009. DOI: 10.21769/BioProtoc.2009.
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