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Isolation of Murine Alveolar Type II Epithelial Cells
小鼠肺泡II型上皮细胞分离   

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Abstract

We have optimized a protocol for isolation of alveolar type II epithelial cells from mouse lung. Lung cell suspensions are prepared by intratracheal instillation of dispase and agarose followed by mechanical disaggregation of the lungs. Alveolar type II epithelial cells are purified from these lung cell suspensions through magnetic-based negative selection using a Biotin-antibody, Streptavidin-MicroBeads system. The purified alveolar type II epithelial cells can be cultured and maintained on fibronectin-coated plates in DMEM with 10% FBS. This protocol enables specific investigation of alveolar type II epithelial cells at molecular and cellular levels and provides an important tool to investigate in vitro the mechanisms underlying lung pathogenesis.

Keywords: Alveolar type II epithelial cells(肺泡Ⅱ型上皮细胞), Lung(肺), Biotin(生物素), Streptavidin(链亲和素), Dispase(分散酶), Agarose(琼脂糖)

Background

Alveolar type II epithelial cells play critical roles in alveolar integrity maintenance, surfactant protein synthesis and secretion, and defense against pulmonary infection of bacteria and viruses. Recent studies using mouse lung cancer models have proven that alveolar type II epithelial cells are a key cell of origin of adenoma/adenocarcinoma induced by chemical carcinogens and oncogenic mutations (Qu et al., 2015; Zhou et al., 2015 and 2017). To further expand our understanding of the role of alveolar type II epithelial cells in lung pathogenesis in vivo, isolation of alveolar type II epithelial cells is needed to allow for a precise mechanism analysis in vitro. Based on previous studies (Corti et al., 1996; Rice et al., 2002), a modified method was used in our laboratory to isolate highly purified, viable and culturable alveolar type II epithelial cells from mice (Zhou et al., 2015; Sun et al., 2016).

Materials and Reagents

  1. Needles (BD, catalog number: 305167 ) or tapes
  2. 10 ml syringe (BD, catalog number: 309604 )
  3. 27 gauge needle (BD, catalog number: 305109 )
  4. Nylon string (Dynarex, catalog number: 3243 )
  5. 22 G x 1” Exel Safelet Catheter (Exel International, catalog number: 26746 )
  6. 1 ml syringe (BD, catalog number: 309659 )
  7. 15 ml tubes (VWR, catalog number: 89039-666 )
  8. 60 mm non-coated cell culture dish (Greiner Bio One International, catalog number: 628160 )
  9. Cell strainer (70 µm) (Fisher Scientific, catalog number: 22-363-548 )
  10. Cell strainer (40 µm) (Fisher Scientific, catalog number: 22-363-547 )
  11. Nylon mesh (25 µm) (ELKO filtering, catalog number: 03-25/19 )
  12. MS column (Miltenyi Biotec, catalog number: 130-042-201 )
  13. Fibronectin-coated plate (Corning, catalog number: 354402 )
  14. Mice (THE JACKSON LABORATORY)
  15. 70% ethanol (Decon Labs, catalog number: 2701 )
  16. Dispase (1 mg/ml dissolved in PBS) (Roche Diagnostics, catalog number: 4942078001 )
  17. 1% low melting point agarose (Dissolved in PBS, autoclaved, aliquoted and stored at 4 °C) (Lonza, catalog number: 50100 )
  18. DMEM (Lonza, catalog number: 12-604F )
  19. DNase I (Roche Diagnostics, catalog number: 10104159001 )
  20. Biotinylated anti-CD45 (Miltenyi Biotec, catalog number: 130-101-952 )
  21. Biotinylated anti-CD16/CD32 (Miltenyi Biotec, catalog number: 130-101-895 )
  22. Streptavidin MicroBeads (Miltenyi Biotec, catalog number: 130-048-101 )
  23. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10437028 )
  24. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9625 )
  25. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  26. Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) (Fisher Scientific, catalog number: BP331-500 )
  27. Potassium phosphate monobasic (KH2PO4) (Acros Organics, catalog number: 205925000 )
  28. Bovine serum albumin (BSA) (MP Biomedicals, catalog number: 199898 )
  29. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E26282-500G )
    Note: This product has been discontinued.
  30. Penicillin-streptomycin (Lonza, catalog number: 17-602E )
  31. Phosphate buffered saline (PBS) (see Recipes)
  32. Labeling buffer (see Recipes)

Equipment

  1. CO2 chamber
  2. Biosafety cabinet
  3. Styrofoam board
  4. Forceps (Roboz Surgical Instrument, catalog number: RS-5135 )
  5. Scissors (Roboz Surgical Instrument, catalog number: RS-6802 )
  6. Water bath incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTM 18000A-1CE )
  7. Shaker (Thermo Fisher Scientific, Thermo ScientificTM, model: BarnsteadTM 2314 )
  8. Centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: IEC CL40R , catalog number: 11210927)
  9. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
  10. Hemocytometer (Hausser Scientific, catalog number: 3110 )

Procedure

  1. Preparation of crude single lung cell suspension
    1. Sacrifice a mouse by CO2 inhalation in a CO2 chamber.
    2. Bring the mouse to a biosafety cabinet.
    3. Dampen the mouse with 70% ethanol.
    4. Place the mouse front side up on dissecting Styrofoam board and fix the arms and legs with needles or tape.
    5. Use scissors to make incision in the skin from abdomen to neck, and tear skin with forceps to expose thoracic cage and neck.
    6. Gently remove the muscle around the neck to expose the trachea.
    7. Carefully cut the ribs to expose the heart and lungs.
    8. Perfuse the lung with 0.9% NaCl, using a 10 ml syringe fitted with a 27 gauge needle, through the right ventricle of heart until it is visually free of blood.
    9. Use forceps to put a ~10 cm-long nylon string under the trachea.
    10. Insert the 22 G x 1” Exel Safelet Catheter into the trachea, remove stylet hub, and tie a knot with a nylon string to secure catheter and trachea together firmly.
    11. Slowly inject 2 ml of dispase into the lung, and allow the lung to collapse for 5 min.
    12. Quickly load a 1 ml syringe with 0.5 ml of 1% low melting point agarose (brought from 4 °C storage, thawed in a 70 °C water bath, and then kept in a 45 °C water bath in a melted status), replace the dispase syringe with the agarose-containing syringe, and gently infuse the lung with the loaded agarose. Leave the syringe in place, and immediately cover the lung with ice and incubate for 2 min to promote the solidification of agarose.
    13. Remove the lung from animal to a 15 ml tube containing 2 ml of dispase, incubate for 45 min at room temperature on a shaker.
      Note: Shaking helps dispase digestion and cell release but the exact speed of shaking is not critical.
    14. Transfer the lung to 7 ml of room-temperature DMEM with 0.01% DNase I in a 60 mm non-coated cell culture dish. The digested tissue is carefully teased from bronchi with scissors and forceps.
    15. The resulting cell suspension is successively filtered through 70 µm and 40 µm cell strainers, and then 25 µm nylon mesh.

  2. Magnetic labeling
    1. Count the filtered cells by a hemocytometer, centrifuge cell suspension at 300 x g for 10 min at 4 °C, and then resuspend in 400 µl of labeling buffer per 107 cells.
    2. Add 5 µl of each biotinylated anti-CD45 and biotinylated anti-CD16/CD32, mix well and incubate for 10 min at 4 °C.
    3. Wash cells with 1 ml of labeling buffer and centrifuge at 300 x g for 10 min at 4 °C.
    4. Resuspend cell pellet in 90 µl of labeling buffer per 107 cells.
      Note: In steps B4-B8, if working with fewer than 107 cells, use the same volume as indicated.
    5. Add 10 µl of Streptavidin MicroBeads per 107 cells.
    6. Mix well and incubate for 15 min at 4 °C.
    7. Wash cells with 1 ml of labeling buffer and centrifuge at 300 x g for 10 min at 4 °C.
    8. Resuspend the cell pellet in 500 µl of labeling buffer.

  3. Magnetic separation
    1. Place an MS column in the magnetic field of a suitable MACS MultiStand.
    2. Prepare column by rinsing with 0.5 ml of labeling buffer.
    3. Apply cell suspension onto the column. Collect flow-through containing unlabelled cells, representing the alveolar type II cells.
    4. Wash column 3 times with 0.5 ml of labeling buffer each. Collect unlabelled cells that pass-through, and combine with the effluent from step C3.

  4. Alveolar type II cell culture
    1. The combined effluents are centrifuged at 300 x g for 10 min at 4 °C. The cell pellet is resuspended to 106 cells per ml in DMEM with 10% FBS, and cultured for 12 h in non-coated cell culture plate in a humidified, 5% CO2 incubator at 37 °C to remove residual mesenchymal cells attached to plate.
      Note: Generally, 4-5 million cells will be retrieved per mouse lung, and ~4 ml medium will be used to resuspend the isolated cells at 106 cells per ml.
    2. Cell suspensions with unattached cells (mainly alveolar type II epithelial cells) are gently collected and centrifuged at 300 x g for 10 min at 4 °C. The pellet is resuspended in DMEM with 10% FBS and cultured on a fibronectin-coated plate. The typical morphology of alveolar type II epithelial cells is showed in Figure 1. Meanwhile, genomic sequencing of cultured alveolar type II epithelial cells is performed to detect the mutations of K-Ras gene (Figure 2).


      Figure 1. Isolated murine alveolar type II epithelial cells cultured in complete DMEM on fibronectin-coated plate for 5 days. Image on the right is the magnification of the red-box area on the left. Scale bars = 20 µm.


      Figure 2. The codon 61 mutation of K-Ras gene in alveolar type II epithelial cell. Q61 codon: CAA (reverse complementary: TTG).

Data analysis

  1. Morphology of isolated alveolar type II epithelial cells.
  2. Genome DNA is extracted from isolated alveolar type II epithelial cells from urethane-treated mice. Then the exon 3 of K-Ras gene is PCR-amplified and subsequently sequenced to detect codon 61 mutations.

Notes

  1. Injection of agarose helps push dispase solution deep into alveoli. The solidified agarose can prevent the reflux of dispase solution, and also reduce the contamination of cells from bronchi, such as clara cells.
  2. If the isolated alveolar type II epithelial cells are to be used for culture, carry out all steps in sterile conditions with autoclaved dissection tools.
  3. Using this protocol, about 4-5 x 106 alveolar type II epithelial cells per mouse can be retrieved. The isolated cells can be cultured and maintained for at least 8 days. It is better to use freshly isolated/cultured alveolar type II epithelial cells. Freezing of the cells for storage is not recommended.

Recipes

  1. Phosphate-buffered saline (PBS) (1 L)
    8.0 g NaCl
    0.2 g KCl
    1.15 g Na2HPO4·7H2O
    0.2 g KH2PO4
    Adjust to pH 7.4
  2. Labeling buffer
    PBS, pH 7.2
    0.5% BSA
    2 mM EDTA

Acknowledgments

The authors thank the members in Xiao-Qu lab for helpful discussion. This study was supported in part by the National Institute of Health (NIH)/National Cancer Institute (NCI) grants R01 CA172090, R21 CA175252, P50 CA090440, P30 CA047904, as well as the American Lung Association (ALA) Lung Cancer Discovery Award LCD 259111 and American Cancer Society (ACS) Fellowship Award PF-12081-01-TBG.

References

  1. Corti, M., Brody, A. R. and Harrison, J. H. (1996). Isolation and primary culture of murine alveolar type II cells. Am J Respir Cell Mol Biol 14(4): 309-315.
  2. Qu Z., Sun F., Zhou J., Li L., Shapiro S. D., Xiao G. (2015). Interleukin-6 prevents the initiation but enhances the progression of lung cancer. Cancer Res 75(16): 3209-15.
  3. Rice, W. R., Conkright, J. J., Na, C. L., Ikegami, M., Shannon, J. M. and Weaver, T. E. (2002). Maintenance of the mouse type II cell phenotype in vitro. Am J Physiol Lung Cell Mol Physiol 283(2): L256-264.
  4. Sun, F., Qu, Z., Xiao, Y., Zhou, J., Burns, T. F., Stabile, L. P., Siegfried, J. M. and Xiao, G. (2016). NF-κB1 p105 suppresses lung tumorigenesis through the Tpl2 kinase but independently of its NF-κB function. Oncogene 35(18): 2299-2310.
  5. Zhou J., Qu Z., Sun F., Han L., Li L., Yan S., Stabile L. P. , Chen L. F. , Siegfried J. M., Xiao G.. (2017). Myeloid STAT3 promotes lung tumorigenesis by transforming tumor immunosurveillance into tumor-promoting inflammation. Cancer Immunol Res 5(3): 257-268.
  6. Zhou, J., Qu, Z., Yan, S., Sun, F., Whitsett, J. A., Shapiro, S. D. and Xiao, G. (2015). Differential roles of STAT3 in the initiation and growth of lung cancer. Oncogene 34(29): 3804-3814.

简介

我们优化了从小鼠肺分离肺泡II型上皮细胞的方案。通过气管内滴注分泌酶和琼脂糖,然后机械解聚肺来制备肺细胞悬浮液。通过使用生物素抗体Streptavidin-MicroBeads系统的磁性阴性选择,从这些肺细胞悬液中纯化肺泡II型上皮细胞。可以将纯化的肺泡II型上皮细胞培养并维持在含有10%FBS的DMEM中的纤连蛋白包被的平板上。该方案能够在分子和细胞水平上对肺泡II型上皮细胞进行特异性研究,并提供了一种重要的工具,用于在体外研究肺发病机制的机制。

背景 肺泡II型上皮细胞在肺泡完整性维持,表面活性蛋白合成和分泌中起关键作用,并防止细菌和病毒的肺部感染。最近使用小鼠肺癌模型的研究已经证明,肺泡II型上皮细胞是由化学致癌物质和致癌突变诱导的腺瘤/腺癌的关键细胞(Qu 等人,2015; Zhou > et al。,2015和2017)。为了进一步扩大我们对肺泡II型上皮细胞在体内肺发病机制中的作用的理解,需要分离肺泡II型上皮细胞以允许体外精确的机理分析, EM>。基于先前的研究(Corti等人,1996; Rice等人,2002),在我们的实验室中使用了一种修饰的方法来分离高度纯化的,可行的和可培养的来自小鼠的肺泡II型上皮细胞(Zhou等人,2015; Sun等人,2016)。

关键字:肺泡Ⅱ型上皮细胞, 肺, 生物素, 链亲和素, 分散酶, 琼脂糖

材料和试剂

  1. 针(BD,目录号:305167)或磁带
  2. 10ml注射器(BD,目录号:309604)
  3. 27号针(BD,目录号:305109)
  4. 尼龙绳(Dynarex,目录号:3243)
  5. 22 G x 1"Exel Safelet导管(Exel International,目录号:26746)
  6. 1 ml注射器(BD,目录号:309659)
  7. 15毫升管(VWR,目录号:89039-666)
  8. 60毫米非涂层细胞培养皿(Greiner Bio One International,目录号:628160)
  9. 细胞过滤器(70μm)(Fisher Scientific,目录号:22-363-548)
  10. 细胞过滤器(40μm)(Fisher Scientific,目录号:22-363-547)
  11. 尼龙网(25μm)(ELKO过滤,目录号:03-25/19)
  12. MS柱(Miltenyi Biotec,目录号:130-042-201)
  13. 纤连蛋白涂层板(Corning,目录号:354402)
  14. 小鼠(杰克逊实验室)
  15. 70%乙醇(Decon Labs,目录号:2701)
  16. 分散液(1mg/ml溶于PBS中)(Roche Diagnostics,目录号:4942078001)
  17. 1%低熔点琼脂糖(溶于PBS,高压灭菌,等分并储存于4℃)(Lonza,目录号:50100)
  18. DMEM(Lonza,目录号:12-604F)
  19. DNase I(Roche Diagnostics,目录号:10104159001)
  20. 生物素化抗CD45(Miltenyi Biotec,目录号:130-101-952)
  21. 生物素化抗CD16/CD32(Miltenyi Biotec,目录号:130-101-895)
  22. 链霉亲和素微珠(Miltenyi Biotec,目录号:130-048-101)
  23. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10437028)
  24. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9625)
  25. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  26. 七水合磷酸氢二钠(Na 2 HPO 4·7H 2 O)(Fisher Scientific,目录号:BP331-500)
  27. 磷酸二氢钾(KH 2 PO 4)(Acros Organics,目录号:205925000)
  28. 牛血清白蛋白(BSA)(MP Biomedicals,目录号:199898)
  29. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E26282-500G)
    注意:本产品已停产。
  30. 青霉素 - 链霉素(Lonza,目录号:17-602E)
  31. 磷酸盐缓冲盐水(PBS)(见食谱)
  32. 标签缓冲液(见配方)

设备

  1. CO 2
  2. 生物安全柜
  3. 泡沫塑料板
  4. 镊子(Roboz手术器械,目录号:RS-5135)
  5. 剪刀(Roboz手术器械,目录号:RS-6802)
  6. 水浴培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Barnstead TM 18000A-1CE)
  7. 振荡器(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Barnstead TM 2314)
  8. 离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:IEC CL40R,目录号:11210927)
  9. MACS MultiStand(Miltenyi Biotec,目录号:130-042-303)
  10. 血细胞计数器(Hausser Scientific,目录号:3110)

程序

  1. 粗制单肺细胞悬液的制备
    1. 在CO 2室中通过CO 2吸入牺牲小鼠。
    2. 将鼠标移到生物安全柜。
    3. 用70%乙醇减压老鼠。
    4. 将鼠标放在解剖泡沫聚苯乙烯板上,用针或胶带固定手臂和腿。
    5. 使用剪刀从腹部到颈部切开皮肤,用镊子撕开皮肤,露出胸笼和颈部。
    6. 轻轻取出颈部肌肉露出气管。
    7. 仔细切割肋骨以暴露心脏和肺部。
    8. 使用装有27号针头的10ml注射器通过心脏右心室灌注0.9%NaCl的肺,直到目视无血。
    9. 使用镊子将〜10厘米长的尼龙绳放在气管下。
    10. 将22 G x 1"Exel Safelet导管插入气管,取出指针针座,并用尼龙线扎结,将导管和气管牢固地固定在一起。
    11. 慢慢地将2ml的分散液注入肺部,并允许肺塌陷5分钟
    12. 快速加载1 ml注射器,加入0.5 ml 1%低熔点琼脂糖(从4°C储存,从70°C水浴中解冻,然后保存在45°C水浴中,熔化状态),更换使用含有琼脂糖的注射器的分散注射器,并用加载的琼脂糖轻轻注入肺。将注射器放在适当位置,立即用冰盖上肺,孵育2分钟以促进琼脂糖凝固。
    13. 将肺从动物中取出至含有2ml分散素的15ml管中,在振荡器上室温孵育45分钟。
      注意:抖动有助于消化和释放细胞,但确切的抖动速度并不重要。
    14. 将肺转移到含有0.01%DNase I的7ml室温DMEM的60mm未包被的细胞培养皿中。消化的组织用剪刀和镊子从支气管小心地挑逗。
    15. 所得到的细胞悬浮液依次通过70μm和40μm细胞过滤器,然后25μm尼龙网过滤
  2. 磁性标签
    1. 通过血细胞计数器计数经过滤的细胞,在4℃以300×g离心细胞悬浮液10分钟,然后每10个细胞重悬于400μl标记缓冲液中。
    2. 加入5μl生物素化抗CD45和生物素化抗CD16/CD32,充分混匀,4℃孵育10分钟。
    3. 用1ml标记缓冲液洗涤细胞并在4℃下以300×g离心10分钟。
    4. 将细胞沉淀重悬于90μl每10个细胞的标记缓冲液中。
      注意:在步骤B4-B8中,如果使用少于10个单元格,请使用与指示相同的音量。
    5. 每10个细胞添加10μl的Streptavidin MicroBeads。
    6. 混匀,4℃孵育15分钟
    7. 用1ml标记缓冲液洗涤细胞并在4℃下以300×g离心10分钟。
    8. 将细胞沉淀重悬于500μl标记缓冲液中。

  3. 磁选
    1. 将MS色谱柱置于合适的MACS MultiStand的磁场中。
    2. 通过用0.5ml标记缓冲液冲洗来制备柱。
    3. 将细胞悬液涂于柱上。收集含有未标记细胞的流通,代表肺泡II型细胞。
    4. 每次用0.5ml标记缓冲液洗涤柱3次。收集通过的未标记细胞,并与步骤C3的流出物结合。

  4. 肺泡II型细胞培养
    1. 将合并的废水在4℃下以300×g离心10分钟。将细胞沉淀物重悬于具有10%FBS的DMEM中的10μg/ml细胞/ml,并在未包被的细胞培养板中在加湿的5%CO 2中培养12小时, sub>培养箱中37℃除去附着在板上的残留间充质细胞 注意:一般来说,每只小鼠肺将收集4-5百万个细胞,并且将使用〜4ml培养基在10℃下重悬悬浮分离的细胞 细胞/ml。
    2. 温和地收集具有未附着细胞(主要是肺泡II型上皮细胞)的细胞悬浮液,并在4℃下以300×g离心10分钟。将沉淀重悬于具有10%FBS的DMEM中并在纤连蛋白包被的平板上培养。肺泡II型上皮细胞的典型形态如图1所示。同时,进行培养的肺泡II型上皮细胞的基因组测序以检测K-Ras基因的突变(图2)。 br />

      图1.在纤维连接蛋白包被的平板上在完全DMEM中培养的隔离的鼠肺泡II型上皮细胞5天。右侧的图像是左侧红盒区域的放大倍数。比例尺= 20μm

      图2.在肺泡II型上皮细胞中 K-Ras 基因的密码子61突变。Q61密码子: CAA(反向互补:TTG)。

数据分析

  1. 孤立肺泡II型上皮细胞的形态学
  2. 从氨基甲酸酯治疗的小鼠的分离的肺泡II型上皮细胞中提取基因组DNA。然后将K-Ra 基因的外显子3进行PCR扩增,随后进行测序以检测61位突变。

笔记

  1. 注射琼脂糖有助于将Dispase溶液深入肺泡。凝固的琼脂糖可以预防分泌酶溶液的回流,也可以减少支气管细胞如克拉拉细胞的污染。
  2. 如果将孤立的肺泡II型上皮细胞用于培养,则在无菌条件下用高压消毒的解剖工具进行所有步骤。
  3. 使用该方案,可以检索每只小鼠约4-5×10 6个肺泡II型上皮细胞。分离的细胞可以培养和维持至少8天。最好使用新鲜分离/培养的肺泡II型上皮细胞。不建议冻结细胞进行储存。

食谱

  1. 磷酸盐缓冲盐水(PBS)(1升)
    8.0克NaCl
    0.2克KCl
    1.15g Na 2 HPO 4< 7H 2< 2><< / 0.2g KH 2 PO 4
    调整至pH 7.4
  2. 标签缓冲区
    PBS,pH 7.2
    0.5%BSA
    2 mM EDTA

致谢

作者感谢小曲实验室的成员进行了有益的讨论。这项研究部分由国立卫生研究院(NIH)/国家癌症研究所(NCI)授权R01 CA172090,R21 CA175252,P50 CA090440,P30 CA047904以及美国肺协会(ALA)肺癌发现奖LCD 259111和美国癌症协会(ACS)奖学金奖PF-12081-01-TBG。

参考

  1. Corti,M.,Brody,AR和Harrison,JH(1996)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/8600933"target = "_blank">鼠肺泡II型细胞的分离和原代培养。 Am J Respir Cell Mol Biol 14(4):309-315。
  2. Qu Z.,Sun F.,Zhou J.,Li L.,Shapiro SD,Xiao G.(2015)。  白细胞介素-6阻止起始,但增强肺癌的进展。癌症研究<75>(16):3209-15。
  3. Rice,WR,Conkright,JJ,Na,CL,Ikegami,M.,Shannon,JM和Weaver,TE(2002)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/12114186"target ="_ blank">体外维护小鼠II型细胞表型。 Am J Physiol Lung Cell Mol Physiol em> 283(2):L256-264。
  4. Sun,F.,Qu,Z.,Xiao,Y.,Zhou,J.,Burns,TF,Stabile,LP,Siegfried,JM和Xiao,G。(2016)。 NF-κB1p105通过Tpl2激酶抑制肺部肿瘤发生,但与NF-κB功能无关。 a>癌基因 35(18):2299-2310。
  5. Zhou J.,Qu Z.,Sun F.,Han L.,Li L.,Yan S.,Stabile LP,Chen LF,Siegfried JM,Xiao G ..(2017)。< a class ="ke- insertfile"href ="https://www.ncbi.nlm.nih.gov/pubmed/28108629"target ="_ blank">髓系STAT3通过将肿瘤免疫监视转化为肿瘤促进炎症来促进肺部肿瘤发生。 > Cancer Immunol R 5(3):257-268。
  6. Zhou,J.,Qu,Z.,Yan,S.,Sun,F.,Whitsett,JA,Shapiro,SD和Xiao,G。(2015)。 STAT3在肺癌的起始和生长中的差异作用。癌基因 34 (29):3804-3814。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sun, F., Xiao, G. and Qu, Z. (2017). Isolation of Murine Alveolar Type II Epithelial Cells. Bio-protocol 7(10): e2288. DOI: 10.21769/BioProtoc.2288.
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