豚鼠鼻黏膜成纤维细胞的分离、培养及鉴定
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摘要
背景:鼻黏膜成纤维细胞可通过分泌多种细胞因子和趋化因子,参与多种鼻部疾病的炎症反应和损伤修复;豚鼠是最适合用于变态反应性疾病研究的实验动物之一。目的:探讨豚鼠鼻黏膜成纤维细胞分离、培养、纯化和鉴定的有效方法。方法:胶原酶消化法分离豚鼠鼻黏膜成纤维细胞,经差速贴壁法纯化后,采用倒置相差显微镜进行形态学观察,波形蛋白免疫细胞化学法鉴定成纤维细胞,并通过MTT检测法绘制细胞生长曲线。结果与结论:①倒置相差显微镜下可见原代鼻黏膜成纤维细胞中有不同形态细胞交杂生长,以类梭形和扁平状细胞为主,细胞成团散落分布;纯化后的成纤维细胞形态均一,多呈长梭形,分布较均匀,呈鱼群状或放射状排列;②免疫细胞化学染色显示,成纤维细胞波形蛋白表达呈阳性;③细胞生长曲线大致呈"S"形。④结果表明,所分离培养的细胞具有典型的成纤维细胞生物学特征。
BACKGROUND: Nasal mucosa fibroblasts are reported to involve in inflammation and wound repair of various nasal diseases by secreting a variety of cytokines and chemokines. Guinea pig is a most suitable experimental animal for the study of allergic diseases. OBJECTIVE: To investigate the effective methods of isolation, culture, purification and identification of guinea pig nasal mucosa fibroblasts. METHODS: Guinea pig nasal mucosa fibroblasts were isolated by collagenase digestion, and purified by differential velocity adherence. The morphology of fibroblasts was observed by inverted phase contrast microscope, fibroblasts were identified by vimentin immunocytochemical staining, and the cell growth was detected by MTT assay to draw the growth curve. RESULTS AND CONCLUSION:(1) Under inverted phase contrast microscope, there were cells with different shapes in the primary nasal mucosa fibroblasts, most of which showed spindle-like and applanate shape, and cells were scattered distribution in cluster. The purified fibroblasts were homogeneous, mainly were long spindle-shaped, and distributed in fish shoal-like and radial-like.(2) Immunocytochemical staining indicated that fibroblasts were positive for vimentin.(3) The cell growth curve appeared to be typical S-shaped.(4) To conclude, the isolated and cultured cells exhibit typical biological characteristics of fibroblasts.
BACKGROUND: Nasal mucosa fibroblasts are reported to involve in inflammation and wound repair of various nasal diseases by secreting a variety of cytokines and chemokines. Guinea pig is a most suitable experimental animal for the study of allergic diseases. OBJECTIVE: To investigate the effective methods of isolation, culture, purification and identification of guinea pig nasal mucosa fibroblasts. METHODS: Guinea pig nasal mucosa fibroblasts were isolated by collagenase digestion, and purified by differential velocity adherence. The morphology of fibroblasts was observed by inverted phase contrast microscope, fibroblasts were identified by vimentin immunocytochemical staining, and the cell growth was detected by MTT assay to draw the growth curve. RESULTS AND CONCLUSION:(1) Under inverted phase contrast microscope, there were cells with different shapes in the primary nasal mucosa fibroblasts, most of which showed spindle-like and applanate shape, and cells were scattered distribution in cluster. The purified fibroblasts were homogeneous, mainly were long spindle-shaped, and distributed in fish shoal-like and radial-like.(2) Immunocytochemical staining indicated that fibroblasts were positive for vimentin.(3) The cell growth curve appeared to be typical S-shaped.(4) To conclude, the isolated and cultured cells exhibit typical biological characteristics of fibroblasts.
引文
[1]中华耳鼻咽喉头颈外科杂志编委会鼻科学组,中华医学会耳鼻咽喉头颈外科学分会鼻科学组.变应性鼻炎诊断和治疗指南(2009年,武夷山)[J].中华耳鼻咽喉头颈外科杂志,2009,44(12):977-978.
[2]Higashino M,Takabayashi T,Takahashi N,et al.Interleukin-19 downregulates interleukin-4-induced eotaxin production in human nasal fibroblasts.Allergol Int.2011;60(4):449-457.
[3]Cho JS,Han IH,Lee HR,Lee HM.Prostaglandin E2 Induces IL-6 and IL-8 Production by the EP Receptors/Akt/NF-κBPathways in Nasal Polyp-Derived Fibroblasts.Allergy Asthma Immunol Res.2014;6(5):449-457.
[4]Park IH,Um JY,Cho JS,et al.Histamine Promotes the Release of Interleukin-6 via the H1R/p38 and NF-κBPathways in Nasal Fibroblasts.Allergy Asthma Immunol Res.2014;6(6):567-572.
[5]Kim JA,Cho JH,Park IH,et al.Diesel Exhaust Particles Upregulate Interleukins IL-6 and IL-8 in Nasal Fibroblasts.PLoS One.2016;11(6):e0157058.
[6]司徒镇强,吴军正.细胞培养[M].西安:界图书出版西安公司,2007:5.
[7]杨志明.组织工程[M].北京:化学工业出版社,2002:41-42,158.
[8]Richert MM,Schwertfeger KL,Ryder JW,et al.An atlas of mouse mammary gland development.J Mammary Gland Biol Neoplasia.2000;5(2):227-241.
[9]李涛,朱慧涛,周杰玉,等.鼻息肉成纤维细胞在常氧与低氧环境下的原代培养及鉴定[J].山东大学耳鼻喉眼学报,2014,20(2):76-80.
[10]刘建国,刘月辉,谢冰斌.丙酸氟替卡松对变应性鼻炎大鼠鼻黏膜中碱性成纤维细胞生长因子蛋白和mRNA表达的影响[J].临床耳鼻咽喉头颈外科杂志,2009,23(14):649-651,655.
[11]夏忠芳,孔维佳,乐建新,等.人正常鼻黏膜及鼻息肉黏膜上皮细胞的原代培养及鉴定[J].华中科技大学学报(医学版),2008,37(4):541-543.
[12]李吉平,王家东,张淳,等.鼻渊舒口服液抑制鼻黏膜成纤维细胞生长的实验研究[J].中国中西医结合耳鼻咽喉科杂志,2006,14(6):344-346.
[13]商爱民,吴靖芳,薛刚,等.张耕大鼠胚胎成纤维细胞的分离培养及生物学特性[J]河北北方学院学报(医学版),2010,27(3):17-20.
[14]王烨,米文娟,韩宇,等.乔莉蜗轴螺旋动脉平滑肌的培养及其鉴定[J].现代生物医学进展,2010,10(9):1624-1627.
[15]崔哲洙,刘艳丽,崔春莲,等.变应性鼻炎与慢性单纯性鼻炎鼻黏膜成纤维细胞中组胺受体蛋白的表达[J].延边大学医学学报,2008,31(4):281-283.
[2]Higashino M,Takabayashi T,Takahashi N,et al.Interleukin-19 downregulates interleukin-4-induced eotaxin production in human nasal fibroblasts.Allergol Int.2011;60(4):449-457.
[3]Cho JS,Han IH,Lee HR,Lee HM.Prostaglandin E2 Induces IL-6 and IL-8 Production by the EP Receptors/Akt/NF-κBPathways in Nasal Polyp-Derived Fibroblasts.Allergy Asthma Immunol Res.2014;6(5):449-457.
[4]Park IH,Um JY,Cho JS,et al.Histamine Promotes the Release of Interleukin-6 via the H1R/p38 and NF-κBPathways in Nasal Fibroblasts.Allergy Asthma Immunol Res.2014;6(6):567-572.
[5]Kim JA,Cho JH,Park IH,et al.Diesel Exhaust Particles Upregulate Interleukins IL-6 and IL-8 in Nasal Fibroblasts.PLoS One.2016;11(6):e0157058.
[6]司徒镇强,吴军正.细胞培养[M].西安:界图书出版西安公司,2007:5.
[7]杨志明.组织工程[M].北京:化学工业出版社,2002:41-42,158.
[8]Richert MM,Schwertfeger KL,Ryder JW,et al.An atlas of mouse mammary gland development.J Mammary Gland Biol Neoplasia.2000;5(2):227-241.
[9]李涛,朱慧涛,周杰玉,等.鼻息肉成纤维细胞在常氧与低氧环境下的原代培养及鉴定[J].山东大学耳鼻喉眼学报,2014,20(2):76-80.
[10]刘建国,刘月辉,谢冰斌.丙酸氟替卡松对变应性鼻炎大鼠鼻黏膜中碱性成纤维细胞生长因子蛋白和mRNA表达的影响[J].临床耳鼻咽喉头颈外科杂志,2009,23(14):649-651,655.
[11]夏忠芳,孔维佳,乐建新,等.人正常鼻黏膜及鼻息肉黏膜上皮细胞的原代培养及鉴定[J].华中科技大学学报(医学版),2008,37(4):541-543.
[12]李吉平,王家东,张淳,等.鼻渊舒口服液抑制鼻黏膜成纤维细胞生长的实验研究[J].中国中西医结合耳鼻咽喉科杂志,2006,14(6):344-346.
[13]商爱民,吴靖芳,薛刚,等.张耕大鼠胚胎成纤维细胞的分离培养及生物学特性[J]河北北方学院学报(医学版),2010,27(3):17-20.
[14]王烨,米文娟,韩宇,等.乔莉蜗轴螺旋动脉平滑肌的培养及其鉴定[J].现代生物医学进展,2010,10(9):1624-1627.
[15]崔哲洙,刘艳丽,崔春莲,等.变应性鼻炎与慢性单纯性鼻炎鼻黏膜成纤维细胞中组胺受体蛋白的表达[J].延边大学医学学报,2008,31(4):281-283.