Hartley豚鼠耳朵成纤维细胞分离培养与鉴定分析
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摘要
本文旨在建立快速获取豚鼠耳朵成纤维细胞的方法,为豚鼠体细胞重编程技术提供起始细胞。首先采用0. 05%Trypsin-EDTA和Collagenase IV两种消化酶对脱毛后的耳朵组织碎块进行消化;然后对获取的细胞进行体外培养和形态学观察,支原体检测,Vimentin蛋白免疫荧光鉴定,测定细胞生长曲线计算细胞倍增时间;用绿色荧光蛋白的逆转录病毒感染后用流式细胞仪检测感染效率,核型鉴定检测病毒对核型的影响。分离的豚鼠耳朵成纤维细胞为贴壁生长细胞,消化后2～3 h即大部分贴壁并基本完全伸展,细胞呈梭形或多角形,胞浆饱满,立体感强,细胞核清晰形态良好,支原体检测阴性。细胞表达成纤维细胞特异性蛋白Vimentin。生长曲线为S形,符合体外培养细胞正常生长增殖规律,细胞倍增时间为24. 85 h。逆转录病毒感染效率达75. 6%,病毒感染后染色体2n=64的细胞占90%,遗传稳定核型正常。本研究提供的体细胞获取方法操作简单,效率高,获取的细胞状态良好,细胞增殖速度快,细胞感染率高,是一种非常合适的豚鼠耳朵成纤维细胞分离手段,为后续进一步研究豚鼠体细胞重编程提供了技术基础。
This experiment was performed to rapidly obtain fibroblasts from guinea pig's ears,and provide initiating cells for somatic cell reprogramming. The ear tissues were minced and digested with 0. 05% Trypsin-EDTA and Collagenase IV. The cells were cultured in vitro and morphologically observed. The cells were detected for mycoplasma and vimentin protein immunofluorescence was performed for characterization. The cell growth curve was drawn to calculate the cell double time. And the infection efficiency was detected by flow cytometry when the cells were infected with retrovirus containing green fluorescent protein. The influence of the virus on the karyotype was detected by karyotype identification.The isolated guinea pig ear fibroblasts were adherent growth cells. Most of them were adherent and fully extended 2 ～ 3 hours after digestion. The cells were fusiform or polygonal,with full and stereoscopic cytoplasm. And the nucleus was clear and in good shape. The mycoplasma detection was negative. The cells expressed fibroblast-specific protein vimentin. The growth curve was S-shaped,which was consistent with the normal growth and proliferation rules of cultured cells in vitro. The cell double time was 24. 85 h. The efficiency of retrovirus infection was more than 70%,and the cells with chromosome 2 n = 64 accounted for 90% after viral infection,with genetic stability and normal karyotype. The method is simple and efficient,producing rapidly proliferated cells in good condition. It is an appropriate method for isolating fibroblasts from guinea pig's ears. The cell line has a high virus infection rate,which can be used for somatic cell reprogramming studies.
This experiment was performed to rapidly obtain fibroblasts from guinea pig's ears,and provide initiating cells for somatic cell reprogramming. The ear tissues were minced and digested with 0. 05% Trypsin-EDTA and Collagenase IV. The cells were cultured in vitro and morphologically observed. The cells were detected for mycoplasma and vimentin protein immunofluorescence was performed for characterization. The cell growth curve was drawn to calculate the cell double time. And the infection efficiency was detected by flow cytometry when the cells were infected with retrovirus containing green fluorescent protein. The influence of the virus on the karyotype was detected by karyotype identification.The isolated guinea pig ear fibroblasts were adherent growth cells. Most of them were adherent and fully extended 2 ～ 3 hours after digestion. The cells were fusiform or polygonal,with full and stereoscopic cytoplasm. And the nucleus was clear and in good shape. The mycoplasma detection was negative. The cells expressed fibroblast-specific protein vimentin. The growth curve was S-shaped,which was consistent with the normal growth and proliferation rules of cultured cells in vitro. The cell double time was 24. 85 h. The efficiency of retrovirus infection was more than 70%,and the cells with chromosome 2 n = 64 accounted for 90% after viral infection,with genetic stability and normal karyotype. The method is simple and efficient,producing rapidly proliferated cells in good condition. It is an appropriate method for isolating fibroblasts from guinea pig's ears. The cell line has a high virus infection rate,which can be used for somatic cell reprogramming studies.
引文
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[24]WANG Q H,PENG Y,CAI X Y,et al.Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages[J].Current Medical Science,2015,35(4):585-590.
[2]FRIKKE-SCHMIDT H,TVEDEN-NYBORG P,LYKKESFELDTJ.l-dehydroascorbic acid can substitute l-ascorbic acid as dietary vitamin C source in guinea pigs[J].Redox Biology,2016,7:8-13.
[3]SCHJOLDAGER J G,TVEDEN-NYBORG P,LYKKESFELDTJ.Prolonged maternal vitamin C deficiency overrides preferential fetal ascorbate transport but does not influence perinatal survival in guinea pigs[J].British Journal of Nutrition,2013,110(09):1573-1579.
[4]KAR M,BAYAR MULUK N,BAFAQEEH S A,et al.Consensus on the methodology for experimental studies in allergic rhinitis[J].International Journal of Pediatric Otorhinolaryngology,2019,121:68-71.
[5]KUMAR M,KRAUSE K K,AZOUZ F,et al.A guinea pig model of Zika virus infection[J].Virology Journal,2017,14(1):1-8.
[6]MORRISON J L,BOTTING K J,DARBY JRT,et al.Guinea pig models for translation of the developmental origins of health and disease hypothesis into the clinic[J].The Journal of Physiology,2018,596(23):5535-5569.
[7]YOUNG Y H.Inner ear test battery in guinea pig models-a review[J].Acta Oto-Laryngologica,2018,138(6):519-529.
[8]TAKAHASHI K,YAMANAKA S.Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors[J].Cell,2006,126(4):663-676.
[9]TAKAHASHI K,TANABE K,OHNUKI M,et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors[J].Cell,2007,131(5):861-872.
[10]ESTEBAN M A,XU J,YANG J,et al.Generation of induced pluripotent stem cell lines from tibetan miniature pig[J].Journal of Biological Chemistry,2009,284(26):17634-17640.
[11]HAN X,HAN J,DING F,et al.Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells[J].Cell Research,2011,21(10):1509-1512.
[12]LIU J,BALEHOSUR D,MURRAY B,et al.Generation and characterization of reprogrammed sheep induced pluripotent stem cells[J].Theriogenology,2012,77(2):338-346.
[13]NAGY K,SUNG HK,ZHANG P,et al.Induced pluripotent stem cell lines derived from equine fibroblasts[J].Stem Cell Reviews and Reports,2011,7(3):693-702.
[14]AFANASSIEFF M,TAPPONNIER Y,SAVATIER P.Generation of induced pluripotent stem cells in rabbits[J].Journal of Biological Chemistry,2010,285(41):31362-31369.
[15]OGOREVC J,OREHEK S,DOVCˇP.Cellular reprogramming in farm animals:an overview of i PSC generation in the mammalian farm animal species[J].Journal of Animal Science and Biotechnology,2016,7:10.
[16]SHU X,PEI D.The function and regulation of mesenchymalto-epithelial transition in somatic cell reprogramming[J].Current Opinion in Genetics&Development,2014,28:32-37.
[17]RAAB S,KLINGENSTEIN M,LIEBAU S,et al.A comparative view on human somatic cell sources for i PSC generation[J].Stem Cells International,2014,2014:768391
[18]MEHRABANI D,MAHBOOBI R,DIANATPOUR M,et al.Establishment,culture,and characterization of guinea pig fetal fibroblast cell[J].Veterinary Medicine International,2014,2014:510328.
[19]FERNANDES I R,RUSSO F B,PIGNATARI G C,et al.Fibroblast sources:where can we get them[J].Cytotechnology,2016,68(2):223-228.
[20]KHAN M,GASSER S.Generating primary fibroblast cultures from mouse ear and tail tissues[J].Journal of Visualized Experiments,2016,(107):1-6.
[21]ZAKHARTCHENKO V,ALBERIO R,STOJKOVIC M,et al.Adult cloning in cattle:potential of nuclei from a permanent cell line and from primary cultures[J].Molecular Reproduction and Development,1999,54(3):264-272.
[22]LUO J,LIANG M M,YANG X G,et al.Establishment and biological characteristics comparison of Chinese swamp buffalo(Bubalus bubalis)fibroblast cell lines[J].In vitro Cellular&Developmental Biology-Animal,2014,50(1):7-15.
[23]LIU C Q,GUO Y,GUAN W J,et al.Establishment and characterization of a fibroblast cell line derived from Mongolian sheep[J].Animal Science Journal,2011,82(2):215-222.
[24]WANG Q H,PENG Y,CAI X Y,et al.Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages[J].Current Medical Science,2015,35(4):585-590.