猪尾小组织块贴壁培养法分离培养成纤维细胞及转染效率的研究
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摘要
目的采取猪尾小组织块贴壁法成功分离培养出原代猪成纤维细胞并进行细胞不同时期的绿色荧光蛋白(green fluorescent protein,GFP)阳性质粒转染效率的研究。方法猪尾小组织块贴壁培养法分离培养出原代猪成纤维细胞后,传代并进行冻存和复苏,比较猪成纤维细胞原代培养、传代培养和复苏细胞存活率。绘制细胞原代培养、传代培养和复苏后的生长情况。分别在猪尾小组织块贴壁培养获得原代成纤维细胞,细胞传代和复苏后进行细胞GFP阳性质粒的Neon电转染,比较不同转染时期细胞存活率和生长情况,转染后24h和48h分别采用荧光显微镜和流式细胞仪检测细胞的GFP质粒阳性表达率。结果猪尾小组织块贴壁培养可以成功分离培养出猪成纤维细胞,细胞冻存后复苏能保持原代细胞生物学特性,原代细胞活力很高,细胞存活率97.670%,传代培养后细胞存活率95.670%,复苏细胞存活率仍在90%以上。原代培养、传代培养和复苏培养细胞生长曲线均呈"S"型,复苏细胞生长略微缓慢。原代培养、传代培养和复苏细胞电转染后细胞存活率均有少许下降,细胞生长增殖能力减弱,但是细胞生长曲线仍呈"S"型曲线。Neon电转染后24h均可获得GFP质粒阳性表达的猪成纤维细胞,荧光显微镜均检测到GFP质粒阳性表达的成纤维细胞,48h后流式细胞仪检测显示原代细胞阳性率8.01%,稍高于传代细胞的7.91%和复苏细胞的7.86%。复苏细胞存活率下降,转染后细胞活力下降,但转染后仍可得到7.86%GFP质粒阳性表达的猪成纤维细胞,原代培养细胞能够获得更高的阳性率,差异有统计学意义。结论猪尾小组织块贴壁培养法可以成功分离培养出成纤维细胞,细胞传代会对细胞活力有影响,冻存后复苏也会影响细胞的存活率和细胞增殖能力。原代细胞、传代细胞和复苏细胞转染均能获得GFP阳性质粒表达。原代培养细胞具有更佳的细胞活力和阳性转染率,进行基因敲除或基因敲入等基因编辑和体细胞克隆研究,选择原代细胞作为转染对象能获得更好的基因编辑成功率。
Objective To examine the transfection efficiency of green fluorescent protein(GFP)positive plasmids in different stages of porcine fibroblasts primarily cultured by adherent culture of small-sized pigtail tissues.Methods The primary porcine fibroblasts were isolated and cultured by adherent culture of small-sized pigtail tissues,and then cryopreserved and resuscitated.The survival rates of fibroblasts from primary culture,subculture and resuscitation were compared.The electro-transfection of GFP-positive plasmids with Neon was performed in fibroblasts after subculture and resuscitation.Cell survival and growth were compared at different transfection stages.The positive expression rate of GFP plasmid was detected by fluorescence microscopy and flow cytometry 24 hand 48 hafter transfection respectively.Results The pigtail fibroblasts could be successfully isolated and cultured by the adherent culture of small-sized pigtail tissues.The primary cell biological characteristics were maintained after cryopreservation.The viability of primary cells was high,with the cell survival rate reaching 97.67%.The cell survival rate was 95.67%after subculture and was still above 90%after resuscitation.The growth curves of cells from primary culture,subculture and resuscitation took on"S"type.The growth of cells after resuscitation was slightly slow.After electro-transfection,the viability of cells from primary culture,subculture and resuscitation was slightly decreased,the cell proliferation was weakened,but the cell growth curve still became an"S"curve.Fluorescence microscopy showed that the porcine fibroblasts with GFP positively expressed were obtained 24 hafter Neon electro-transfection.Flow cytometry revealed that 48 hafter the electro-transfection,the GFP-positive rate of primary cells was 8.01%,slightly higher than that of subcultured cells(7.91%)and resuscitated cells(7.77%).The survival rate of resuscitated cells was decreased,and the cell viability decreased after transfection.However,7.77% GFP-positive rate could be obtained in resuscitated cells after transfection,with the primarily cultured cells having a higher positive rate and the difference being statistically significant.Conclusion The fibroblasts could be successfully isolated and cultured by the adherent culture of small-sized pigtail tissues.The cell viability was affected by cell passaging,cryopreservation and resuscitation.GFP-positive plasmid could be obtained from primarily cultured,subcultured and resuscitated cells after transfection.The primarily cultured cells showed better cell viability and positive transfection rates.The success rate can be improved by choosing primarily cultured cells for gene knockout or gene knock-in in gene editing and somatic cell cloning researches.
Objective To examine the transfection efficiency of green fluorescent protein(GFP)positive plasmids in different stages of porcine fibroblasts primarily cultured by adherent culture of small-sized pigtail tissues.Methods The primary porcine fibroblasts were isolated and cultured by adherent culture of small-sized pigtail tissues,and then cryopreserved and resuscitated.The survival rates of fibroblasts from primary culture,subculture and resuscitation were compared.The electro-transfection of GFP-positive plasmids with Neon was performed in fibroblasts after subculture and resuscitation.Cell survival and growth were compared at different transfection stages.The positive expression rate of GFP plasmid was detected by fluorescence microscopy and flow cytometry 24 hand 48 hafter transfection respectively.Results The pigtail fibroblasts could be successfully isolated and cultured by the adherent culture of small-sized pigtail tissues.The primary cell biological characteristics were maintained after cryopreservation.The viability of primary cells was high,with the cell survival rate reaching 97.67%.The cell survival rate was 95.67%after subculture and was still above 90%after resuscitation.The growth curves of cells from primary culture,subculture and resuscitation took on"S"type.The growth of cells after resuscitation was slightly slow.After electro-transfection,the viability of cells from primary culture,subculture and resuscitation was slightly decreased,the cell proliferation was weakened,but the cell growth curve still became an"S"curve.Fluorescence microscopy showed that the porcine fibroblasts with GFP positively expressed were obtained 24 hafter Neon electro-transfection.Flow cytometry revealed that 48 hafter the electro-transfection,the GFP-positive rate of primary cells was 8.01%,slightly higher than that of subcultured cells(7.91%)and resuscitated cells(7.77%).The survival rate of resuscitated cells was decreased,and the cell viability decreased after transfection.However,7.77% GFP-positive rate could be obtained in resuscitated cells after transfection,with the primarily cultured cells having a higher positive rate and the difference being statistically significant.Conclusion The fibroblasts could be successfully isolated and cultured by the adherent culture of small-sized pigtail tissues.The cell viability was affected by cell passaging,cryopreservation and resuscitation.GFP-positive plasmid could be obtained from primarily cultured,subcultured and resuscitated cells after transfection.The primarily cultured cells showed better cell viability and positive transfection rates.The success rate can be improved by choosing primarily cultured cells for gene knockout or gene knock-in in gene editing and somatic cell cloning researches.
引文
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[17]张庆晓,王慧利,孟春花,等.电穿孔法转染猪胎儿成纤维细胞条件优化[J].江苏农业科学,2012,40(12):202-204.
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[23]Reardon S.New life for pig-to-human transplants[J].Nature,2015,527(7577):152-154.
[24]Butler J R,Martens G R,Estrada J R,et al.Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles:an alternative to direct complement regulation[J].Transgenic Res,2016,25(5):751-759.
[25]陈云飞,王轶,杨洪吉.猪作为供体的异种肝移植的研究与进展[J].中国组织工程研究,2012,16(5);899-902.
[26]Cooper D K C,Wijkstrom M,Hariharan S,et al.Selection of patients for initial clinical trials of solid organ xenotransplantation[J].Transplantation,2017,101(7):1551-1558.
[27]Murthy R,Bajona P,Bhama J K,et al.Heart xenotransplantation:historical background,experimental progress,and clinical prospects[J].Ann Thorac Surg,2016,101(4):1605-1613.
[2]Niemann H,Lucas-Hashn A.Somatic cell nuclear transfer cloning:practical applications and current legislation[J].Reprod Domest Anim,2012,47(Suppl 5):2-10.
[3]McLaren A.Cloning:pathways to a pluripotent future[J].Science,2000,288(5472):1775-1780.
[4]Gomez M C,Pope C E,Ricks D M,et al.Cloning endangered fields using hererospecific donor oocytes and interspecies embryo transfer[J].Reprod Fertil Dev,2009,21(1):76-82.
[5]Mastromonaco G F,King W A.Cloning in companion animal,non-domestic and endanger species:can the technology become a practical reality?[J].Reprod Fertil Dev,2007,19(6):748-761.
[6]Li Z,Shi J,Liu D,et al.Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning[J].Cell Reprogram,2013,15(1):35-42.
[7]Lee G S,Kim H S,Hyun S H,et al.Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein[J].Theriogenology,2005,63(4):973-991.
[8]刘西梅,华再东,任红艳,等.猪成纤维细胞的简易分离培养[J].湖北农业科学,2017,56(4):702-703,726.
[9]信吉阁,成文敏,潘伟荣,等.猪胎儿成纤维细胞和耳皮肤成纤维细胞培养方法的研究[J].黑龙江畜牧兽医,2013,5(9):1-4.
[10]于玲珠,刘娣,孙兴参,等.猪耳皮肤成纤维细胞的培养[J].细胞生物学杂志,2003,25(4):258-260.
[11]李忠秋,刘春龙,汪亮,等.民猪耳皮成纤维细胞分离培养及生物学特性[J].畜牧与饲料科学,2017,38(4):5-7.
[12]赵彦玲,吴庆侠,强巴央宗,等.藏猪耳部成纤维细胞的分离与培养[J].广东农业科学,2013,40(17):130-132.
[13]Liu C,Guo Y,Lu T,et al.Establishment and genetic chareaceristics analysis of in vitro culture a fibroblast cell line derived from wuzhishan mniature pig[J].Cryobiology,2014,68(2):281-287.
[14]杨菲菲,屠平光,马美蓉,等.不同仔猪血清浓度对金华猪耳缘组织成纤维细胞体外培养的影响[J].畜禽业,2018,29(9):5-6.
[15]Bai C,Li C,Jin D,et al.Establishment of characterization of a fibroblast line from landrace[J].Artif Cells Blood substit Immobil Biotechnol,2010,38(3):129-135.
[16]张放,周载鑫,张丹丹,等.电穿孔法转染人原代成纤维细胞条件的优化[J].第二军医大学学报,2017,38(10):1311-1314.
[17]张庆晓,王慧利,孟春花,等.电穿孔法转染猪胎儿成纤维细胞条件优化[J].江苏农业科学,2012,40(12):202-204.
[18]Chang E N,Scudamore C H,Chung S W.Transplantation:focus on kidney,liver and islet cells[J].Can J Surg,2004,47(2):122-129.
[19]Lauerer M,Kaiser K,Nagel E.Organ transplantation in the face of donor shortage-ethical implication with a focus on liver allocation[J].Visc Med,2016,32(4):278-285.
[20]Ekser B,Ezzelarab M,Hara H,et al,Clinical xenotransplantation:the next medical revolution[J].Lancet,2012,379(9816):672-683.
[21]Ekser B,Markmann J F,Tector A J.Current status of pig liver xenotransplantation[J].Int J Surg,2015,23(Pt B):240-246.
[22]Perkel J M.Xenotransplantation makes a comeback[J].Nat Biotechnol,2016,34(1):3-4.
[23]Reardon S.New life for pig-to-human transplants[J].Nature,2015,527(7577):152-154.
[24]Butler J R,Martens G R,Estrada J R,et al.Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles:an alternative to direct complement regulation[J].Transgenic Res,2016,25(5):751-759.
[25]陈云飞,王轶,杨洪吉.猪作为供体的异种肝移植的研究与进展[J].中国组织工程研究,2012,16(5);899-902.
[26]Cooper D K C,Wijkstrom M,Hariharan S,et al.Selection of patients for initial clinical trials of solid organ xenotransplantation[J].Transplantation,2017,101(7):1551-1558.
[27]Murthy R,Bajona P,Bhama J K,et al.Heart xenotransplantation:historical background,experimental progress,and clinical prospects[J].Ann Thorac Surg,2016,101(4):1605-1613.