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  

Research of Isolation and Culture of Fibroblasts by Adherent Culture of Small-sized Pigtail Tissues and the Transfection Efficiency

作　　者：张树华[1] 张帆[1] Christopher Burlak 陈庆[1] Zhang Shuhua;Zhang Fan(Department of Hepatobiliary Surgery,Union Hospital,Tongji Medical College,Huazhong University of Science of Technology,Wuhan 430022,China;Schulze Diabetes Itjstitute,Department of Surgery,University of Minnesota,Minneapolis,MN 55455,USA)

机构地区：[1]华中科技大学同济医学院附属协和医院肝胆外科,武汉430022 [2]美国明尼苏达州立大学SDI研究中心,美国明尼阿波利斯55455

出　　处：《华中科技大学学报（医学版）》2019年第1期15-20,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

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 obta

关 键 词：猪成纤维细胞 贴壁培养 生长曲线 电转染 质粒 绿色荧光蛋白 

分 类 号：R329-33[医药卫生—人体解剖和组织胚胎学]