[1]李 雅,荣守华,智艳芳,等.正常宫颈上皮细胞原代培养体系的改良*[J].郑州大学学报(医学版),2017,(03):370-375.[doi:10.13705/j.issn.1671-6825.2017.03.032]
 LI Ya,RONG Shouhua,ZHI Yanfang,et al.An improved culture system for the primary culture of normal cervical epithelial cells[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2017,(03):370-375.[doi:10.13705/j.issn.1671-6825.2017.03.032]
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正常宫颈上皮细胞原代培养体系的改良*()
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《郑州大学学报(医学版)》[ISSN:1671-6825/CN:41-1340/R]

卷:
期数:
2017年03期
页码:
370-375
栏目:
应用研究
出版日期:
2017-05-15

文章信息/Info

Title:
An improved culture system for the primary culture of normal cervical epithelial cells
作者:
李 雅荣守华智艳芳樊婷婷邱 翠李肖甫#
郑州大学第三附属医院细胞室 郑州 450052
Author(s):
LI YaRONG ShouhuaZHI YanfangFAN TingtingQIU CuiLI Xiaofu
Department of Cytopathology,the Third Affiliated Hospital,Zhengzhou University,Zhengzhou 450052
关键词:
原代培养 宫颈上皮细胞 中性蛋白酶 角质细胞无血清培养基
Keywords:
primary culture cervical epithelial cell dispase dK-SFM
分类号:
R711.74
DOI:
10.13705/j.issn.1671-6825.2017.03.032
摘要:
目的:探索一种高效、可重复的正常宫颈上皮细胞原代培养方法。方法:取60例正常宫颈组织,分成2组,分别采用Ⅱ型中性蛋白酶联合0.25 g/mL胰蛋白酶-EDTA(中性蛋白酶联合法)及Ⅰ型胶原酶消化分离后进行原代培养,比较两种分离效果; 观察采用中性蛋白酶联合法消化不同时间对分离效果的影响。观察添加体积分数5%FBS dK-SFM培养基与单纯dK-SFM培养基培养原代宫颈上皮细胞的生长曲线差异,并用形态学、广谱角蛋白免疫细胞化学染色鉴定细胞来源。结果:正常宫颈上皮细胞原代培养总成功率为40%。中性蛋白酶联合法成功率、分离所得细胞密度、原代细胞融合时间及细胞纯度均高于Ⅰ型胶原酶消化法(P<0.05)。中性蛋白酶消化20 h的分离效果优于消化16和24 h(P<0.05)。体积分数5%FBS dK-SFM培养基原代培养的宫颈上皮细胞增殖活性高于单纯dK-SFM培养基(P<0.05)。细胞生长状况良好,可在体外传代至5~6代,并可冻存与复苏,免疫细胞化学染色显示广谱角蛋白表达阳性。结论:Ⅱ型中性蛋白酶联合0.25 g/mL胰蛋白酶-EDTA分离法和体积分数5% FBS dK-SFM培养基培养原代正常宫颈上皮细胞,高效、可重复性好。
Abstract:
Aim: To explore an effective and repeatable method for the primary culture of normal cervical epithelial cells.Methods: A total of 60 normal human ectocervical tissue were collected and divided into two groups, primarily cultured using combined type Ⅱdispase and 0.25 g/mL trypsin-EDTA digestion method and type Ⅰ collagenase digestion method separately, and to compare the effectiveness of isolation, furthermore, to observe the detached effectiveness of combined dispase digestion method after digestion for different time. Cervical epithelial cells were cultured in 5% FBS dK-SFM and dK-SFM alone, respectively, to observe the growth of the primary cervical cells. The cultured cells were identified by the morphological analysis and pancytokeratin was identified by immunocytochemistry.Results: The total success rate of the primary culture in this study was 40%. The success rate, density, primary cell fusion time and purity of cells of the dispase digestion group were all higher than those of collagenase digestion group(P<0.05). The dispase digestion for 20 h was more effective than digestion for 16 and 24 h(P<0.05). The cell growth curve showed that the cells cultured in 5% FBS dK-SFM grew better and had a higher viability than cells cultured in dK-SFM alone(P<0.05). The primary cultured cells grew well and could be subcultured for five to six generations,and they also could be cryopreserved and resuscitated. Pancytokeratin positive staining was determined by immunocytochemistry.Conclusion: It is an effective and repeatable method for primary culture of normal cervical epithelial cells by using combined type Ⅱ dispase and 0.25 g/mL trypsin-EDTA digestion and 5% FBS dK-SFM medium.

参考文献/References:

[1] SCHÖNTHAL AH, WARREN DW, STEVENSON D, et al. Proliferation of lacrimal gland acinar cells in primary culture.Stimulation by extracellular matrix,EGF,and DHT [J].Exp Eye Res,2000,70(5):639
[2] 王雪峰,何援利,刘木彪,等.携带Bcl-2基因的慢病毒对原代培养的人卵巢颗粒细胞的影响[J].解放军医学杂志,2012,37(8):760
[3] 曹颖,金哲,于妍妍,等.成人宫颈上皮细胞的原代培养及鉴定[J].现代生物医学进展,2012,12(2):204
[4] 江静,邓齐,马营营,等.成人宫颈上皮细胞的原代培养及影响因素[J].中国妇幼保健,2014,29(10):1495
[5] 王雪银,刘玉珍,陈昭日,等.人正常宫颈上皮细胞的体外分离及细胞活性比较[J].现代妇产科进展,2014,23(3):188
[6] LIU YZ,LÜ XP,PAN ZX,et al.Establishment of a novel method for primary culture of normal human cervical keratinocytes[J].Chin Med J,2013,126(17):3344
[7] STENN KS,LINK R,MOELLMANN G,et al.Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type Ⅳ collagenase[J].J Invest Dermatol,1989,93(2):287
[8] TREMELLEN G, MOTTERSHEAD B, TREMELLEN K, et al. TGF-β mediates proinflammatory seminal fluid signaling in human cervical epithelial cells[J].J Immunol,2012,189(2):1024
[9] SHARKEY DJ,MACPHERSON AM,TREMELLEN KP.Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells[J].Mol Hum Reprod,2007,13(7/8):491
[10]PALANTAVIDA S,GUZ NV,WOODWORTH CD,et al.Ultrabright fluorescent mesoporous silica nanoparticles for prescreening of cervical cancer[J].Nanomedicine,2013,9(8):1255
[11]PEEHL DM,HAM RG.Growth and differentiation of human keratinocytes without a feeder layer or conditioned medium[J].In Vitro,1980,16(6):516
[12]BOYCE ST,HAM RG.Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture[J].J Invest Dermatol,1983,81(1 Suppl):33s
[13]YOKOO S,YAMAGAMI S,USUI T,et al.Human corneal epithelial equivalents for ocular surface reconstruction in a complete serum-free culture system without unknown factors[J].Invest Ophthalmol Vis Sci,2008,49(6):2438
[14]JEAN J,BERNARD G,DUQUE-FERNANDEZ A,et al.Effects of serum-free culture at the air-liquid interface in a human tissue-engineered skin substitute[J].Tissue Eng Part A,2011,17(7/8):877
[15]赵超,白丽霞,屠铮,等.人乳头状瘤病毒16型E6、E7基因转染的人宫颈上皮永生化细胞系的建立及鉴定[J].中国妇产科临床杂志,2006,7(4):278

备注/Memo

备注/Memo:
*河南省医学科技攻关计划资助项目 201503112
#通信作者,男,1964 年7 月生,硕士,教授,研究方向:肿瘤细胞病理学,E-mail:lixiaofu1964@163.com
更新日期/Last Update: 2017-05-20