摘要： 目的 探讨一种高效、可重复的宫颈上皮细胞原代培养方法。方法 本研究选取4例正常宫颈组织和4例宫颈鳞状细胞癌组织，并分别采用Ⅱ型中性蛋白酶联合0.25%胰蛋白酶-EDTA（中性蛋白酶联合法）及Ⅰ型胶原酶消化法分离后进行原代培养。通过CD326、P40以及Ki-67+P16双染进行免疫荧光染色鉴定细胞来源。结果 正常宫颈上皮细胞原代培养14 d，汇合度可达70%~80%，传代可至4~5代，且荧光染色CD326、P40均表达强阳性，P16表达弱阳性，Ki-67仅在个别细胞核中表达；宫颈鳞状细胞癌细胞原代培养14 d，汇合度可达70%~80%，传代可至5~6代，荧光染色CD326和P40均表达强阳性，P16表达阳性，Ki-67广泛表达于细胞核。结论 Ⅱ型中性蛋白酶联合0.25%胰蛋白酶-EDTA分离法及5% FBS的KFSM培养基可高效、多次重复原代培养正常宫颈上皮细胞；Ⅰ型胶原酶消化分离法及5% FBS的KFSM培养基培养可高效、多次重复原代培养宫颈鳞状细胞癌细胞。

原代正常宫颈及宫颈癌细胞的培养方法

吴思, 路一平, 王馨艺, 孙峥嵘

中国医科大学附属盛京医院生物样本库, 沈阳 110004

收稿日期:2022-05-01发布日期:2022-11-09
通讯作者:孙峥嵘E-mail:sunzr@sj-hospital.org
作者简介:吴思（1990-），女，技师，本科.
基金资助:国家自然科学基金（82000076）


关键词: 正常宫颈组织, 宫颈癌组织, 原代细胞培养
Abstract: Objective To explore an efficient and repeatable method to obtain a primary culture of cervical epithelial cells.Methods We selected four samples of normal cervical tissue and four samples of cervical squamous cell carcinoma tissue. These tissues were lysed using the neutral protease combined method（typeⅡneutral protease combined with 0.25% trypsin-EDTA）and the typeⅠcollagenase digestion method. In addition，immunofluorescence staining with CD326，P40，and Ki-67+P16 double staining was used to identify the origin of the cell.Results In the primary culture，the confluence of normal cervical epithelial cells could reach about 70%-80% until the 14th day，and could be passaged through 4-5 generations. Moreover，CD326 and P40 fluorescent staining were strongly positive and P16 was weakly positive；however，Ki-67 was only expressed in a few nuclei. Similarly，the confluence of cervical squamous cell carcinoma cells（in the primary culture）could reach about 70%-80% until the 14th day，and could be passaged through 5-6 generations. Moreover， CD326 and P40 fluorescent staining was strongly positive，P16 was positive，and Ki-67 was widely expressed in nuclei. Conclusion The typeⅡ neutral protease combined with 0.25% trypsin-EDTA separation method and KFSM medium with 5% FBS can be used to efficiently and repeatedly culture normal cervical epithelial cells. TypeⅠcollagenase digestion separation method and KFSM culture with 5% FBS substance culture can be used to efficiently and repeatedly replicate the primary culture of cervical squamous epithelial cancer cells.
Key words: normal cervical tissue, cervical cancer tissue, primary cell culture
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