细胞培养中珍贵贴壁细胞污染挽救方法的评价

首都医科大学学报 ›› 2011, Vol. 32 ›› Issue (1): 129-134.

细胞培养中珍贵贴壁细胞污染挽救方法的评价

王鸿^1，2，张伟^1，2*，孟娜^1，2

1. 首都医科大学附属北京同仁医院耳鼻咽喉头颈外科； 2. 教育部耳鼻咽喉头颈外科重点实验室，北京市耳鼻咽喉科研究所

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2011-02-21 发布日期:2011-02-21
通讯作者: 张伟

Evaluation of Methods for Rescuing Novel Adherent Cell Line Contaminatedby Bacteria in Cell Culture

WANG Hong^1,2, ZHANG Wei^1,2*, MENG Na^1,2

1. Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University;2. Key Laboratory of Otorhinolaryngology Head and Neck Surgery, Beijing Institute of Otorhinolaryngology

Received:1900-01-01 Revised:1900-01-01 Online:2011-02-21 Published:2011-02-21
Contact: ZHANG Wei

摘要/Abstract

摘要：

目的细菌污染在细胞培养工作中在所难免，特别是珍稀细胞系受到细菌污染，污染后能否逆转，哪种方法可以挽救污染细胞既有效又对细胞生物学特性影响小，是本文探讨的目的。
方法分别采用抗生素除菌法、抗生素联合巨噬细胞吞噬法和裸鼠体内接种除菌法清除人喉鳞状细胞癌细胞系（laryngeal squamous carcinoma-1，LSC-1）的污染细菌, 并对除菌效果做无菌检验，对细胞性状做免疫组织化学角蛋白染色和流式细胞仪DNA倍体分析，并与第3代细胞比较。
结果 ① 在3种敏感抗生素中，32 mg/L的头孢哌酮/舒巴坦抗菌效果最好，细胞可传代3次；头孢哌酮/舒巴坦联合巨噬细胞吞噬法处理，细胞可传代5~6次，但抗生素撤离后上述2种方法培养的细胞液中均再次出现细菌，细胞逐渐死亡。裸鼠体内接种法的细胞形态最好，传代8次以上，冻存、复苏后仍能生长繁殖，并可在无抗生素培养基中生长，上清液细菌培养为阴性；② 免疫组织化学角蛋白染色，抗生素法和抗生素联合巨噬细胞法所得细胞的角蛋白表达比例较低，而裸鼠体内接种法得到的细胞角蛋白表达较高，与该细胞系第3代比例基本相同；③ DNA倍体分析，裸鼠体内接种法的细胞仍保持有二倍体和异倍体核型，与第3代细胞基本相同，而抗生素处理的细胞只保留有二倍体核型。
结论裸鼠体内接种除菌法能彻底清除喉癌细胞系中的污染细菌，并能保持肿瘤细胞的来源特征和增生活性，是细胞系去除污染细菌最为理想的方法。

关键词: 贴壁细胞系, 污染, 挽救

Abstract:

Objective To compare the methods for rescuing the cell line contaminated by bacteria.
Methods Antibiotics or antibiotic combined with macrophage or animal inoculation were used to eliminate the bacteria in laryngeal squamous carcinoma cell line LSC-1. Then cytokeratin(CK AE1/AE3) and DNA ploidy in LSC-1 cells were detected by immunohistochemical and flow cytometer analysis.
Results ① Cefoperazone/Sulbactam at a dose of 32 mg/L was the best one in three sensitive antibiotics and made LSC-1 cells to survive three passages. Cefoperazone/Sulbactam combined with macrophage made LSC-1 cells to survive 5~6 passages. But in the two methods, bacteria appeared again when Cefoperazone/Sulbactam was eliminated and the cells gradually died. LSC-1 cells that experienced animal inoculation survived over 8 passages with well growth, and bacterial culture was negative. ② The positive rate of CK AE1/AE3 in the cells undergone animal inoculation was higher than that of the other two groups and the same as that of the third passage of LSC-1 largely. ③ DNA ploidy analysis showed that LSC-1 cells undergone animal inoculation had diploid and aneuploid karyotypes and were consistent with the third passage of LSC-1. However LSC-1 treated with Cefoperazone/Sulbactam only expressed diploid.
Conclusion Using animal inoculation to eliminate contaminating bacteria in cell line is a useful method. Not only the bacteria can be cleared thoroughly but also the original characteristics and malignancy of the cell may be reserved.

Key words: adherent cell lines, contamination, rescue

中图分类号:

Q 813.1⁺1

王鸿;张伟;孟娜. 细胞培养中珍贵贴壁细胞污染挽救方法的评价[J]. 首都医科大学学报, 2011, 32(1): 129-134.

WANG Hong;ZHANG Wei;MENG Na;. Evaluation of Methods for Rescuing Novel Adherent Cell Line Contaminatedby Bacteria in Cell Culture[J]. Journal of Capital Medical University, 2011, 32(1): 129-134.

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