摘要
目的支原体污染是细胞培养过程中最为常见也较为棘手的问题,因此,解决细胞支原体污染问题存在必要性和迫切性。但是目前可用的商品化去除支原体试剂选择少,价格贵,本研究拟探索一种操作简单,成本低且去除支原体效果佳的抗生素联合用药方案。方法选取四种抗生素药物联合用药:红霉素、氯霉素、泰乐菌素和环丙沙星,连续12天处理培养支原体污染的C3A肝癌细胞。首先通过倒置显微镜观察加药处理后,C3A细胞内颗粒、形态等变化;加药处理C3A细胞后通过CCK-8法检测其活率。其次,进行支原体聚合酶链式反应(PCR)检测和荧光染色,评价抗生素联合用药方案去除支原体的有效性。最后,实时荧光定量PCR(qPCR)方法检测C3A细胞主要功能基因角蛋白(CK18)、白蛋白(ALB)、氨甲酰磷酸合成酶l(CPS1)和细胞色素P450酶系1A2(CYP1A2)等功能基因的表达恢复情况。结果经抗生素联合用药处理后,细胞形态更为规整,胞内颗粒显著减少,形态透亮折光度增强等特征变化,对C3A细胞活率无明显影响,抗生素联合用药组PCR电泳结果显示没有支原体DNA条带,同时荧光结果表明没有出现其余点状或者串点的支原体荧光,支原体去除后功能基因CK18、ALB、CPS1和CYP1A2相对表达量均升高。结论新的抗生素联合用药方案能够达到支原体去除效果,C3A细胞活率维持较好,去除支原体后细胞主要功能基因表达可得到恢复提升。
Objective Mycoplasma contamination can cause a wide range of troublesome problems associated with cell culture in biology research,it is therefore necessary to keep cell culture free of mycoplasma contamination.However,there are few options to choose the available commercial mycoplasma removal drugs which are also expensive.It is hope to explore a new antibiotic treatment which is low cost,simple-operation,and effective in mycoplasma removal.Methods A new antibiotic treatment was combined with four antibiotic drugs including erythromycin,chloramphenicol,tylosin and ciprofloxacin,which was applied in C3 A cell line contaminated by mycoplasma for 12 days.First of all,we observed the changes of the particles and morphology of C3 A cell after treatment of the new combination antibiotic drugs through an inverted microscope;the viability of C3 A cell was detected by cell counting kit-8(CCK-8)assay after treatment.Moreover,polymerase chain reaction(PCR)detection and fluorescent staining of mycoplasma were performed to evaluate whether the new combination antibiotic drugs could remove mycoplasma contamination.Finally,real-time quantitative PCR(qPCR)method was used to define the recovery of the main functional genes expression including cytokeratin 18(CK18),albumin(ALB),carbamoyl phosphate synthetase I(CPS1)and human cytochrome P4501 A2(CYP1 A2).Results After the treatment of new combination antibiotic drugs,more regular and uniform cell morphological changes were showed,the subsequent reductions in intracellular particles and enhanced morphological transparency were observed,furthermore no significant effect on the C3 A cell viability occurred.Mycoplasma DNA band was not presented by PCR detection,and the fluorescence results indicated that the C3 A cell kept away from other spots or cross-points of mycoplasma fluorescence.After the removal of mycoplasma,the relative expression level of CK18,ALB,CPS1 and CYP1 A2 increased.Conclusion The result of mycoplasma removal could be achieved by the new combination antibiotic drugs treatment with well-maintained viability and restoration of functional genes expression in C3 A cell.
作者
黎少
梁永康
冯思桦
彭青
高毅
LI Shao;LIANG Yongkang;FENG Sihua;PENG Qing;GAO Yi(Zhujiang Hospital of Southern Medical University,Guangzhou 510280,China;不详)
出处
《现代医院》
2021年第2期301-305,共5页
Modern Hospitals
基金
国家重点研究计划项目(2018YFC1106400)
广东省自然科学基金项目(2014A030312013)
广州市科技计划项目(201803010086)。
关键词
抗生素处理
支原体去除
支原体检测
基因表达
Antibiotic treatment
Mycoplasma removal
Mycoplasma detection
Gene expression
