细胞库支原体检测体系以及支原体去除方法的建立

发布时间：2018-10-16 12:18

【摘要】：生命科学迎来了繁荣发展的时代。从生物石油到药品、医疗,无一不和生命科学息息相关。细胞株作为生命科学研究的重要材料显得尤为重要。无污染的细胞株是得到可靠实验数据的前提。作为国家级细胞株保藏中心,监测细胞株的质量一直是本细胞库重点工作之一。细胞株的微生物污染源一般有细菌、真菌、支原体。细菌和真菌污染细胞培养物时导致培养基变色,镜下可以看到菌体。但细胞株感染支原后培养基通常不会有颜色变化,更不能在普通显微镜下观察到。支原体会影响培养细胞的各项参数,甚至导致细胞株死亡,因此建立细胞库支原体检测体系以及支原体去除方法非常必要。本研究结合本库自身情况选用最易操作的三种方法:DNA染色、直接培养法、PCR,对本细胞库保藏的常用的100种细胞株进行支原体检测。根据各不同方法的优缺点建立适用于本细胞库的支原体检测流程体系:入库前PCR试剂盒预检、入库后三项全检、日常两项监控、供应中自建PCR检测。论文在有支原体污染的细胞株中选取珍贵、稀有、且国内研究者急需求的细胞株为研究对象,同时使用BM-Cyclin和MC-210两种药剂去除支原体。在支原体去除后的一周、一个月、一年的时间段里,重复检测支原体污染,用以评价两种药剂效果和优缺点。通过对比,最终确定BM-Cyclin为细胞库去除支原体污染的首选药剂。
[Abstract]:Life science ushered in an era of prosperity and development. Everything from biooil to medicine is closely related to life sciences. As an important material in life science research, cell line is very important. Non-polluting cell lines are a prerequisite for reliable experimental data. As a national cell line preservation center, monitoring the quality of cell line has been one of the key tasks of the cell bank. Bacteria, fungi, and mycoplasma are the main sources of microbial pollution in cell lines. Bacteria and fungi contaminate the cell culture resulting in discoloration of the medium, which can be seen under the microscope. However, the culture medium did not change in color after infection, and could not be observed under ordinary microscope. Mycoplasma can affect the parameters of cultured cells and even lead to cell line death. Therefore, it is necessary to establish the detection system of mycoplasma and the removal method of mycoplasma. In this study, we selected the three most convenient methods: DNA staining, direct culture, and PCR, to detect mycoplasma of 100 common cell lines preserved in our library. According to the merits and demerits of different methods, the flow system of mycoplasma detection suitable for this cell library was established: pre-examination of PCR kit before storage, three full tests after entering the library, two daily monitoring items, and self-built PCR detection in supply. In this paper, the rare and precious cell lines contaminated by mycoplasma were selected as the research objects. BM-Cyclin and MC-210 were used to remove mycoplasma at the same time. Mycoplasma contamination was repeatedly detected one week, one month and one year after mycoplasma was removed to evaluate the efficacy, advantages and disadvantages of the two agents. By comparison, BM-Cyclin was determined as the first choice for removing mycoplasma contamination from cell banks.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R446;R375

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