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目的人胚胎干细胞(hESCs,human embryonic stem cells)应用广泛,但来源有限。前期检测发现本室培养的H9 hESCs有支原体污染。本研究拟探讨高效支原体杀灭剂plasmocin去除hESCs支原体污染的可能性,及其对hES增殖、分化的影响。方法常规培养hESCs细胞,用切割法进行传代。分别用低(12.5μg/ml)、中(25μg/ml)、高(37.5μg/ml)浓度plasmocin处理hESCs 2～3周,用PCR法检测支原体,用结晶紫染色观察hESCs克隆大小,显微镜下观察hESCs分化状况。结果低、中、高浓度plasmocin处理2周均不能杀灭hESCs的支原体,延长高浓度plasmocin处理时间至3周,亦未能杀灭支原体。实验发现,plasmocin呈剂量依赖性抑制hESCs增殖,且高浓度plasmocin可诱导hESCs分化,而高浓度bFGF(40 ng/ml)则能部分阻断plasmocin抑制增殖和诱导分化的作用。结论plasmocin未能去除hESCs的支原体污染,可能与支原体产生抗药性及hESCs的克隆生长特性有关。
The purpose of human embryonic stem cells (hESCs, human embryonic stem cells) is widely used, but the source is limited. Pre-test found that cultured H9 hESCs mycoplasma contamination. This study was to investigate the possibility of mycoplasma hESCs elimination by plasmocin, a highly efficient mycoplasma killing agent, and its effect on the proliferation and differentiation of hES. Methods The hESCs were cultured routinely and passaged by cutting. HESCs were treated with low (12.5μg / ml), medium (25μg / ml) and high (37.5μg / ml) concentrations of plasmocin for 2 to 3 weeks respectively. The mycoplasma was detected by PCR and the size of hESCs was observed by crystal violet staining. Observe the differentiation status of hESCs. Results Low, medium and high concentrations of plasmocin could not kill mycoplasma hESCs for 2 weeks, prolonged high-dose plasmocin treatment time to 3 weeks, also failed to kill mycoplasma. It was found that plasmocin inhibited the proliferation of hESCs in a dose-dependent manner, and high concentrations of plasmocin induced the differentiation of hESCs. However, high concentration of bFGF (40 ng / ml) partially blocked the inhibitory effect of plasmocin on proliferation and differentiation. Conclusion Plasmocin failed to remove mycoplasma contamination of hESCs, which may be related to the resistance of mycoplasma and clonal growth of hESCs.