细胞培养中支原体污染的危害

支原体污染发生后危害巨大，能改变细胞的DNA,RNA及蛋白表达，改变细胞、篡改数据，造成实验数据不准确、不稳定或每次实验都得出不同结论或相反的结论。支原体污染不除，实验结果势必或多或少产生偏差，故进行实验前，确定细胞为mycoplasma-free至关重要，实验结果之数据才有意义 所以从2013年开始，《Nature》期刊已正式要求投稿的文章，如涉及细胞培养都要进行支原体检测。相信会有越来越多的高水平期刊将做出同样的支原体检测要求。 以下是支原体对一些相关实验的影响

1.细胞生物学：支原体污染将直接导致zui常用的MTT细胞毒性实验结果的严重错误！

•  结果：由于支原体对MTT的额外还原作用，对阿霉素（Doxorubicin, 一种抗肿瘤药物）的抗性，支原体污染和非污染的细胞二者相差15倍！（Due to an additional reduction of tetrazolium by mycoplasmas, contaminated cells appeared up to 15 fold resistant to doxorubicin.）
• 参考文献：Falsification of tetrazolium dye (MTT) based cytotoxicity assay results due to mycoplasma contamination of cell cultures. Anticancer Res. 1999 Mar-Apr; 19(2A):1245-8.

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2.免疫学：支原体污染本身将可以直接诱导树突状细胞的成熟！这对于目前国内外普遍开展的人体免疫细胞的肿瘤治疗将是灾难性的。

•   结果：（1）The capacity of these cell lines to induce DC maturation was due to their contamination by mycoplasma. Our results reveal that DC are able to sense mycoplasma infection and mature as they do in response to most viruses and bacteria.（2）Further investigation demonstrated that the changes in DC phenotype and functions were due to the presence of mycoplasmas in these two cell lines; eradication of mycoplasmas compley abolished the observed effects, and importantly, pure mycoplasmas in the absence of tumor cell supernatants were able to produce the same effects.
• 参考文献：（1）Dendritic cell maturation is induced by mycoplasma infection but not by necrotic cells. Eur. J. Immunol. 2000. 30: 705–708。（2）Mycoplasma-mediated alterations of in vitro generation and functions of human dendritic cells. J Biomed Sci. 2005;12(1):31-46.

3.神经生物学：支原体污染本身可以直接降解淀粉样多肽Amyloid-beta (Abeta)！

• 结果：These data show that mycoplasmas degrade Abeta and thus may represent a significant source of variability when comparing extracellular Abeta levels in different cell lines.
• 参考文献：（1）Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants. BMC Res Notes. 2008 Jun 30;1:38. （2）Neuroprotective effects of Mycoplasma hyorhinis against amyloid-β-peptide toxicity in SH-SY5Y human neuroblastoma cells are mediated by calpastatin upregulation in the mycoplasma-infected cells. Neurochem Int. 2011 Mar;58(4):497-503.

4.生物化学：支原体污染可以直接影响L-arginine的代谢！

• 结果：This demonstrates that infection with M. hyorhinis leads to different effects on gene regulation of the murine and human iNOS gene. Our study underlines the importance of routine checking of cell cultures for mycoplasma contamination, particularly in studies on NO-mediated effects or inflammatory processes.
• 参考文献: Impact of Mycoplasma hyorhinis infection on L-arginine metabolism: differential regulation of the human and murine iNOS gene. Biol Chem. 2005 Oct;386(10):1055-63.

5.病理学：支原体污染可以直接刺激前列腺素E2（Prostaglandin E2）的产生！

• 结果：Mycoplasma fermentans (MF) also stimulated PGE(2) production. The co-infection of mycoplasma and Chlamydia resulted in an additive effect in the production of PGE(2). Thus it is important to use host cells and Chlamydia free of mycoplasma contamination for the analysis of Chlamydia-induced prostaglandin production.
• 参考文献:Production of prostaglandin E2 in monocytes stimulated in vitro by Chlamydia trachomatis, Chlamydophila pneumoniae, and Mycoplasma fermentans. Microb Pathog. 2004 Sep;37(3):155-61.

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6.基因的表达与调控：支原体污染可以显著改变数百个基因的表达水平！

• 结果：支原体污染后与无污染的同一细胞系相比，表达差异达2倍以上的基因就有200多个（Even so, more than 200 genes were identified as being up- or down-regulated, demonstrating that mycoplasma infection has a profound effect on gene expression patterns.）。有些基因的表达甚至相差十几倍！（A variety of pathways are af­fected, and both up-regulation (up to 10-fold) and down-regulation (up to 12-fold) were seen.）
• 参考文献: Miller CJ, Kassem HS, Pepper SD, Hey Y, Ward TH, Margison GP. Mycoplasma infection significantly alters microarray gene expression profiles. Biotechniques. 2003 Oct;35(4):812-4.

?基于以上的危害，细胞培养实验中保证您的细胞无支原体污染客不容缓