支原體污染發(fā)生后危害巨大，能改變細(xì)胞的DNA,RNA及蛋白表達(dá)，改變細(xì)胞、篡改數(shù)據(jù)，造成實(shí)驗(yàn)數(shù)據(jù)不準(zhǔn)確、不穩(wěn)定或每次實(shí)驗(yàn)都得出不同結(jié)論或相反的結(jié)論。支原體污染不除，實(shí)驗(yàn)結(jié)果勢必或多或少產(chǎn)生偏差，故進(jìn)行實(shí)驗(yàn)前，確定細(xì)胞為mycoplasma-free至關(guān)重要，實(shí)驗(yàn)結(jié)果之?dāng)?shù)據(jù)才有意義 所以從2013年開始，《Nature》期刊已正式要求投稿的文章，如涉及細(xì)胞培養(yǎng)都要進(jìn)行支原體檢測。相信會(huì)有越來越多的高水平期刊將做出同樣的支原體檢測要求。 以下是支原體對一些相關(guān)實(shí)驗(yàn)的影響
1.細(xì)胞生物學(xué)：支原體污染將直接導(dǎo)致zui常用的MTT細(xì)胞毒性實(shí)驗(yàn)結(jié)果的嚴(yán)重錯(cuò)誤！
- 結(jié)果：由于支原體對MTT的額外還原作用，對阿霉素（Doxorubicin, 一種抗腫瘤藥物）的抗性，支原體污染和非污染的細(xì)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.）
- 參考文獻(xiàn)：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.免疫學(xué)：支原體污染本身將可以直接誘導(dǎo)樹突狀細(xì)胞的成熟！這對于目前國內(nèi)外普遍開展的人體免疫細(xì)胞的腫瘤治療將是災(zāi)難性的。
- 結(jié)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.
- 參考文獻(xiàn)：（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.神經(jīng)生物學(xué)：支原體污染本身可以直接降解淀粉樣多肽Amyloid-beta (Abeta)！
- 結(jié)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.
- 參考文獻(xiàn)：（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.生物化學(xué)：支原體污染可以直接影響L-arginine的代謝！
- 結(jié)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.
- 參考文獻(xiàn): 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.病理學(xué)：支原體污染可以直接刺激前列腺素E2（Prostaglandin E2）的產(chǎn)生！
- 結(jié)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.
- 參考文獻(xiàn):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.基因的表達(dá)與調(diào)控：支原體污染可以顯著改變數(shù)百個(gè)基因的表達(dá)水平！
- 結(jié)果：支原體污染后與無污染的同一細(xì)胞系相比，表達(dá)差異達(dá)2倍以上的基因就有200多個(gè)（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.）。有些基因的表達(dá)甚至相差十幾倍！（A variety of pathways are affected, and both up-regulation (up to 10-fold) and down-regulation (up to 12-fold) were seen.）
- 參考文獻(xiàn): 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，細(xì)胞培養(yǎng)實(shí)驗(yàn)中保證您的細(xì)胞無支原體污染客不容緩
