巴龙霉素对携带线粒体DNA C1494T突变的细胞系生长的影响

Effect of paromomycin on growth of the cell lines carrying mitochondrial DNA C1494T mutation

目的用细胞学方法,分析线粒体DNA12SrRNA基因中C1494T突变在氨基糖甙类抗生素聋发病机理中的作用。方法从携有线粒体DNAC1494T突变的母系遗传性氨基糖甙类抗生素性耳聋的中国大家系选择部分成员,另外从遗传背景相同的正常中国人群选择对照个体,分别建立淋巴细胞系;并通过细胞融合技术,将淋巴细胞系的线粒体分别融合到缺乏线粒体DNA的!0206细胞中,建立相应的转线粒体细胞系;家系成员与对照个体的淋巴细胞系和转线粒体细胞系,分别在不含/含有氨基糖甙类抗生素(巴龙霉素)的培养液中培养,以倍增时间(doublingtime,DT)作为细胞生长特性的评价标准,通过计算在正常和含有氨基糖甙类抗生素的培养液中倍增时间的比值,比较氨基糖甙类抗生素对细胞生长的影响。结果携有线粒体DNAC1494T突变家系成员较对照个体的淋巴细胞系的倍增时间比值平均增加了24%,但不同家系成员的细胞倍增时间比值的增加程度不同,自10%至50%不等;而当细胞核遗传背景相同后,家系成员较对照个体的转线粒体细胞系的倍增时间比值增长30%,并且来自不同表型的家系成员的细胞倍增时间比值基本相同。结论线粒体DNAC1494T突变可以造成细胞对氨基糖甙类抗生素的超敏性,但其效应要受到核基因的调控。

Objective To analysis the effect of mitochondrial 12S rRNA C1494T mutation on the aminoglycoside toxicity. Methods Lymphoblastoid cell lines were derived from several matrilineal relatives of a large Chinese family carrying the C1494T mutation, and from control members lacking the mutation. Alternatively, eybrid cell lines were constructed by transferring mitoehondria from lymphoblastoid cell lines derived from the Chinese family into human mtDNA-less（p^0） cells. The cells were incubated in the DMEM or RPMI 1640 media containing/without paromomyein. The growth properties were measured with cell population doubling time （DT）. And the effects of aminoglycoside were compared with the ratio of DT in the presence and absence of paromomycin. Results Exposure to high concentration of paromomyein caused a variable but significant average increase in doubling time in the C1494T mutation-carrying lymphoblastoid cell lines derived from four symptomatic individuals and two asymptomatic individuals in this Chinese family, when compared to four unrelated control cell lines. However, under a constant nuclear background, the very significant/nearly identical increase in the ratio of doubling times in DMEM medium in the presence/absence of high concentration of paromomycin was observed in symptomatic or asymptomatic cybrid cell lines carrying the C1494T mutation as compared to the average rate in control cell lines. Conclusion The 12S rRNA C1494T mutation led to the sensitivity to aminoglycosides, but nuclear background plays a critical role in aminoglycoside toxicity associated with the C1494T mutation.