论文部分内容阅读
目的探讨三维细胞培养系统(RCCS)模拟微重力环境对铜绿假单胞菌(PA)毒力编码基因表达及菌体蛋白质谱的影响。方法应用RCCS建立PA ATCC 27853模拟微重力培养体系,实验组旋转瓶轴心与地面平行模拟微重力环境,对照组即重力组旋转瓶轴心与地面垂直;采用实时荧光定量聚合酶链反应(Real-time PCR)检测c DNA样本中毒力编码基因外毒素(tox A)、弹性蛋白酶(las B)、鼠李脂糖(rhl A)、绿脓菌素(phza2)mRNA的相对含量;利用表面增强激光解吸电离飞行时间质谱技术及CM10芯片检测PA菌体蛋白质谱。结果 RCCS模拟微重力处理14 d后,Real-time PCR检测实验组c DNA样本中tox A、las B、rhl A、phza2毒力编码基因相对含量均低于对照组(P<0.05);表面增强激光解吸电离飞行时间质谱检测发现有82种小分子菌体蛋白质[荷比在(2~20)×103Da范围内],两组42种菌体蛋白相对含量表达差异有统计学意义,实验组20种蛋白质相对含量表达高于对照组,22种蛋白质相对含量表达低于对照组(P<0.05)。结论 RCCS模拟微重力培养条件下PA毒力编码基因表达和菌体蛋白质谱发生显著变化。
Objective To investigate the effects of three-dimensional cell culture system (RCCS) on the expression of virulence-encoding genes of Pseudomonas aeruginosa (PA) and microbial protein profiles in simulated microgravity environment. Methods The RCCS was used to establish a simulated microgravity culture system of PA ATCC 27853. In the experimental group, the axis of the rotating bottle and the ground were simulated in parallel to the microgravity. The axis of the rotating bottle in the control group was perpendicular to the ground. The real-time fluorescent quantitative polymerase chain reaction time PCR was used to detect the relative contents of tox A, las B, rhl A and phza2 mRNA in c DNA samples. Laser desorption / ionization time of flight mass spectrometry and detection of PA bacterial protein by CM10 chip. Results The relative contents of toxA, lasB, rhlA and phza2 virulence genes in c DNA samples of experimental group were lower than that of control group (P <0.05) after RCCS simulated microgravity treatment for 14 days. The results of laser desorption ionization time-of-flight mass spectrometry showed that there were 82 kinds of small-molecule protein [the ratio of charge was in the range of (2 ~ 20) × 103Da], and there was significant difference in the relative content of 42 kinds of bacteria between two groups. The relative content of the protein was higher than that of the control group, and the relative content of 22 proteins was lower than that of the control group (P <0.05). Conclusion The expression of PA virulence gene and the protein spectrum of PA in simulated microgravity culture in RCCS were significantly changed.