RCCS模拟微重力对铜绿假单胞菌毒力编码基因和蛋白质谱的影响

Effects of Simulated Microgravity with RCCS on the Expression of Virulence Encoding Genes and Proteinic Profiles of Pseudomonas Aeruginosa Mycoprotein

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 simulated microgravity with the rotary cell culture system （RCCS） on the expression of virulence encoding genes and proteinic profiles of Pseudomonas aeruginosa （PA） mycoprotein. Methods The cultured system of PA ATCC 27853 under simulated microgravity were established using RCCS, and the control group was oriented to achieving normal earth gravity, while experiment group simulated microgravity. The relative contents of toxA, lasB, rhlA and phza2 mRNA virulence encoding genes in cDNA samples were detected by real-time PCR （polymerase chain reaction） method. The PA tropina profiles were detected using the surface-enhanced laser desorption ionization time of flight mass spectrometry and CMIO chip. Results After being cultured for 14 d, the relative contents of toxA, lasB, rhlA and phza2 mRNA virulence encoding genes in cDNA samples were significantly lower than those in control group by real-time PCR detection （P 〈 0. 05） ; 82 types of micromolecule tropina from 2 × 10^3 to 20 × 10^3 dalton range were discovered by surface-enhanced laser desorption-ionization time-mass spectrometry （SELD1-TOF-MS） method in control and experiment groups ; the differences of 42 types of micromolecule tropina expressions of relative contents in the two groups were statistically significant, and 20 types expressions Of relative contents in the experiment group were higher than those in control group, and 22 types＇expression of relative contents were lower than those in the control group （ P 〈 0. 05 ）. Conclusion The expression of virulence encoding genes and proteinic profiles of PA mycoprotein can be effectively affected after being cultured by RCCS simulated microgravity.