铜绿假单胞菌制剂对大鼠C6胶质瘤细胞作用的实验研究

The Effect of Pseudomonas Aeruginosa Injectionon on Rat C6 Glioma Cells in Vitro

目的研究铜绿假单胞菌注射液(PA-MSHA)对体外培养的大鼠C6胶质瘤细胞的增殖抑制机制.方法体外培养大鼠C6胶质瘤细胞,测定细胞活力、细胞贴壁率,绘制生长曲线,计算细胞倍增时间,确定最佳细胞接种浓度及药物干预区间.倒置显微镜观察药物作用后细胞形态学变化,MTT比色法测定不同时间、不同浓度PA-MSHA对C6胶质瘤细胞的增殖抑制率,计算细胞存活率及半数抑制浓度(IC50).结果 C6细胞在常规培养条件下呈贴壁生长,生长状态良好,每次传代前活力测定,存活率均>95%.于2 h、4 h、6 h、8 h、10 h、12 h测定贴壁率分别为20%、68%、82%、85%、92%、98%,符合本实验要求.细胞生长曲线提示5×103/孔的浓度为96孔板内最佳接种浓度.细胞接种后24～72 h为细胞倍增时间.倒置显微镜下观察药物作用后细胞形态学变化明显.MTT比色法结果提示,细胞生长抑制率与药物作用时间和剂量正相关.计算24 h和48 h IC50值分别为(5.80±1.79)×108 cfu/mL和(3.90±2.14)×108 cfu/mL.结论铜绿假单胞菌(PA-MSHA)制剂对体外培养的C6胶质瘤细胞具有增殖抑制作用,且呈时间剂量依赖性.

Objective To research the proliferation inhibition action of Pseudomonas aeruginosa（PA-MSHA）preparation on C6 glioma cells in vitro.Methods We cultured C6 glioma cells in vitro,determined the cell viability and cell attachment rate,drew the growth curve,calculated cell doubling time,and determined the optimum inoculation concentration of cells and the range of drug intervention.We also observed the morphorlogical changes of cells with inverted microscope,detected the proliferation inhibition rate of C6 glioma cells at different times after treated with different concentrations of PA-MSHA with MTT colorimetric assay,and calculated the cell survival rate and the half inhibitory concentration（IC50）.Results C6 glioma cells grew well in adherent way under conventional culture conditions.The cell viability was determinated before each passage,and the survival rate were always over 95%.The attachment rate was respectively 20%,68%,82%,85%,92% and 98% at different time points including 2 h,4 h,6 h,8 h,10 h and 12 h,in accordance with the requirements of this experiment.The growth curve of cells suggested the best concentration was 5× 103/well in 96 well-plates.The cells doubling proliferation time was 24 h to 72 h after the cells were inoculated in 96-well plates.We found obvious morphological changes of cells under inverted microscope.MTT assay results suggested that cell growth inhibition rate was time and dose-related with PA-MSHA.The IC50 values for 24h and 48h were respectively（5.80±1.79）× 10^8 cfu/mL and（3.90±2.14）× 10^8 cfu/ml.Conclusion Pseudomonas aeruginosa（PA-MSHA）preparation can inhibit the proliferation of C6 cells in vitro in a time and dose-dependent manner.