刚地弓形虫VEG株速殖子感染宿主细胞最优接种比的曲线拟合分析

目的探讨不同浓度梯度的刚地弓形虫速殖子体外感染对侵蚀宿主细胞能力的影响及二者之间的最优接种比。方法依照耗材规格和建议上液量,将宿主Vero细胞依次铺入96孔、24孔、12孔、6孔细胞板和25T培养瓶后培养12 h;常规纯化和计数VEG株速殖子并倍比稀释出8个梯度,分别用来感染上述细胞,使首个感染量与宿主细胞铺入数相等;继续培养4 d后全量收集培养物,经DNA提取和Real-time PCR定量检测,以7种回归模型对所得速殖子数的均值进行拟合分析(n=3),选出最佳函数类型并求极值,以此明确弓形虫感染与宿主细胞间的接种关系。结果 Real-time PCR定量结果显示,VEG株速殖子最初感染量及所用耗材培养规格对弓形虫侵蚀宿主细胞的能力均有影响,并且四次多项式函数为弓形虫感染宿主细胞4 d时的最佳拟合曲线方程;经求导和求极值发现,同种耗材培养条件下,弓形虫VEG株速殖子感染数与宿主Vero细胞间存在最优接种比例关系。结论 96孔、24孔、12孔、6孔细胞板和25T培养瓶内弓形虫速殖子与宿主细胞间的接种比例依次为1/7.20、1/5.32、1/4.53、1/4.03和1/43.02。

Optimal inoculation ratios of Toxoplasma gondii VEG tachyzoites to their host cells using curve regression methods

To investigate the effect of different gradients of Toxoplasma gondii tachyzoites to the growth of their host cells in vitro and their inoculation ratios,Vero cells were spread into 96-,24-,12-,and 6-well plates or 25 T bottles according to the recommended volumes were cultivated for 12 hours at 37℃in the air containing 5%CO 2.The purified tachyzoites of T.gondii VEG strain were counted and diluted into eight gradients with the serial two-fold method,and were used to infect the cells above,guaranteeing the number of parasites in the first well or bottle were equal to the host cells.After cultivation for 4 days,the products were collected and used to extract DNA for the Real-time PCR detections.According to the obtained number of T.gondii(n=3),seven kinds of curves were matched based on the common regression models to determine the best type of function which belong to,and their extremum values were calculated to confirm the inoculation ratios between the parasite and its host cells.The results of Real-time PCR showed that the ability of T.gondii VEG strain for eroding its host cells were relevant to the incipient infection amount of this parasite and the specification of consumables.In addition,we found that the quartic polynomial function was the best regression equation for the 4 day-cultivated tachyzoites,and the optimal inoculation ratios between the parasite and its host cells,that is,1/7.20 for 96-,1/5.32 for 24-,1/4.53 for 12-,and 1/4.03 for 6-well plate or 1/43.02 for 25 T bottle,were finally obtained in this study via calculating the second derivative and extremum of these equations.