乐复能在小鼠L929细胞中的抗病毒活性测定

发布时间：2018-06-09 21:08

本文选题：干扰素 + 乐复能　；参考：《中南大学》2014年硕士论文

【摘要】：目的：检验乐复能是否具有种间交叉生物活性；测定乐复能在VSV感染的小鼠成纤维细胞系L929中的抗病毒活性；探讨其进一步进行临床前的人类疾病小鼠模型研究的可行性。方法：培养小鼠成纤维细胞系L929,将其制备成细胞悬液,接种于96孔板中置温箱中过夜培养。以不同的浓度起始,连续对倍稀释乐复能和重组小鼠干扰素α1(MuIFN-α1),并依次加入96孔板的培养细胞中,设置正常细胞对照孔,继续培养24小时。弃培养液后,在乐复能组、MuIFN-α1组添加含有VSV病毒的完全培养基(MOI:0.1),并设置正常细胞对照组和病毒对照组,连续培养36小时。吸出细胞培养液,0.85%NaCl漂洗后拍干,添加细胞染色液室温下染色2小时。自来水漂净染色液,室温下完全干燥后,每孔加入2-甲氧基乙醇(2-methoxyethanol),静置45分钟脱色,酶标仪检测各细胞孔的吸光值。实验重复三次,结果取平均值,并计算保护率。以药物浓度的对数值为横轴,保护率为纵轴分别绘制乐复能和MuIFN-α1在L929-VSV系统中的回归分析图,并通过线性回归方法计算两种药物的50%最大效应浓度(EC50)。结果：①乐复能组和MuIFN-α1组细胞的OD550值均明显升高。计算两组药物对小鼠L929细胞的保护率,均随着药物浓度的增高而增大,两者呈正相关(乐复能：r=0.972, p0.001; MuIFN-α=0.950,p0.001)。通过药物浓度和细胞保护率相关曲线发现,相同浓度下,MuIFN-α1在小鼠L929细胞中的对细胞的保护率较乐复能高,即抗病毒活性更明显。②通过线性回归的方法计算出乐复能和MuIFN-α1的EC50值分别为：170.752pg和38.810pg。由此推算乐复能在L929-VSV系统中抗病毒活性为6.32×106IU/mg。证实乐复能在VSV病毒感染的小鼠L929细胞中具有良好的抗病毒活性,提示其具有较强的种间交叉作用。结论：①乐复能和MuIFN-α1均对感染VSV病毒的小鼠L929细胞具有保护作用,且保护率与药物浓度呈正相关；②相同药物浓度下,MuIFN-α1对小鼠L929细胞的保护率较乐复能高,MuIFN-α1的抗病毒活性高于乐复能；③乐复能在VSV病毒感染的小鼠L929细胞中具有较强的种间交叉作用,可用于乐复能小鼠动物实验。
[Abstract]:Objective: to test whether lofenin has interspecific cross biological activity and to determine its antiviral activity in VSV infected mouse fibroblast cell line L929. Methods: the mouse fibroblast cell line L929 was cultured and the suspension was prepared and cultured overnight in a 96-well plate. At the beginning of different concentrations, the cells were continuously diluted with Lefergic and recombinant mouse interferon 伪 1, MuIFN- 伪 1, and added in turn to 96 well plate culture cells. The normal control cells were set up and cultured for 24 hours. After the culture medium was abandoned, the complete medium containing VSV virus was added to the MuIFN- 伪 1 group, and the normal cell control group and the virus control group were set up for 36 hours. The cell culture medium (0.85 NaCl) was bleached and dried, and the cells were stained at room temperature for 2 hours. After completely drying at room temperature, 2-methoxyethanolol was added to each pore, and the dye was decolorized for 45 minutes. The absorptivity of each cell was measured by enzyme labeling instrument. The experiment was repeated three times, the results were averaged and the protection rate was calculated. The logarithmic values of drug concentration were taken as horizontal axis and the protection rate was used as longitudinal axis to draw the regression analysis of L929-VSV system for L929-VSV system with L929-VSV system and MuIFN- 伪 1, respectively. The 50% maximum effective concentration (MEC) of the two drugs was calculated by linear regression method. Results the OD550 values of cells in the two groups were significantly higher than those in the control group and MuIFN- 伪 1 group. The protective rate of the two drugs on L929 cells increased with the increase of drug concentration, and there was a positive correlation between the two groups (Le Fu Neng 0.972, p 0.001; MuIFN- 伪 0.950 p 0.001). It was found that the protective rate of MuIFN- 伪 1 in mouse L929 cells at the same concentration was higher than that in L929 cells. The EC50 values of Lefeng and MuIFN- 伪 1 were calculated by linear regression method, and the EC50 values were: 1: 170.752pg and 38.810pg. respectively. The antiviral activity of L929-VSV was estimated to be 6.32 脳 10 ~ (6) IU / mg. It was confirmed that L929 cells infected with VSV virus had good antiviral activity, which indicated that L929 cells infected with VSV virus had strong interspecific cross effect. Conclusion both of them have protective effect on L929 cells infected with VSV virus. The protective rate of MuIFN- 伪 1 on L929 cells was higher than that of L929 cells at the same drug concentration, and the antiviral activity of MuIFN- 伪 1 was higher than that of L929 cells. 3 lofenac has strong interspecific cross effect in L929 cells infected with VSV virus, and it can be used in the animal experiment of L929 cells infected with VSV virus.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】