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目的研究重组人精氨酸酶对人脑胶质瘤细胞U87的杀伤作用。方法培养人脑胶质瘤细胞U87,将对数生长期人脑胶质瘤细胞U87随机分为12组:对照组仅加等量的培养液,其余11组实验组以倍比稀释的方式分别给予质量浓度为0.2×10~(-3),0.5×10~(-3),0.1×10~(-2),0.2×10~(-2),0.4×10~(-2),0.8×10~(-2),1.6×10~(-2),3.2×10~(-2),6.3×10~(-2),1.25×10-1,0.25 U·mL~(-1)的重组人精氨酸酶,用甲基噻唑蓝四氮唑盐(MTT)法检测各组人脑胶质瘤细胞U87细胞活力的变化;倒置显微镜观察U87细胞经重组人精氨酸酶作用后的形态学变化;Western blotting法检测精氨酸琥珀酸合成酶(ASS)的表达。重组人精氨酸酶作用于人脑胶质瘤细胞U87时,分成补充L-精氨酸组和不补充L-精氨酸组,用MTT法检测人脑胶质瘤细胞U87细胞活力的变化。结果不同质量浓度的重组人精氨酸酶作用U87细胞72 h后,U87细胞的活力均降低且呈明显的剂量依赖性,0.2×10~(-2),0.8×10~(-2),1.6×10~(-2),0.25 U·mL~(-1)实验组的细胞活力分别为75.12%,52.28%,44.09%,38.14%,差异有统计学意义(P<0.05)。通过倒置显微镜观察,重组人精氨酸酶作用U87细胞72 h后,0.2×10~(-2),1.6×10~(-2),1.25×10-1U·mL~(-1)实验组的细胞密度随着药物浓度升高而显著降低,镜下可见U87细胞形态发生明显的变形,皱缩,坏死。Western blotting实验结果表明,U87细胞的ASS蛋白表达缺陷。当重组人精氨酸酶作用于U87细胞同时,补充部分L-精氨酸时的细胞活力为60.53%,未补充L-精氨酸组的细胞活力为47.48%,差异有统计学意义(P<0.01)。结论由于人脑胶质瘤细胞U87细胞合成精氨酸的关键酶ASS蛋白表达缺陷,重组人精氨酸酶通过耗竭外源性精氨酸,对人脑胶质瘤细胞U87产生显著的杀伤效应。
Objective To study the killing effect of recombinant human arginase on human glioma U87 cells. Methods Human glioma U87 cells were cultured and the logarithmic growth phase human glioma cells U87 were randomly divided into 12 groups: the control group only added equal amount of culture solution, and the other 11 groups were diluted by multiples The cells were treated with 0.2 × 10 ~ (-3), 0.5 × 10 ~ (-3), 0.1 × 10 ~ (-2), 0.2 × 10 ~ (-2), 0.4 × 10 ~ (-2) × 10 -2, 1.6 × 10 -2, 3.2 × 10 -2, 6.3 × 10 -2, 1.25 × 10 -1 and 0.25 U · mL -1, respectively. Of recombinant human arginase, methylthiazolyl tetrazolium salt (MTT) method was used to detect the activity of U87 cells in each group of human glioma cells; inverted microscope observation of U87 cells after recombinant human arginase Morphological changes were observed. The expression of arginine succinate synthase (ASS) was detected by Western blotting. Recombinant human arginase acts on human glioma U87 cells and is divided into L-arginine group and L-arginine group. MTT assay was used to detect the activity of U87 cells in human glioma cells . Results U87 cells treated with recombinant human arginase of different concentrations for 72 h reduced the viability of U87 cells in a dose - dependent manner with a dose - dependent manner. U87 cells exhibited a dose - dependent manner with 0.2 × 10 -2, 0.8 × 10 -2, The cell viability in the experimental groups of 1.6 × 10 -2 and 0.25 U · mL -1 were 75.12%, 52.28%, 44.09% and 38.14%, respectively. The difference was statistically significant (P <0.05). Observed by inverted microscope, U87 cells were treated with recombinant human arginase for 72 h, then treated with 0.2 × 10 ~ (-2), 1.6 × 10 ~ (-2), 1.25 × 10-1 U · mL ~ (-1) Of the cell density with the drug concentration was significantly lower, microscopic morphology of U87 cells showed significant deformation, shrinkage, necrosis. Western blotting results showed that the expression of ASS protein in U87 cells was deficient. When recombinant human arginase was applied to U87 cells, the cell viability was 60.53% when supplemented with L-arginine and 47.48% with no L-arginine supplementation (P <0.05). The difference was statistically significant (P <0.01). Conclusion Recombinant human arginase exerts a significant killing effect on U87 human glioma cells by depleting exogenous arginine due to the defective expression of ASS protein, a key enzyme in the synthesis of arginine, in human glioma U87 cells .