重组人精氨酸酶对人脑胶质瘤细胞U87的杀伤作用研究

Cytotoxicity effect of recombinant human arginase on human glioma cells U87 in vitro

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 explore the cytotoxicity effect of recombinant human arginase on human glioma cells in vitro. Methods The human glioma cells U87 in the logarithmic phase of growth were randomly divided into the control group and test groups in different concentrations of recombinant human arginase which changed from 0. 2 × 10^（-3）to 0. 25U·mL^（-1）after multiple proportion dilution. Cell viability was detected by MTT assay. After treatment of recombinant human arginase,the morphological changes of the U87 cells were observed under inverted microscope. The protein expression of argininosuccinate synthetase（ ASS）,key enzymes of synthesizing arginine from citrulline in the urea cycle,were examined by Western blotting assay in U87 cells. Then,U87 cells were treated with the combination of recombinant human arginase and exogenous L- arginine,recombinant human arginase alone and exogenous L- arginine alone,respectively,while MTT assay was used to detected cell viability. Results After treatment with recombinant human arginase,the cytotoxicity was remarkably induced in U87 cells in a dose- dependent way,and the cell viability in0. 2 × 10^（-2）U·mL^（-1）test group was 75. 12%,with the 0. 8 × 10^（-2）U·mL^（-1）test group was 52. 28%,the 1. 6 × 10^（-2）U·mL^（-1）test group was 44. 09%,the 0. 25 U·mL^（-1）test group was 38. 14%. The differences in each two groups were statistically significant（ P〈0. 05）. By inverted microscope,we observed the density of U87 cells were significantly decreased after treatment with 0. 2 × 10^（-2）,1. 6 × 10^（-2）and 1. 25 × 10- 1U·mL^（-1）of recombinant human arginase for 72 h,respectively,and cells became deformed,round,shrinkage and even lysis. Western blotting analysis showed that U87 cells were ASS deficient. Furthermore,when U87 cells were supplemented with exogenous L- arginine,the cytotoxicity induced by recombinant human arginase could be partially rescued（ cell viability： 60. 53%）,compared with using recombinant human arginase alone（ cell viability： 47. 48%）,and the difference between groups was statistically significant（ P〈0. 01）. Conclusion Recombinant human arginase could induce significant cytotoxicity which was involved in arginine depletion in ASS- negative U87 cells.