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目的:探讨槐定碱对神经胶质瘤U87细胞增殖和侵袭的抑制作用以及U87细胞DNA拓扑异构酶I(DNA TOP I)、EGFR-酪氨酸激酶(EGFR-TK)、基质金属蛋白酶2(MMP2)和氨肽酶N(APN)活性的影响。方法:将不同浓度槐定碱加入到神经胶质瘤U87细胞株中,利用MTT法测定槐定碱对U87细胞和人脑正常星型胶质HEB细胞生长的抑制作用,酶标仪法检测槐定碱对细胞凋亡蛋白caspase-3活性,采用Transwell小室法分析U87细胞的侵袭能力;采用非放射性NF-κB EMSA试剂盒测定U87细胞中NF-κB表达。结果:随着槐定碱浓度的增加(5、10、25、50、100μmol/L),神经胶质瘤U87细胞生长抑制率不断的增加,但HEB细胞的生长并未受到明显的抑制[(11.23±1.18)%vs(2.43±0.29)%、(22.48±3.21)%vs(3.65±0.42)%、(43.21±4.09)%vs(4.03±0.55)%、(57.31±5.09)%vs(5.21±0.43)%、(77.98±6.98)%vs(7.22±0.78)%,均P<0.05];与空白组比较,槐定碱存在下的U87细胞侵袭能力明显降低[(87.43±7.33)%、(65.12±6.16)%、(50.63±4.56)%、(35.32±4.04)%、(23.46±2.32)%vs(120.32±9.32),均P<0.05]。槐定碱对DNA TOP I、EGFR-TK、APN和MMP-2的半抑制率IC_(50)分别为(22.43±2.21)、(31.25±3.09)、(6.32±0.32)和(8.23±0.63)μmol/L,但U87细胞中凋亡蛋白caspase-3活性呈增强趋势;槐定碱能够下调NF-κB信号的表达。结论:低毒性的槐定碱可能通过降低DNA TOP I、EGFR-TK、APN和MMP-2活性,并下调NF-κB信号通路和激活凋亡caspase-3酶联反应的方式,对神经胶质瘤U87细胞的侵袭、增殖和信号通路产生抑制作用。
OBJECTIVE: To investigate the inhibitory effect of sophoridine on proliferation and invasion of glioma U87 cells as well as DNA TOP I, EGFR-TK, (MMP2) and aminopeptidase N (APN) activity. Methods: Different concentrations of sophoridine were added to glioma U87 cell line. MTT assay was used to determine the inhibitory effect of sophoridine on the growth of U87 cells and normal human glia HEB cells. The alkaline phosphatase activity of caspase-3 was detected by Transwell chamber assay. The expression of NF-κB in U87 cells was determined by using non-radioactive NF-κB EMSA kit. Results: With the increase of sophoridine concentration (5, 10, 25, 50 and 100 μmol / L), the growth inhibition rate of glioma U87 cells increased continuously, but the growth of HEB cells was not significantly inhibited [ 11.23 ± 1.18)% vs (2.43 ± 0.29)%, (22.48 ± 3.21)% vs (3.65 ± 0.42)%, (43.21 ± 4.09)% vs (4.03 ± 0.55)%, (57.31 ± 5.09)% vs (P <0.05). Compared with the blank group, the invasion ability of U87 cells in the presence of sophoridine was significantly reduced (87.43 ± 7.33)%, (77.98 ± 6.98)% vs (7.22 ± 0.78)%, (65.12 ± 6.16)%, (50.63 ± 4.56)%, (35.32 ± 4.04)%, (23.46 ± 2.32)% vs (120.32 ± 9.32), all P <0.05]. The IC50 values of sophoridine on DNA TOP I, EGFR-TK, APN and MMP-2 were (22.43 ± 2.21), (31.25 ± 3.09), (6.32 ± 0.32) and (8.23 ± 0.63) μmol / L, but the activity of caspase-3 in U87 cells tended to increase. Sophoridine could down-regulate the expression of NF-κB. Conclusion: Sophoridine with low toxicity may inhibit glioma via down-regulating the activity of TOP I, EGFR-TK, APN and MMP-2 and down-regulating NF-κB signaling pathway and activating caspase-3, The invasion, proliferation and signal pathways of U87 cells have inhibitory effects.