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目的:血管的新生与髓母细胞瘤的生长、侵袭和转移密切相关,本实验旨在观察姜黄素对髓母细胞瘤Daoy细胞的生长及其血管生长因子的影响,并探讨可能的机制。方法:体外培养髓母细胞瘤Daoy细胞,然后经不同浓度(5、10、20、40μmol/L)的姜黄素作用不同时间(24、48、72 h)后,通过MTT法检测姜黄素对Daoy细胞生长增殖的影响;ELISA法检测上清液中血管内皮生长因子(vascular endothelial growth factor,VEGF)的生成水平,Western blot检测Daoy细胞中Wnt/β-catenin通路中重要分子β-catenin以及VEGF蛋白水平的表达。结果:MTT结果示空白对照组、DMSO组、5μmol/L组、10μmol/L组、20μmol/L组和40μmol/L组作用24 h后,细胞的抑制率分别为(4.2±0.3)%、(4.3±0.5)%、(6.1±0.3)%、(6.9±0.7)%、(30.5±1.7)%和(40.9±2.4)%,差异具有统计学意义(F_(浓度)=151.997,P=0.000);当20、40μmol/L的姜黄素作用48 h和72 h后,Daoy细胞的增殖抑制率分别为(41.3±2.0)%、(51.2±2.9)%、(48.4±1.6)%和(60.9±3.2)%,相较于作用24 h,差异也具有统计学意义(F_(时间)=281.531,P=0.000;F_(浓度×时间)=269.728,P=0.000)。ELISA结果显示:空白对照组、DMSO对照组、5μmol/L组、10μmol/L组、20μmol/L组、40μmol/L组细胞上清液中VEGF的含量分别为(237.5±2.5)、(248.4±3.0)、(235.6±7.1)、(230.8±3.8)、(198.2±9.9)、(150.5±3.6)pg/m L,差异具有统计学意义(F_(浓度)=30.921,P=0.000)。当20μmol/L和40μmol/L的姜黄素作用48 h和72 h后,Daoy细胞上清液中生成的VEGF含量分别为(170.3±2.7)、(124.6±3.1)、(140.1±13.9)和(111.5±11.6)pg/m L,与作用24 h比较,差异也具有统计学意义(F_(时间)=69.234,P=0.000;F_(浓度×时间)=185.173,P=0.000)。Western blot结果显示,空白对照组、DMSO组、5μmol/L组、10μmol/L组、20μmol/L组和40μmol/L组Daoy细胞内β-catenin蛋白水平表达相对吸光度(absorbance,A)值分别为(0.654±0.021)、(0.873±0.034)、(0.869±0.018)、(0.857±0.024)、(0.472±0.035)和(0.351±0.019)(F_(浓度×时间)=27.813,P=0.000);VEGF蛋白水平的相对A值为(0.781±0.027)、(0.801±0.025)、(0.792±0.034)、(0.750±0.024)、(0.324±0.015)和(0.201±0.012)(F_(浓度)=74.826,P=0.000)。结果还显示,当姜黄素浓度为20μmol/L时,随着药物作用时间的延长,Daoy细胞内β-catenin和VEGF蛋白表达的相对A值明显降低,差异具有统计学意义,呈时间依赖性(F=19.156,P=0.001;F=100.282,P=0.000)。结论:姜黄素可通过下调Wnt/β-catenin通路及下游靶基因VEGF的表达进而抑制Daoy细胞增殖,这或将为姜黄素治疗髓母细胞瘤提供新的理论依据
OBJECTIVE: The neovascularization is closely related to the growth, invasion and metastasis of medulloblastoma. The purpose of this study was to observe the effect of curcumin on the growth and the angiogenic growth factor of medulloblastoma Daoy cells and to explore the possible mechanism. Methods: Daoy cells of medulloblastoma were cultured in vitro. Curcumin at different concentrations (5, 10, 20 and 40 μmol / L) for 24 h, 48 h and 72 h, respectively, Cell growth and proliferation. The level of VEGF in the supernatant was detected by ELISA. The expression of β-catenin and VEGF protein in Wnt / β-catenin pathway in Daoy cells was detected by Western blot Horizontal expression. Results: MTT results showed that the cell inhibitory rates were (4.2 ± 0.3)% and (4.2 ± 0.3)% respectively in the blank control group, the DMSO group, the 5μmol / L group, the 10μmol / L group, the 20μmol / L group and the 40μmol / 4.3 ± 0.5%, 6.1 ± 0.3%, 6.9 ± 0.7%, 30.5 ± 1.7% and 40.9 ± 2.4%, respectively. The difference was statistically significant (F_ (concentration) = 151.997, P = 0.000 ). The inhibitory rates of proliferation of Daoy cells were (41.3 ± 2.0)%, (51.2 ± 2.9)%, (48.4 ± 1.6)% and (60.9)% respectively after 48 h and 72 h treatment with 20 and 40 μmol / L curcumin ± 3.2%). The difference was also statistically significant (F_ (time) = 281.531, P = 0.000; F_ (concentration × time) = 269.728, P = 0.000) compared with 24 hours. The results of ELISA showed that the contents of VEGF in the supernatant of the control group, DMSO control group, 5μmol / L group, 10μmol / L group, 20μmol / L group and 40μmol / L group were (237.5 ± 2.5) (P <0.05), (235.6 ± 7.1), (230.8 ± 3.8), (198.2 ± 9.9) and (150.5 ± 3.6) pg / m L, respectively. The difference was statistically significant (F_ (concentration) = 30.921, P = 0.000). The levels of VEGF in Daoy cell supernatant were (170.3 ± 2.7), (124.6 ± 3.1), (140.1 ± 13.9) and (140.1 ± 13.9), respectively, when treated with 20μmol / L and 40μmol / L curcumin for 48 and 72 h 111.5 ± 11.6 pg / m L). The difference was statistically significant (F_ (time) = 69.234, P = 0.000; F_ (concentration × time) = 185.173, P = 0.000). Western blot results showed that the relative absorbance (A) values of β-catenin protein expression in Daoy cells in the blank control group, DMSO group, 5μmol / L group, 10μmol / L group, 20μmol / L group and 40μmol / L group were (0.654 ± 0.021), (0.873 ± 0.034), (0.869 ± 0.018), (0.857 ± 0.024), (0.472 ± 0.035) and 0.351 ± 0.019 (F_ (concentration × time) = 27.813, P = 0.000) The relative A values of VEGF protein level were (0.781 ± 0.027), (0.801 ± 0.025), (0.792 ± 0.034), (0.750 ± 0.024), (0.324 ± 0.015) and (0.201 ± 0.012) , P = 0.000). The results also showed that when curcumin concentration was 20μmol / L, the relative A value ofβ-catenin and VEGF protein expression in Daoy cells was significantly decreased with the prolongation of drug effect, the difference was statistically significant and time-dependent F = 19.156, P = 0.001; F = 100.282, P = 0.000). Conclusion: Curcumin can inhibit the proliferation of Daoy cells by down-regulating the expression of Wnt / β-catenin pathway and downstream target gene VEGF, which may provide a new theoretical basis for curcumin treatment of medulloblastoma