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目的观察天然冰片对体外血肿瘤屏障(blood tumor barrier,BTB)模型通透性和丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPKs)信号转导通路相关激酶表达与激活的影响。方法大鼠C6脑胶质瘤细胞与人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)共培养建立BTB模型后,设空白组,冰片低、中、高剂量组(25、50、100μg/m L),每组7个时点(0、10、30、60、120、180、240 min),每个时点3个样本。空白组在冰片组给药同时更换空白培养基,冰片组给不同剂量冰片,给药后不同时间点收集细胞。辣根过氧化物酶(horseradish peroxidase,HRP)流量测定BTB通透性,Western blot检测细胞外信号调节激酶(extracellular signal regulated protein kinase,ERK),磷酸化ERK(phosphorylation extracellular signal regulated protein kinase,P-ERK),P38MAPK,磷酸化P38MAPK(phosphor-P38,P-P38MAPK),氨基末端激酶(c-Jun N-terminal kinase,JNK)和磷酸化JNK(phosphorylation c-Jun N-terminal kinase,P-JNK)的表达。结果与本组0 min比较,低、中、高剂量组各时间点通透率升高(P<0.01),P-ERK表达先升高,在30 min达极高值,逐渐恢复初始水平(P>0.05)。与空白组比较,低剂量组10~240 min HRP通透率升高(P<0.01),30、60 min PERK蛋白表达升高(P<0.05),低、中、高剂量组180 min P-JNK表达升高(P<0.05),240 min P-JNK表达降低(P<0.05)。与低剂量组比较,中剂量组10~180 min P-ERK表达升高(P<0.05),30~180 min通透率升高(P<0.05),180 min和240 min P-JNK表达降低(P<0.05)。与中剂量组比较,高剂量组10~180 min通透率升高(P<0.05),10~180 min P-ERK表达升高(P<0.05),180 min和240 min P-JNK表达降低(P<0.05)。结论冰片通过激活MAPKs信号通路的ERK磷酸化进而可逆性下调相关蛋白的表达,达到调节可逆性BTB开放的效果。
Objective To observe the effect of natural borneol on the permeability and the expression of kinases in mitogen-activated protein kinase (MAPKs) signal transduction pathway in the in vitro blood tumor barrier (BTB) model. Methods After BTB model was established by co-culture of C6 glioma cells and human umbilical vein endothelial cells (HUVECs), a blank group, a borneol low, medium and high dose group (25, 50 and 100 μg / m L) with 7 time points (0, 10, 30, 60, 120, 180, 240 min) and 3 samples at each time point. Blank group in the borneol group administration while replacing the blank medium, borneol group to different doses of borneol, after administration of cells collected at different time points. The BTB permeability of horseradish peroxidase (HRP) was measured by flow cytometry. The expressions of extracellular signal regulated protein kinase (ERK) and phosphorylated ERK (phosphorylated ERK) ERK, P38MAPK, phospho-P38MAPK, JNK and P-JNK, expression. Results Compared with 0 min in this group, the permeability increased at each time point (P <0.01), the expression of P-ERK increased first, reached the extremely high level at 30 min, and gradually recovered to the initial level P> 0.05). Compared with the blank group, the permeation rate of HRP increased from 10 to 240 min in low dose group (P <0.01), and increased in 30 and 60 min (P <0.05) JNK expression increased (P <0.05), and the expression of P-JNK decreased at 240 min (P <0.05). Compared with the low dose group, the expression of P-ERK increased from 10 to 180 min (P <0.05) and from 30 to 180 min (P <0.05), and decreased from 180 min to 240 min (P <0.05). Compared with the middle dose group, the P-ERK expression was increased at 10 ~ 180 min (P <0.05), and the P-ERK expression at 180 min and 240 min was decreased (P <0.05). CONCLUSION: Borneol can down-regulate the expression of related proteins by activating the phosphorylation of ERK in MAPKs signaling pathway, which can regulate the opening of reversible BTB.