摘要目的：评价缺氧诱导因子?1α（ HIF?1α）在七氟醚预处理减轻大鼠皮质神经元凋亡中的作用及其与Slit2∕Robo信号通路的关系。方法新生SD大鼠，培养原代皮质神经元，以1×106∕ml的细胞密度接种于6孔板（2 ml∕孔）或96孔板（100μl∕孔），采用随机数字表法，分为4组（ n＝24）：正常对照组（ C组）、缺氧复氧组（ A∕R组）、七氟醚预处理组（ SP组）和HIF?1α抑制剂2?甲氧基雌二醇组（ H组）。采用氧糖缺失90 min 复氧复糖24 h的方法制备神经元缺氧复氧损伤模型；SP 组通入2．0％七氟醚2 h，随后采用PBS洗涤3次，每次5 min，结束后立即进行缺氧复氧；H组加入5μmol∕L 2?甲氧基雌二醇孵育72 h时进行七氟醚预处理。采用膜联蛋白Ⅴ∕碘化丙啶双染流式细胞术检测细胞凋亡情况，采用比色法检测乳酸脱氢酶（ LDH ）漏出水平，分别采用免疫印迹法和实时定量荧光PCR法测定Slit2、Robo1和Robo4及其mRNA的表达水平。结果与C组比较，A∕R组LDH漏出率和细胞凋亡率升高，Slit2和Robo1及其mRNA的表达上调（ P＜0．05），Robo4及其mRNA的表达差异无统计学意义（ P＞0．05）；与A∕R组比较，SP组和H组LDH漏出率和细胞凋亡率降低，SP组Slit2和Robo1及其mRNA的表达上调（ P＜0．05），Robo4及其mRNA的表达差异无统计学意义（ P＞0．05）；与SP组比较，H组LDH漏出率和细胞凋亡率升高，Slit2和Robo1及其mRNA的表达下调（ P＜0．05）。结论 HIF?1α介导了七氟醚预处理减轻大鼠皮质神经元凋亡的过程，其机制与激活Slit2∕Robo1信号通路有关，而与Slit2∕Robo4信号通路无关。

abstractsObjective To evaluate the role of hypoxia inducible factor?1α ( HIF?1α) in reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning and the relationship with Slit2∕Robo signaling pathway. Methods Primary cortical neurons obtained from neonatal Sprague?Dawley rats were seeded in 6?well (2 ml∕well) or 96?well plates (100 μl∕well) at a density of 1×106∕ml, and randomly divided into 4 groups ( n=24 each ) using a random number table: control group ( C group ) , anoxia?reoxygenation ( A∕R ) group, sevoflurane preconditioning group ( SP group ) and HIF?1α inhibitor 2?methoxyestradiol group ( H group ) . The neurons were subjected to O2?glucose deprivation for 90 min followed by restoration of O2?glucose supply for 24 h. In group SP, the neurons were exposed to 2%sevoflurane for 2 h followed by 5 min washout with phosphate buffered saline for 3 times, and then sevoflurane preconditioning was performed immediately. In group H, sevoflurane preconditioning was performed with 5μmol∕L 2?methoxyestradiol at 72 h of incubation. The apoptosis in neurons was assessed using AnnexinⅤ?FITC∕PI assay, and apoptosis rate ( AR) was calculated. The amount of lactic dehydrogenase ( LDH) released was measured using colorimetric method. The expression of Slit2, Robo1 and Robo4 mRNA and protein was detected by fluorescent quantitative real?time polymerase chain reaction or Western blot. Results Compared with group C, the amount of LDH released and AR were significantly increased, Silt2 and Robo1 mRNA and protein expression was up?regulated, and no significant change was found in Robo4 mRNA and protein expression in A∕R group. Compared with group A∕R, the amount of LDH released and AR were significantly decreased in SP and H groups, and Silt2 and Robo1 mRNA and protein expression was up?regulated, and no significant change was found in Robo4 mRNA and protein expression in SP group. Compared with group SP, the amount of LDH released and AR were significantly increased, and Silt2 and Robo1 mRNA and protein expression was down?regulated in H group. Conclusion HIF?1α mediates reduction of apoptosis in cortical neurons of rats by sevoflurane preconditioning, and the mechanism is associated with Slit2∕Robo1 signaling pathway, but not with Slit2∕Robo4 signaling pathway.