中华麻醉学杂志
中華痳醉學雜誌
중화마취학잡지
CHINESE JOURNAL OF ANESTHESIOLOGY
2015年
5期
550-554
,共5页
孙文波%张立民%康立娜%吴金光%吕建敏%黄冬冬%孙秀伟
孫文波%張立民%康立娜%吳金光%呂建敏%黃鼕鼕%孫秀偉
손문파%장립민%강립나%오금광%려건민%황동동%손수위
缺氧诱导因子1,α亚基%麻醉药,吸入%细胞低氧%神经元%细胞凋亡%预处理
缺氧誘導因子1,α亞基%痳醉藥,吸入%細胞低氧%神經元%細胞凋亡%預處理
결양유도인자1,α아기%마취약,흡입%세포저양%신경원%세포조망%예처리
Hypoxia-inducible factor 1,alpha subunit%Apoptosis regulatory proteins%Anesthetics,inhalation%Cell hypoxia%Neurons%Apoptosis%Preconditioning
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。
目的：評價缺氧誘導因子?1α（ HIF?1α）在七氟醚預處理減輕大鼠皮質神經元凋亡中的作用及其與Slit2∕Robo信號通路的關繫。方法新生SD大鼠，培養原代皮質神經元，以1×106∕ml的細胞密度接種于6孔闆（2 ml∕孔）或96孔闆（100μl∕孔），採用隨機數字錶法，分為4組（ n＝24）：正常對照組（ C組）、缺氧複氧組（ A∕R組）、七氟醚預處理組（ SP組）和HIF?1α抑製劑2?甲氧基雌二醇組（ H組）。採用氧糖缺失90 min 複氧複糖24 h的方法製備神經元缺氧複氧損傷模型；SP 組通入2．0％七氟醚2 h，隨後採用PBS洗滌3次，每次5 min，結束後立即進行缺氧複氧；H組加入5μmol∕L 2?甲氧基雌二醇孵育72 h時進行七氟醚預處理。採用膜聯蛋白Ⅴ∕碘化丙啶雙染流式細胞術檢測細胞凋亡情況，採用比色法檢測乳痠脫氫酶（ LDH ）漏齣水平，分彆採用免疫印跡法和實時定量熒光PCR法測定Slit2、Robo1和Robo4及其mRNA的錶達水平。結果與C組比較，A∕R組LDH漏齣率和細胞凋亡率升高，Slit2和Robo1及其mRNA的錶達上調（ P＜0．05），Robo4及其mRNA的錶達差異無統計學意義（ P＞0．05）；與A∕R組比較，SP組和H組LDH漏齣率和細胞凋亡率降低，SP組Slit2和Robo1及其mRNA的錶達上調（ P＜0．05），Robo4及其mRNA的錶達差異無統計學意義（ P＞0．05）；與SP組比較，H組LDH漏齣率和細胞凋亡率升高，Slit2和Robo1及其mRNA的錶達下調（ P＜0．05）。結論 HIF?1α介導瞭七氟醚預處理減輕大鼠皮質神經元凋亡的過程，其機製與激活Slit2∕Robo1信號通路有關，而與Slit2∕Robo4信號通路無關。
목적：평개결양유도인자?1α（ HIF?1α）재칠불미예처리감경대서피질신경원조망중적작용급기여Slit2∕Robo신호통로적관계。방법신생SD대서，배양원대피질신경원，이1×106∕ml적세포밀도접충우6공판（2 ml∕공）혹96공판（100μl∕공），채용수궤수자표법，분위4조（ n＝24）：정상대조조（ C조）、결양복양조（ A∕R조）、칠불미예처리조（ SP조）화HIF?1α억제제2?갑양기자이순조（ H조）。채용양당결실90 min 복양복당24 h적방법제비신경원결양복양손상모형；SP 조통입2．0％칠불미2 h，수후채용PBS세조3차，매차5 min，결속후립즉진행결양복양；H조가입5μmol∕L 2?갑양기자이순부육72 h시진행칠불미예처리。채용막련단백Ⅴ∕전화병정쌍염류식세포술검측세포조망정황，채용비색법검측유산탈경매（ LDH ）루출수평，분별채용면역인적법화실시정량형광PCR법측정Slit2、Robo1화Robo4급기mRNA적표체수평。결과여C조비교，A∕R조LDH루출솔화세포조망솔승고，Slit2화Robo1급기mRNA적표체상조（ P＜0．05），Robo4급기mRNA적표체차이무통계학의의（ P＞0．05）；여A∕R조비교，SP조화H조LDH루출솔화세포조망솔강저，SP조Slit2화Robo1급기mRNA적표체상조（ P＜0．05），Robo4급기mRNA적표체차이무통계학의의（ P＞0．05）；여SP조비교，H조LDH루출솔화세포조망솔승고，Slit2화Robo1급기mRNA적표체하조（ P＜0．05）。결론 HIF?1α개도료칠불미예처리감경대서피질신경원조망적과정，기궤제여격활Slit2∕Robo1신호통로유관，이여Slit2∕Robo4신호통로무관。
Objective 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.
