[1]李雪花,李文媛,李兴江,等.转录因子Krüpple样因子7对新生大鼠海马神经元缺血再灌注损伤的影响[J].新乡医学院学报,2019,36(3):213-217.[doi:10.7683/xxyxyxb.2019.03.003]
　LI Xue-hua,LI Wen-yuan,LI Xing-jiang,et al.Effect of Krüppel-like factor 7 on ischemia-reperfusion injury of hippocampal neurons in neonatal rats[J].Journal of Xinxiang Medical University,2019,36(3):213-217.[doi:10.7683/xxyxyxb.2019.03.003]
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转录因子Krüpple样因子7对新生大鼠海马神经元缺血再灌注损伤的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
36
期数:
2019年3
页码:
213-217
栏目:
基础研究
出版日期:
2019-03-05

文章信息/Info

Title:
Effect of Krüppel-like factor 7 on ischemia-reperfusion injury of hippocampal neurons in neonatal rats
作者:
李雪花1; 李文媛2; 3; 李兴江2; 3; 孙广大2; 3; 赵庭琪4; 王　莹2; 3
（1.阜新市中心医院神经内科，辽宁　阜新　123000；2.牡丹江医学院解剖学教研室，黑龙江　牡丹江　157011；3.牡丹江医学院神经组织工程研究所，黑龙江　牡丹江　157011；4.牡丹江医学院本科2016级，黑龙江　牡丹江　157011)
Author(s):
LI Xue-hua1; LI Wen-yuan2; 3; LI Xing-jiang2; 3; SUN Guang-da2; 3; ZHAO Ting-qi4; WANG Ying2; 3
(1.Department of Neurology,Fuxin Central Hospital,Fuxin 123000,Liaoning Province,China;2.Department of Anatomy,Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China;3.Institute of Neural tissue Engineering,Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China;4.Grade 2016,Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China)
关键词:
Krüpple样因子7; 缺血再灌注损伤; 海马神经元; 凋亡
Keywords:
Krüppel-like factor 7; ischemia-reperfusion injury; hippocampal neurons; apoptosis
分类号:
R338
DOI:
10.7683/xxyxyxb.2019.03.003
文献标志码:
A
摘要:
目的　探讨转录因子 Krüpple样因子7（KLF7）对新生大鼠海马神经元缺血再灌注损伤的影响。方法　取新生Sprauge-Dawley大鼠海马组织制备海马神经元单细胞悬液，按每孔1×106个细胞接种于聚赖氨酸包被6 孔细胞培养板，细胞贴壁继续培养7 d后分为正常组、氧糖剥夺/复氧（OGD/R)组和OGD/R+KLF7组。正常组培养7 d的原代大鼠海马神经元按每孔1×106个细胞于含胎牛血清的DMEM/F12培养基中进行培养，置于含体积分数5%CO2的培养箱中37 ℃下培养3 d；OGD/R组培养7 d的原代大鼠海马神经元用磷酸盐缓冲液洗3次，用移液器将原培养液置换为无糖平衡盐溶液，置于三气缺氧细胞培养箱（体积分数1%O2、94%N2、5%CO2）中37 ℃下培养4 h，然后应用 4.5 g·L-1 葡萄糖培养基代替无糖平衡盐溶液，置于含体积分数5%CO2的常温培养箱中培养24 h；OGD/R+ KLF7组培养成功的OGD/R模型细胞加入Lenti-KLF7慢病毒进行转染，置于含体积分数5%CO2的培养箱中37 ℃下孵育3 d。应用倒置相差显微镜观察各组海马神经元的生长状态，实时荧光定量聚合酶链反应检测各组细胞中KLF7、NGF、Caspase-3、Bcl-2和Bax mRNA表达，细胞计数试剂盒检测各组细胞活性，Hoechst33342/磺化丙啶双染法检测各组细胞凋亡情况。结果　3组细胞中KLF7、NGF、Caspase-3、Bax和Bcl-2 mRNA相对表达量比较差异均有统计学意义(F=237.702、24.031、476.505、112.900、89.467，P<0.05)；OGD/R组和OGD/R+KLF7组细胞中KLF7、NGF、Caspase-3、Bax和Bcl-2 mRNA相对表达量显著高于正常组（P<0.05）；OGD/R+KLF7组细胞中KLF7、NGF和Bcl-2 mRNA相对表达量显著高于OGD/R组（P<0.05），Caspase-3和Bax mRNA相对表达量显著低于OGD/R组（P<0.05）。正常组、OGD/R组和OGD/R+ KLF7组细胞活性分别为1.080±0.090、0.499±0.049和0.727±0.115，3组细胞活性比较差异有统计学意义（F=42.710，P<0.05）；OGD/R组和OGD/R+ KLF7组细胞活性显著低于正常组（P<0.05），OGD/R+KLF7组细胞活性显著高于OGD/R组（P<0.05）。正常组、OGD/R组、OGD/R+ KLF7组细胞凋亡率分别为（40.6±6.3）%、(76.7±4.3)%和(58.5±4.4)%，3组细胞凋亡率比较差异有统计学意义（F=36.822，P<0.05）；OGD/R组和OGD/R+ KLF7组细胞凋亡率显著高于正常组(P<0.05)，OGD/R+ KLF7组细胞凋亡率显著低于OGD/R组（P<0.05）。结论　KLF7对OGD/R诱导的海马神经元缺血再灌注细胞损伤具有保护作用，其机制可能与介导NGF表达，进而调控Caspase-3、Bcl-2和Bax细胞凋亡信号通路有关。
Abstract:
Objective　To investigate the effect of Krüppel-like factors 7(KLF7) on ischemia-reperfusion injury of hippocampal neurons in neonatal rats.Methods　The hippocampal tissues of newborn Sprauge-Dawley rats were isolated to prepare the single cell suspension of hippocampal neuron,the single cell suspension was inoculated on a polylysine-coated 6-well cell culture plate(1×106 cells per well),and adherent cells were cultured for 7 days.The cells were divided into normal group,oxygen glucose deprivation(OGD)/reoxygenation(R) group and OGD/R+KLF7 group.The primary hippocampal neurons cultured for 7 days in the normal group were cultured in DMEM/F12 medium containing fetal bovine serum (1×106 cells per well),and continue to culture in the incubator containing 5%CO2 for 3 days at 37 ℃.In the OGD/R group,the primary hippocampal neurons cultured for 7 days were washed three times with phosphate buffer solution,then the original culture solution was replaced by sugar-free equilibrium salt solution with a transposer;the cells were cultured in a three-gas hypoxic cell incubator (volume fraction 1%O2,94%N2,5%CO2) for 4 hours at 37 ℃;the sugar-free equilibrium salt solution was replaced by 4.5 g·L-1 dextrose culture-medium,then the cells were cultured in a room temperature incubator containing with 5%CO2 for 24 hours.The OGD/R cells in the OGD/R+KLF7 group were transfected with Lenti-KLF7 lentivirus and incubated in the incubator containing 5%CO2 for 3 days at 37 ℃.The growth of hippocampal neurons in each group was observed by inverted phase contrast microscopy.The expression of KLF7,NGF,Caspase-3,Bcl-2 and Bax mRNA was detected by real-time fluorescence quantitative polymerase chain reaction.The cell viability was measured by cell counting kit-8 method,and the apoptosis was detected by Hoechst 33342/propidium iodine double staining.Results　There was significant difference in the relative expressions of KLF7,NGF,Caspase-3,Bax and Bcl-2 mRNA in cells among the three groups (F=237.702,24.031,476.505,112.900,89.467;P<0.05).The relative expressions of KLF7,NGF,Caspase-3,Bax and Bcl-2 mRNA in cells in the OGD/R group and OGD/R+KLF7 group were significantly higher than those in the normal group (P<0.05).The relative expressions of KLF7,NGF and Bcl-2 mRNA in cells in the OGD/R+KLF7 group were significantly higher than those in the OGD/R (P<0.05).The relative expressions of Caspase-3 and Bax mRNA in cells in the OGD/R+KLF7 group were significantly lower than those in the OGD/R group (P<0.05).The cell activity in the normal group,OGD/R group and OGD/R+KLF7 group was 1.080±0.090,0.499±0.049 and 0.727±0.115,respectively;there was significant difference among the three groups (F=42.710,P<0.05).The cell viability in the OGD/R group and OGD/R+KLF7 group was significantly lower than that in the normal group (P<0.05),and the cell viability in the OGD/R+KLF7 group was significantly higher than that in the OGD/R group (P<0.05).The apoptotic rate in the normal group,OGD/R group and OGD/R+KLF7 group was （40.6±6.3）%,(76.7±4.3)% and (58.5±4.4)%,respectively;there was significant difference among the three groups (F=36.822,P<0.05).The apoptotic rate in the OGD/R group and OGD/R+KLF7 group was significantly higher than that in the normal group (P<0.05),and the apoptotic rate in the OGD/R+KLF7 group was significantly lower than that in the OGD/R group (P<0.05).Conclusion　KLF7 can protect hippocampal neurons from OGD/R-induced ischemia-reperfusion injury,and its mechanism may be related to mediating NGF expression and regulating the apoptosis signaling pathways of Caspase-3,Bcl-2 and Bax.

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更新日期/Last Update: 2019-03-05