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  

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 赵庭琪 王莹[2,3] LI Xue-hua;LI Wen-yuan;LI Xing-jiang;SUN Guang-da;ZHAO Ting-qi;WANG Ying(Department of Neurology,Fuxin Central Hospital,Fuxin 123000,Liaoning Province,China;Department of Anatomy,Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China;Institute of Neural tissue Engineering,Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China;Grade 2016,Mudanjiang Medical University,Mudanjiang 157011,Heilongjiang Province,China)

机构地区：[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

出　　处：《新乡医学院学报》2019年第3期213-217,223,共6页Journal of Xinxiang Medical University

基　　金：国家自然科学基金资助项目(编号:81870977);黑龙江省自然科学基金资助项目(编号:LC2017040);黑龙江省卫生计生委立项科研课题(编号:2018-379);黑龙江省大学生创新创业训练计划项目(编号:201810229030);牡丹江医学院大学生科研立项重点项目(编号:2018-28)

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1×10~6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1×10~6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%CO-_2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%O_2、94%N_2、5%CO_2)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-2mRNA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�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

关 键 词：Krüpple样因子7 缺血再灌注损伤 海马神经元 凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]