腺苷激酶基因修饰的间充质干细胞移植对氯化锂-匹罗卡品颞叶癫痫模型癫痫发作和海马神经元的保护性作用  

Protective effects of adenosine kinase gene-modified mesenchymal stem cell transplantation on the seizures and hippocampus neuron damage of LiCl-pilocarpine induced temporal lobe epilepsy

作　　者：万学焱 王嫚[2] 周青 Wan Xueyan;Wang Man;Zhou Qing(Department of Neurosurgery,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Department of Neurology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院神经外科,武汉430030 [2]华中科技大学同济医学院附属同济医院神经内科,武汉430030

出　　处：《中华实验外科杂志》2024年第6期1233-1236,共4页Chinese Journal of Experimental Surgery

基　　金：湖北省自然科学基金青年项目(2022CFB714)。

摘　　要：目的探讨抑制腺苷激酶表达的间充质干细胞(ADK--MSCs)脑内移植对氯化锂-匹罗卡品模型癫痫发作和海马神经元损伤的影响。方法利用包含抑制腺苷激酶表达的慢病毒载体转染小鼠骨髓间充质干细胞,制作抑制腺苷激酶表达的间充质干细胞(ADK--MSCs),检测其腺苷分泌量,将其移植入准备进行氯化锂-匹罗卡品造模的小鼠海马区。实验分组对照组,氯化锂-匹罗卡品造模组TLE组,和ADK--MSCs移植后氯化锂-匹罗卡品造模组ADK--MSCs组。造模28 d后,使用视频脑电图监测造模后自发性癫痫发作。使用尼氏染色和活性半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3免疫组织化学评估海马神经元的损伤和凋亡。造模45 d后,观察移植ADK--MSCs细胞的存活情况。两组间比较采用t检验。结果1×10^(5)个ADK--MSCs细胞上清中的腺苷浓度为12.1 ng/ml。ADK--MSCs组自发性癫痫发作频率为(8.20±2.38)次/周,明显低于TLE组[(16.40±3.20)次/周,t=4.584,P<0.01]。尼氏染色显示400倍放大视野中ADK--MSCs组CA1区存活神经元为(91.00±6.51)个,明显高于TLE组[(71.80±5.06)个,t=5.199,P<0.001];CA3区存活神经元ADK--MSCs组[(64.20±7.98)个]明显高于TLE组[(45.75±7.63)个,t=3.511,P<0.01]。活性Caspase-3免疫染色显示ADK--MSCs组海马锥体神经元阳性细胞明显低于TLE组,蛋白质印迹法(Western blot)分析证实ADK--MSCs组活性Caspase-3蛋白表达明显低于TLE组(n=3,t=4.302,P<0.05)。造模45 d后,通过荧光显微镜可观察到海马区存活的绿色荧光蛋白(GFP)阳性绿色荧光ADK--MSCs细胞。结论抑制ADK表达的间充质干细胞脑内移植在氯化锂-匹罗卡品颞叶癫痫模型中具有抗癫痫发作和减轻神经损伤的作用。Objective To explore the effects of adenosine kinase(ADK)-inhibited mouse bone marrow mesenchymal stem cells(MSCs)intracerebral transplantation on spontaneous recurrent seizures and hippocampus neuron in LiCl-pilocarpine epilepsy model.Methods Lentiviral vectors containing miRNA inhibiting ADK were used to produce ADK knockdown bone MSCs(ADK--MSCs).The adenosine secretion amount was analyzed.The ADK--MSCs were transplanted into the hippocampus of LiCl-pilocarpine mouse model.The mice in control group were injected by saline,those in TLE group were injected by pilocarpine,and those in ADK--MSCs group were transplanted by ADK--MSCs before pilocarpine kindling.At 28 d after kindling,video monitoring EEG was used to observe seizures.Nissl staining was used to assess hippocampus survival neurons.Immunohistochemistry and Western blotting were used to evaluate apoptosis in hippocampus.The survival ADK--MSCs were observed by fluorescence microscope at 45 d after kindling.Statistical differences between means of two groups were analyzed using Student’s t test.Results The concentration of ADK in the supernatant of 1×10^(5)ADK--MSCs was 12.1 ng/ml.The ADK--MSCs group had lower seizures frequency as(8.20±2.38)times per week,than(16.40±3.20)times per week in the TLE group(t=4.584,P<0.01).Nissl staining showed that ADK--MSCs group had more survival neurons in CA1 region(91.00±6.51 vs.71.80±5.06,t=5.199,P<0.001)and CA3 region(64.20±7.98 vs.45.75±7.63,t=3.511,P<0.01)than TLE group.The immunohistochemical positive cells of active cysteinyl aspartate-specific protease(Caspase)-3 were decreased in the ADK--MSCs group compared to the TLE group.Western blotting confirmed that the expression of active Caspase-3 in hippocampus was reduced in the ADK--MSCs group compared to the TLE group(n=3,t=4.302,P<0.05).At 45 d after kindling,survival green fluorescent protein(GFP)positive transplanted ADK--MSCs were observed in the hippocampus.Conclusion Intracerebral transplantation of ADK-inhibited MSCs can suppress spontaneous recurrent

关 键 词：颞叶癫痫 腺苷激酶 间充质干细胞 细胞移植 

分 类 号：R742.1[医药卫生—神经病学与精神病学]