摘要
目的通过建立小胶质细胞-神经元共培养系统,制备体外神经细胞炎症模型。方法选用小鼠BV-2小胶质细胞、NSC34运动神经元、HT-22海马神经元。实验Ⅰ采用不同浓度LPS(10、100、500和1000 ng/ml)刺激BV-2小胶质细胞,提取小胶质细胞培养上清液即条件培养基,分别培养两种神经元,采用CCK-8法选取导致神经元活力明显下降50%的LPS浓度用于Transwell共培养系统的建立。实验Ⅱ将小胶质细胞接种于Transwell上室,神经元种于下室。采用随机数字表法分为2组(n=12):对照组和LPS组,小胶质细胞分别用细胞培养基、LPS培养或孵育6 h,随后更换新鲜培养基继续培养12 h,合并上、下室细胞继续培养。BV-2-NSC34 Transwell共培养系统共培养12 h,BV-2-HT-22 Transwell共培养系统共培养24 h。采用ELISA法检测神经元培养液IL-1β、IL-18浓度,采用流式细胞术检测神经元凋亡率,采用qRT-PCR检测神经元Bcl-2、Bax的mRNA表达,采用Western blot法检测神经元cleaved caspase-3、Bcl-2、Bax的表达。结果实验ⅠBV-2-NSC34 Transwell共培养系统中LPS刺激浓度为10 ng/ml,BV-2-HT-22 Transwell共培养系统中LPS刺激浓度为1000 ng/ml。实验Ⅱ与对照组比较,LPS组神经元培养液IL-1β和IL-18浓度、神经元凋亡率升高,Bax及其mRNA表达上调,Bcl-2及其mRNA表达下调,cleaved caspase-3表达上调(P<0.05或0.01)。结论通过条件培养基技术与Transwell共培养技术成功建立小胶质细胞-神经元共培养系统,为术后认知功能障碍相关神经细胞炎症模型的建立提供实验方案。
Objective To develop an in vitro neuroinflammation model by establishing a microglia-neuron co-culture system.Methods Mouse microglia(BV-2),motor neurons(NSC34)and hippocampal neurons(HT-22)were selected.This experiment was performed in two parts.ExperimentⅠBV-2 microglia were stimulated with different concentrations of lipopolysaccharide(LPS,10,100,500 and 1000 ng/ml).Microglia culture supernatant(Conditioned Medium)was extracted and two types of neurons were cultured separately.The concentration of LPS that resulted in a significant 50%decrease in neuronal viability was selected using the CCK-8 method for establishment of the Transwell co-culture system.ExperimentⅡMicroglia were cultured in the upper chamber of Transwell,and neurons were seeded in the lower chamber.Microglia were divided into 2 groups(n=12 each)using the random number table method:control group and LPS group.In control group and LPS group,microglia were cultured for 6 h with cell culture medium and LPS,respectively,then the medium was replaced with fresh medium,microglia were continuously incubated for 12 h,and then the cells in the upper and lower chambers were combined.The cells were incubated using the BV-2-NSC34 Transwell co-culture system for 12 h and using the BV-2-HT-22 Transwell co-culture system for 24 h.The concentrations of interleukin-1beta(IL-1β)and IL-18 in neuronal culture supernatant were measured by enzyme-linked immunosorbent assay,the apoptotic rate of neurons was determined by flow cytometry,the expression of Bcl-2 and Bax mRNA in neurons was detected by quantitative real-time polymerase chain reaction,and the expression of cleaved caspase-3,Bcl-2 and Bax in neurons was detected by Western blot.Results ExperimentⅠLPS concentration for stimulation was 10 ng/ml in BV-2-NSC34 Transwell co-culture system and 1,000 ng/ml in BV-2-HT-22 Transwell co-culture system.ExperimentⅡCompared with control group,the concentrations of IL-1βand IL-18 and apoptotic rate of neurons were significantly increased,Bax protein and mRNA expression was up-regulated,Bcl-2 protein and mRNA expression was down-regulated,and cleaved caspase-3 expression was up-regulated in LPS group(P<0.05 or 0.01).Conclusions The microglia-neuron co-culture system is successfully established by the conditioned medium technique and Transwell co-culture system,which provides an experimental protocol for establishment of neuroinflammation models associated with postoperative cognitive dysfunction.
作者
马宝育
张振江
张蕊
刘婕
赵永军
Ma Baoyu;Zhang Zhenjiang;Zhang Rui;Liu Jie;Zhao Yongjun(Shandong Provincial Medicine and Health Key Laboratory of Clinical Anesthesia School of Anesthesiology,Weifang Medical University,Weifang 261053,China;Department of Thoracic Surgery,The First Affiliated Hospital of Weifang Medical University,Weifang 261000,China)
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2022年第4期416-420,共5页
Chinese Journal of Anesthesiology
基金
山东省自然科学基金(ZR2017MH066)。