铁超载对小鼠肝脏原代细胞凋亡的影响OACSTPCD
Effect of iron overload on apoptosis in primary cells from mouse livers
目的 建立稳定的小鼠肝脏原代细胞同步分离技术,研究铁超载对肝脏原代细胞凋亡的影响.方法 通过胶原酶消化、percoll密度梯度离心和CD146磁珠分选等步骤,同步分离纯化小鼠肝实质细胞、肝窦内皮细胞和枯否细胞,并使用流式细胞术、免疫荧光染色等鉴定细胞类型.体外培养各类型细胞,使用0、25、50、100 μmol/L的柠檬酸铁铵刺激24 h建立铁超载模型,通过普鲁士蓝染色观察细胞铁含量,流式细胞术测定细胞存活率和线粒体膜电位水平.结果 肝脏原代细胞同步分离技术效率稳定,肝实质细胞得率为(4.0±0.5)×107细胞/只,存活率为(76.33±0.67)％;肝窦内皮细胞得率为(5.0±1.0)×106细胞/只,存活率和纯度分别为(93.63±0.25)％和(93.40±0.46)％;枯否细胞得率为(1.5±0.5)×106细胞/只,存活率和纯度分别为(98.33±0.12)％和(88.30±2.02)％.获得的细胞数量多、活力好、纯度高,能满足后续实验要求.与空白对照组相比,柠檬酸铁铵处理的3种类型细胞中铁含量显著增加.在柠檬酸铁铵的刺激下,肝实质细胞的存活率从(73.97±5.54)％下降至(54.10±1.68)％,JC-1聚合物平均荧光强度从326.33±30.37下降至155.00±6.56,JC-1单体平均荧光强度从1700.00±144.04上升至3713.33±81.82;肝窦内皮细胞的存活率从(90.60±1.74)％下降至(78.03±2.15)％,JC-1聚合物平均荧光强度从502.33±5.51下降至372.33±4.04,JC-1单体平均荧光强度从750.00±67.51上升至1340.00±36.39;枯否细胞的存活率从(94.23±1.44)％下降至(88.37±1.56)％,JC-1聚合物平均荧光强度从652.67±25.66下降至478.00±12.49,JC-1单体平均荧光强度从1984.33±80.65上升至3062.33±245.20.结论 建立了稳定的小鼠肝脏原代细胞同步分离技术,并证实铁超载可增加肝实质细胞、肝窦内皮细胞和枯否细胞的凋亡.
Objective To investigate the impact of iron overload on apoptosis in primary mouse liver cells via a synchronous separation technology.Methods Hepatocyte(HC),liver sinusoidal endothelial cell(LSEC),and Kupffer cell(KC)were isolated and purified with collagenase,percoll density gradient centrifugation,and CD146 magnetic beads.Cell types were identified using flow cytometry and immunofluorescence staining.Cells of different types were cultured in vitro,and an iron overload model was established by treating the mice with 0,25,50 and 100 μmol/L ferric ammonium citrate(FAC)for 24 h.The iron content was quantified using Prussian blue staining,while cell viability and mitochondrial membrane potential were assessed by flow cytometry.Results The synchronous separation technology of primary liver cells exhibited stable efficiency.The yield of HC was(4.0±0.5)×107 cells per mouse,exhibiting an effective survival rate of(76.33±0.67)％.The yield of LSEC was(5.0±1.0)×106 cells per mouse,with a survival rate of(93.63±0.25)％and a purity level of(93.40±0.46)％.The yield of KC was(1.5±0.5)×106 cells per mouse while a high survival rate of(98.33±0.12)％and a purity level of(88.30±2.02)％were maintained.The obtained cells were large in number,with good vitality and high purity,which could meet the requirements of subsequent experiments.Treatment with FAC significantly elevated iron contents in different types of cells when compared with the control group.Upon stimulation of FAC,the survival rate of HC decreased from(73.97±5.54)％to(54.10±1.68)％,the mean fluorescence intensity of JC-1 aggregates decreased from 326.33±30.37 to 155.00±6.56,JC-1 monomer increased from 1700.00±1 44.04 to 3713.33±81.82.The survival rate of LSEC decreased from(90.60±1.74)％to(78.03±2.15)％,the mean fluorescence intensity of JC-1 aggregates decreased from 502.33±5.51 to 372.33±4.04,and JC-1 monomer increased from 750.00±67.51 to 1340.00±36.39.The survival rate of KC decreased from(94.23±1.44)％to(88.37±1.56)％,the mean fluorescence intensity of JC-1 aggregates decreased from 652.67±25.66 to 478.00±12.49,and JC-1 monomer increased from 1984.33±80.65 to 3062.33±245.20.Conclusion A robust and reliable simultaneous isolation technique of primary mouse HC,LSEC,and KC has been established.Moreover,our finding demonstrates that iron overload significantly enhances apoptosis levels in HC,LSEC and KC.
苏贤;王博;李东东;王蕾;祁凤英;付秋霞;阎少多
军事科学院军事医学研究院,北京 100850
临床医学
铁超载肝实质细胞肝窦内皮细胞枯否细胞细胞凋亡
iron overloadhepatocyteliver sinusoidal endothelial cellKupffer cellapoptosis
《军事医学》 2024 (006)
445-452 / 8
国家自然科学基金项目(82170228),北京市自然科学基金项目(7232108)
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