CAJ | 학술논문

目的构建稳定过表达人解偶联蛋白2 (UCP2)的张氏肝细胞株,观察UCP2对线粒体膜电位(MMP)和活性氧(ROS)的作用. 方法将含有人UCP2 cDNA全长的重组质粒(pcDNA3.1-hU CP2)转染张氏肝细胞系,pcDNA3.1空载体作为对照.Zeocin筛选稳定表达UCP2的细胞株,Western blot和免疫细胞化学鉴定UCP2蛋白表达.利用不同剂量UCP2抑制剂京尼平(25、50、100μmol/L),预处理稳定表达UCP2的细胞株.荧光分光光度法检测MMP和ROS水平变化.数据分析用单因素方差分析和q检验(Newman-keuls法),P＜0.05为差异有统计学意义.结果 pcDNA3.1-hU CP2成功转染张氏肝细胞,UCP2相对表达量约为对照组的1.6倍.过表达细胞罗丹明123和2 ′,7 ′ -二氯氢化荧光素二脂荧光强度(分别为11.11±2.76和4.97±0.62)均明显低于正常对照组张氏肝细胞(分别为15.56±2.55和6.14±1.25,q值分别为4.80和3.35,P＜0.01和P＜ 0.05)和空载体对照组肝细胞(分别为16.11±2.93和6.23±1.13,q值分别为5.40和3.60,P＜0.01和P＜ 0.05)；空载体组上述两指标与正常对照组相比,差异均无统计学意义.京尼平低、中、高剂量组与过表达组相比,罗丹明123的荧光强度(分别为14.89±2.89,17.89±2.93,24.00±2.55,q值分别为4.08,7.33和13.93,P值均＜0.01)和2 ′,7 ′-二氯氢化荧光素二脂的荧光强度(分别为9.16±0.78,10.84±1.09, 11.83±1.25,q值分别为12.00,16.83和19.67,P值均＜0.01)均明显升高,并呈现剂量依赖性.结论成功构建稳定过表达人U CP2的张氏肝细胞株,UCP2表达水平及活性变化可通过MMP和ROS影响线粒体功能.
목적 구건은정과표체인해우련단백2 (UCP2)적장씨간세포주,관찰UCP2대선립체막전위(MMP)화활성양(ROS)적작용. 방법 장함유인UCP2 cDNA전장적중조질립(pcDNA3.1-hU CP2)전염장씨간세포계,pcDNA3.1공재체작위대조.Zeocin사선은정표체UCP2적세포주,Western blot화면역세포화학감정UCP2단백표체.이용불동제량UCP2억제제경니평(25、50、100μmol/L),예처리은정표체UCP2적세포주.형광분광광도법검측MMP화ROS수평변화.수거분석용단인소방차분석화q검험(Newman-keuls법),P＜0.05위차이유통계학의의.결과 pcDNA3.1-hU CP2성공전염장씨간세포,UCP2상대표체량약위대조조적1.6배.과표체세포라단명123화2 ′,7 ′ -이록경화형광소이지형광강도(분별위11.11±2.76화4.97±0.62)균명현저우정상대조조장씨간세포(분별위15.56±2.55화6.14±1.25,q치분별위4.80화3.35,P＜0.01화P＜ 0.05)화공재체대조조간세포(분별위16.11±2.93화6.23±1.13,q치분별위5.40화3.60,P＜0.01화P＜ 0.05)；공재체조상술량지표여정상대조조상비,차이균무통계학의의.경니평저、중、고제량조여과표체조상비,라단명123적형광강도(분별위14.89±2.89,17.89±2.93,24.00±2.55,q치분별위4.08,7.33화13.93,P치균＜0.01)화2 ′,7 ′-이록경화형광소이지적형광강도(분별위9.16±0.78,10.84±1.09, 11.83±1.25,q치분별위12.00,16.83화19.67,P치균＜0.01)균명현승고,병정현제량의뢰성.결론 성공구건은정과표체인U CP2적장씨간세포주,UCP2표체수평급활성변화가통과MMP화ROS영향선립체공능.
Objective To establish the Chang liver cell line stably overexpressing human uncoupling protein 2 (UCP2) and observe the effect of UCP2 on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS).Methods The Chang liver cell line was transfected with recombinant plasmid containing full-langth human UCP2 cDNA (pcDNA3.1-hUCP2) or pcDNA3.1 empty vector.The stable cell line was established by antibiotic screening with Zeocin.UCP2 expression was detected by Western blotting and immunocytochemistry.The UCP2 overexpressing cells were pretreated with genipin at various doses (25,50 and 100 tnol/L).MMP and intracellular ROS were detected by fluorescence spectrophotometry.Results The total normalized protein content in UCP2 overexpressing cells was 1.6-fold higher than that in unmanipulated normal cells.The fluorescence intensities of Rhodamine123 and DCFH-DA in UCP2overexpressing Chang liver cells (11.11 ± 2.76 and 4.97 ± 0.62,respectively) were significantly lower than those in unmanipulated normal cells (15.56 ± 2.55,P ＜ 0.01 and 6.14 ± 1.25,P ＜ 0.05,respectively) and in cells transfected with empty vector (16.11 ± 2.93,P ＜ 0.01 and 6.23 ± 1.13,P ＜ 0.05,respectively).Treatment of UCP2 overexpressing cells with 25,50 and 100 μnol/L genipin caused a dose-dependent increase in fluorescence intensities of Rhodamine123 (14.89 ± 2.89,17.89 ± 2.93 and 24.00 ± 2.55,respectively,allP＜0.01) and DCFH-DA (9.16 ± 0.78,10.84 ± 1.09 and 11.83 ± 1.25,respectively,allP ＜ 0.01).Conclusions The Chang liver cell line stably overexpressing UCP2 was established successfully.Using this cell system,UCP2 was found to play a role in mitochondrial function by regulating MMP and ROS.