CAJ | 학술논문

目的：比较在高糖和低糖培养条件下，经半硫芥（CEES）染毒后支气管上皮细胞（16HBE）能量代谢的变化。方法采用高糖（4．5 mg/ml）和低糖（1．1 mg/ml）两种不同培养基培养16HBE细胞。 MTS法比较细胞增殖和CEES染毒后6 h细胞活性差异；用不同剂量CEES染毒两组细胞，24 h后HPLC检测胞内ATP、ADP、AMP含量，并计算ATP/ADP比值、腺苷酸池（总腺嘌呤核苷酸， total adenine nucleotides ， TAN）及能荷值（能量负荷值，energy charge， EC），Western印迹检测线粒体能量代谢功能酶COX-10和ISCU变化；流式细胞仪检测0．5 mmol/L CEES染毒后5、8、12、24 h时相点线粒体膜电位（MMP）变化。结果低糖培养的16HBE细胞的增殖明显增加，能量代谢指标ADP、TAN、COX-10和ISCU显著高于高糖组。高于0．5 mmol/L染毒剂量的CEES能显著降低高糖、低糖培养的16HBE细胞的活性，且在1．0 mmol/L下两组间差异有显著意义。高糖组在0．5、1．0 mmol/L染毒24 h后，ATP、ADP、TAN显著增高，而ATP/ADP比值及EC显著降低。低糖组在1．0 mmol/L染毒24 h后，ADP、AMP、TAN显著低于染毒前，而ATP/ADP比值及EC显著高于染毒前。在0．5 mmol/L CEES染毒下，高糖组线粒体膜电位在8～12 h显著增加，其后至24 h时恢复至正常水平。低糖组MMP在5 h有一过性显著降低，8 h后与染毒前已无显著差异。高糖培养时，16HBE细胞的氧化磷酸化相关蛋白COX-10和ISCU蛋白水平显著低于低糖培养的细胞；0．5～1．0 mmol/L CEES染毒24 h后，两者的水平显著升高，与低糖组已无显著差异。1．0 mmol/L CEES染毒24 h后，低糖组COX-10和ISCU水平显著降低。结论糖浓度差异会影响16 HBE细胞的能量代谢、增殖和CEES损伤后能量应激反应。高糖可能通过增加细胞的应激反应能力抵抗CEES的细胞毒性作用。
목적：비교재고당화저당배양조건하，경반류개（CEES）염독후지기관상피세포（16HBE）능량대사적변화。방법채용고당（4．5 mg/ml）화저당（1．1 mg/ml）량충불동배양기배양16HBE세포。 MTS법비교세포증식화CEES염독후6 h세포활성차이；용불동제량CEES염독량조세포，24 h후HPLC검측포내ATP、ADP、AMP함량，병계산ATP/ADP비치、선감산지（총선표령핵감산， total adenine nucleotides ， TAN）급능하치（능량부하치，energy charge， EC），Western인적검측선립체능량대사공능매COX-10화ISCU변화；류식세포의검측0．5 mmol/L CEES염독후5、8、12、24 h시상점선립체막전위（MMP）변화。결과저당배양적16HBE세포적증식명현증가，능량대사지표ADP、TAN、COX-10화ISCU현저고우고당조。고우0．5 mmol/L염독제량적CEES능현저강저고당、저당배양적16HBE세포적활성，차재1．0 mmol/L하량조간차이유현저의의。고당조재0．5、1．0 mmol/L염독24 h후，ATP、ADP、TAN현저증고，이ATP/ADP비치급EC현저강저。저당조재1．0 mmol/L염독24 h후，ADP、AMP、TAN현저저우염독전，이ATP/ADP비치급EC현저고우염독전。재0．5 mmol/L CEES염독하，고당조선립체막전위재8～12 h현저증가，기후지24 h시회복지정상수평。저당조MMP재5 h유일과성현저강저，8 h후여염독전이무현저차이。고당배양시，16HBE세포적양화린산화상관단백COX-10화ISCU단백수평현저저우저당배양적세포；0．5～1．0 mmol/L CEES염독24 h후，량자적수평현저승고，여저당조이무현저차이。1．0 mmol/L CEES염독24 h후，저당조COX-10화ISCU수평현저강저。결론당농도차이회영향16 HBE세포적능량대사、증식화CEES손상후능량응격반응。고당가능통과증가세포적응격반응능력저항CEES적세포독성작용。
Objective To compare the changes in energy metabolism in 2-chloroethyl ethryl sulfide(CEES)-poisoned bronchial epithelial cell 16HBE cultured in media at different glucose concentrations .Methods Bronchial epithelial cell 16HBE was cultured in high (4.5 mg/ml) or low (1.1 mg/ml) glucose medium and exposed to a sulfur mustard simulant CEES of 0.2, 0.5, 1.0 mmol/L.Cell growth and cytotoxicity were tested using MTS .ATP, ADP and AMP were detected by HPLC and the value of ATP/ADP, total adenine nucleotides ( TAN) and energy charge ( EC) was subsequently calculat-ed.Mitochondrial oxidative phosphorylation-related proteins, COX-10 and ISCU, were detected using Western blotting . Rhodamine 123 was applied to detect the mitochondrial membrane potential using flow cytometry .Results Low glucose accelerated the growth and energy metabolism of 16HBE cells in regular culture , and the contens of ADP , TAN, COX-10 and ISCU in low glucose group were significantly higher than those in high glucose group .CEES exposure (≥0.5 mmol/L) significantly affected cell viability in both high and low glucose groups , with significant difference between the two groups exposed to 1.0 mmol/L CEES.In high glucose group, 24 h after 0.5 or 1.0 mmol/L CEES exposure, the contents of ATP, ADP and TAN were significantly increased , while ATP/ADP and EC decreased .In low glucose group , ADP, AMP and TAN significantly decreased, while ATP/ADP and EC increased 24 h after 1.0 mmol/L CEES exposure.The mi-tochondrial membrane potential (MMP) also changed differently after 0.5 mmol/L CEES exposure.MMP in high glucose group marginally increased at 3 h, and significantly increased at 8-12 h (P<0.05), and returned to normal at 24 h. MMP in low glucose group showed a transient decrease at 5 h (P<0.01), and back to normal at 8 h.The protein levels of COX-10 and ISCU were significantly increased in high glucose group 24 h after 0.5-1.0 mmol/L CEES exposure , but sig-nificantly decreased in low one 24 h after 1.0 mmol/L CEES exposure .Conclusion When 16HBE is cultured at a high or low glucose concentration , the cell growth, stress responses and energy metabolism including MMP , COX-10, ISCU and ATP production are in different status before or after CEES exposure .High glucose could protect against CEES exposure .