华中科技大学学报（医学版）
華中科技大學學報（醫學版）
화중과기대학학보（의학판）
ACTA UNIVERSITATIS MEDICINAE TONGJI
2010年
1期
87-90,97
,共5页
申玲玲%顾久玲%黄巍%赵云斌
申玲玲%顧久玲%黃巍%趙雲斌
신령령%고구령%황외%조운빈
高效液相色谱法%内标法%肿瘤坏死因子-α转化酶%抑制作用
高效液相色譜法%內標法%腫瘤壞死因子-α轉化酶%抑製作用
고효액상색보법%내표법%종류배사인자-α전화매%억제작용
high-performance liquid chromatography%internal standard method%tumor necrosis factor-α converting enzyme%inhibitory effect
目的 对高效液相色谱法(HPLC)测定抑制剂对肿瘤坏死因子-α转化酶(TACE)的抑制作用进行方法 改进,以提高其准确度和普及性,并用于测定抑制剂GM6001及抑制剂A对TACE的抑制作用.方法 TACE与多肽底物经37℃孵育15 min后,加入Ala-Dpa作为内标物,用高效液相色谱法进行分析.以55%甲醇水溶液为流动相,检测波长353 nm.根据剩余底物与内标物的色谱峰面积比,从校准曲线中得出底物的转化量,据此计算TACE的酶活性.结果底物与内标物的色谱峰面积比与底物浓度呈良好线性关系,相关系数为0.996 8,线性范围10 μmol/L 至 400 μmol/L.精密度试验表明,采用内标法定量,精密度得到显著改善.经测定,抑制剂GM6001及抑制剂A对TACE的半数抑制浓度IC50分别为317.5 nmol/L和175.8 nmol/L.结论 以Ala-Dpa作内标物,HPLC测定TACE 酶活性,为TACE抑制剂的筛选提供了一种更准确、更实用的方法 .
目的 對高效液相色譜法(HPLC)測定抑製劑對腫瘤壞死因子-α轉化酶(TACE)的抑製作用進行方法 改進,以提高其準確度和普及性,併用于測定抑製劑GM6001及抑製劑A對TACE的抑製作用.方法 TACE與多肽底物經37℃孵育15 min後,加入Ala-Dpa作為內標物,用高效液相色譜法進行分析.以55%甲醇水溶液為流動相,檢測波長353 nm.根據剩餘底物與內標物的色譜峰麵積比,從校準麯線中得齣底物的轉化量,據此計算TACE的酶活性.結果底物與內標物的色譜峰麵積比與底物濃度呈良好線性關繫,相關繫數為0.996 8,線性範圍10 μmol/L 至 400 μmol/L.精密度試驗錶明,採用內標法定量,精密度得到顯著改善.經測定,抑製劑GM6001及抑製劑A對TACE的半數抑製濃度IC50分彆為317.5 nmol/L和175.8 nmol/L.結論 以Ala-Dpa作內標物,HPLC測定TACE 酶活性,為TACE抑製劑的篩選提供瞭一種更準確、更實用的方法 .
목적 대고효액상색보법(HPLC)측정억제제대종류배사인자-α전화매(TACE)적억제작용진행방법 개진,이제고기준학도화보급성,병용우측정억제제GM6001급억제제A대TACE적억제작용.방법 TACE여다태저물경37℃부육15 min후,가입Ala-Dpa작위내표물,용고효액상색보법진행분석.이55%갑순수용액위류동상,검측파장353 nm.근거잉여저물여내표물적색보봉면적비,종교준곡선중득출저물적전화량,거차계산TACE적매활성.결과저물여내표물적색보봉면적비여저물농도정량호선성관계,상관계수위0.996 8,선성범위10 μmol/L 지 400 μmol/L.정밀도시험표명,채용내표법정량,정밀도득도현저개선.경측정,억제제GM6001급억제제A대TACE적반수억제농도IC50분별위317.5 nmol/L화175.8 nmol/L.결론 이Ala-Dpa작내표물,HPLC측정TACE 매활성,위TACE억제제적사선제공료일충경준학、경실용적방법 .
Objective A high-performance liquid chromatography(HPLC)method was modified and used to determine the inhibitory effects of GM6001 and inhibitor A on tumor necrosis factor-α converting enzyme(TACE).Methods TACE and polypeptides substrate were incubated for 15 min at 37°C.Ala-Dpa was added as internal standard of quantitative analysis.Then the solution was analyzed by HPLC.The 55% methanol aqueous solution was used as the mobile phase.The wavelength of detector was 353 nm.The ratio of the peak area of remaining substrate to that of internal standard was determined.And the amounts of inverted substrate could be obtained from calibration curve.The TACE activity could be calculated.Results The relative peak areas of substrate were linearly increased depending on the growth of substrate concentration.The correlation coefficient was 0.996 8 and linear range was from 10 to 400 μmol/L.Precision experiments indicated that the precision was improved obviously by using internal standard method in the determination of TACE activity by HPLC.The values of 50% inhibitory concentration IC50 of GM6001 and inhibitor A determined by the newly proposed method were 317.5 and 175.8 nmol/L,respectively.Conclusion The HPLC method assaying TACE activity with Ala-Dpa as internal standard is more accurate,and more practical for screening of TACE inhibitors.
