中华临床感染病杂志
中華臨床感染病雜誌
중화림상감염병잡지
CHINESE JOURNAL OF CLINICAL INFECTIOUS DISEASES
2014年
4期
328-332
,共5页
冯婷婷%胡华%秦爱兰%孙蔚%吴南屏%甘建和
馮婷婷%鬍華%秦愛蘭%孫蔚%吳南屏%甘建和
풍정정%호화%진애란%손위%오남병%감건화
抗HIV药%药物筛选试验%流式细胞术%逆转录聚合酶链反应%半数抑制浓度
抗HIV藥%藥物篩選試驗%流式細胞術%逆轉錄聚閤酶鏈反應%半數抑製濃度
항HIV약%약물사선시험%류식세포술%역전록취합매련반응%반수억제농도
Anti-HIV agents%Drug screening assays%Flow cytometry%Reverse transcriptase polymerase chain reaction%Inhibitory concentration 50
目的 用细胞共培养和流式细胞仪检测建立新型抗HIV-1药物筛选方法,并判断这种方法在筛选抗HIV-1药物中的应用前景.方法 把JLTRG细胞和H9/HTLV-ⅢB细胞按照不同比例共培养24,48,72和96 h.在荧光显微镜下观察细胞绿色荧光强度和密度,同时用流式细胞仪测定绿色荧光的表达,并确定培养体系中最佳细胞比例和培养时间.通过选出的最佳条件结合药物半衰期检测恩夫韦肽(T20)和依非韦伦(EFV)的有效性及药物半数抑制量(IC50).采用HIV-1 P24抗原荧光定量PCR法检测病毒载量,并采用Spearman秩相关性分析法判断其与药物浓度及平均荧光强度之间的相关性.结果 实验结果显示,JLTRG细胞与H9/HTLV-ⅢB细胞按照10∶1的细胞数量比例共培养72 h为最佳培养比例和时间.以T20和EFV对共培养体系进行验证,结果显示随着T20和EFV浓度的改变,共培养体系中JLTRG细胞感染HIV-1的程度不同,其IC50分别为10 nmol/L和5 nmol/L.T20和EFV药物浓度与平均荧光强度以及病毒载量呈负相关(r=-1,-0.986和-1,-1,P＜0.01);平均荧光强度与病毒载量呈正相关(r=0.986和1,P＜0.01).结论 本研究建立的新型抗HIV-1药物筛选方法具有耗时短、操作方便等优点,可为抗HIV-1药物的筛选提供新的选择.
目的 用細胞共培養和流式細胞儀檢測建立新型抗HIV-1藥物篩選方法,併判斷這種方法在篩選抗HIV-1藥物中的應用前景.方法 把JLTRG細胞和H9/HTLV-ⅢB細胞按照不同比例共培養24,48,72和96 h.在熒光顯微鏡下觀察細胞綠色熒光彊度和密度,同時用流式細胞儀測定綠色熒光的錶達,併確定培養體繫中最佳細胞比例和培養時間.通過選齣的最佳條件結閤藥物半衰期檢測恩伕韋肽(T20)和依非韋倫(EFV)的有效性及藥物半數抑製量(IC50).採用HIV-1 P24抗原熒光定量PCR法檢測病毒載量,併採用Spearman秩相關性分析法判斷其與藥物濃度及平均熒光彊度之間的相關性.結果 實驗結果顯示,JLTRG細胞與H9/HTLV-ⅢB細胞按照10∶1的細胞數量比例共培養72 h為最佳培養比例和時間.以T20和EFV對共培養體繫進行驗證,結果顯示隨著T20和EFV濃度的改變,共培養體繫中JLTRG細胞感染HIV-1的程度不同,其IC50分彆為10 nmol/L和5 nmol/L.T20和EFV藥物濃度與平均熒光彊度以及病毒載量呈負相關(r=-1,-0.986和-1,-1,P＜0.01);平均熒光彊度與病毒載量呈正相關(r=0.986和1,P＜0.01).結論 本研究建立的新型抗HIV-1藥物篩選方法具有耗時短、操作方便等優點,可為抗HIV-1藥物的篩選提供新的選擇.
목적 용세포공배양화류식세포의검측건립신형항HIV-1약물사선방법,병판단저충방법재사선항HIV-1약물중적응용전경.방법 파JLTRG세포화H9/HTLV-ⅢB세포안조불동비례공배양24,48,72화96 h.재형광현미경하관찰세포록색형광강도화밀도,동시용류식세포의측정록색형광적표체,병학정배양체계중최가세포비례화배양시간.통과선출적최가조건결합약물반쇠기검측은부위태(T20)화의비위륜(EFV)적유효성급약물반수억제량(IC50).채용HIV-1 P24항원형광정량PCR법검측병독재량,병채용Spearman질상관성분석법판단기여약물농도급평균형광강도지간적상관성.결과 실험결과현시,JLTRG세포여H9/HTLV-ⅢB세포안조10∶1적세포수량비례공배양72 h위최가배양비례화시간.이T20화EFV대공배양체계진행험증,결과현시수착T20화EFV농도적개변,공배양체계중JLTRG세포감염HIV-1적정도불동,기IC50분별위10 nmol/L화5 nmol/L.T20화EFV약물농도여평균형광강도이급병독재량정부상관(r=-1,-0.986화-1,-1,P＜0.01);평균형광강도여병독재량정정상관(r=0.986화1,P＜0.01).결론 본연구건립적신형항HIV-1약물사선방법구유모시단、조작방편등우점,가위항HIV-1약물적사선제공신적선택.
Objective To establish and assess a new screening method for anti-HIV-1 drugs.Methods JLTRG cells were co-cultured with different proportions of H9/HTLV-Ⅲ B cells for 24,48,72 and 96 h.Intensity and density of green fluorescent protein were observed under fluorescence microscope,and were tested using flow cytometry.The optimal proportion of cells in co-culture system and the culture time were determined.The effectiveness of Enfuvirtide (T20) and Efavirenz (EFV),and their half maximal inhibitory concentrations (IC50) were determined by using cell co-culture system and half life of drugs.HIV load was detected using RT-PCR for HIV-1 p24 antigen,and its correlations with drug concentration and mean fluorescent intensity were analyzed by Spearman rank correlation analysis.Results Experiments demonstrated that JLTRG cells co-cultured with H9/HTLV-ⅢB cells at the proportion of 10 ∶ 1 for 72 hours was the best.Along with the concentrations of T20 and EFV changed,JLTRG cells were infected with HIV-1 in different degrees,and the IC50s of T20 and EFV were 10 nmol/L and 5 nmol/L,respectively.The concentrations of T20 and EFV were negatively correlated with mean fluorescent intensity and viral load (r =-1,-0.986 and-1,-1,P ＜ 0.01); and mean fluorescent intensity was positively correlated with viral load (r =0.986 and 1,P ＜ 0.01).Conclusion The drug screening method established in this study is efficient and easy to operate,which provides a new option for anti-HIV-1 drug screening.
