CAJ | 학술논문

目的采用不同方法转染原代人外周血淋巴细胞,以寻找一种简便、高效的淋巴细胞转染方法.方法采用聚蔗糖-泛影葡胺(Ficoll-Hypaque)分离法从健康人外周血中分离出单个核细胞,锥虫蓝检测细胞存活率.单个核细胞在6孔板内培养2h后吸出悬浮的淋巴细胞至24孔板中继续培养.分别采用电穿孔介导载体质粒(PEGFP-N1)转染活化的淋巴细胞、慢病毒(LVGFP)单次感染及慢病毒重复感染方法分别转染静息或活化的淋巴细胞,在荧光显微镜下观察感染后不同时间点细胞绿色荧光蛋白(GFP)表达情况,同时收集细胞用流式细胞术检测GFP阳性细胞的比例,比较不同条件下慢病毒感染淋巴细胞的效率.结果通过Ficoll-Hypaque分离的单个核细胞的纯度可达95％,其活细胞率＞95％,获得的淋巴细胞占90％～ 95％,形态较均一.“2 100V、10 ms、1个脉冲”电穿孔淋巴细胞后可见散在荧光,但随着时间推移荧光逐渐减弱,72 h基本看不到荧光.慢病毒单次感染静息期淋巴细胞48 h后无绿色荧光,流式细胞术检测GFP阳性细胞＜1％.慢病毒单次感染增殖期淋巴细胞可见绿色荧光,且随着病毒浓度增大,荧光逐渐增强,GFP阳性细胞在1％～5％之间.慢病毒重复感染增殖期淋巴细胞可见较强绿色荧光,GFP阳性细胞在5％～10％左右,随着时间推移荧光逐渐增强.结论慢病毒重复感染增殖期淋巴细胞,能有效地将外源基因导入淋巴细胞内稳定表达.
목적 채용불동방법전염원대인외주혈림파세포,이심조일충간편、고효적림파세포전염방법.방법 채용취자당-범영포알(Ficoll-Hypaque)분리법종건강인외주혈중분리출단개핵세포,추충람검측세포존활솔.단개핵세포재6공판내배양2h후흡출현부적림파세포지24공판중계속배양.분별채용전천공개도재체질립(PEGFP-N1)전염활화적림파세포、만병독(LVGFP)단차감염급만병독중복감염방법분별전염정식혹활화적림파세포,재형광현미경하관찰감염후불동시간점세포록색형광단백(GFP)표체정황,동시수집세포용류식세포술검측GFP양성세포적비례,비교불동조건하만병독감염림파세포적효솔.결과 통과Ficoll-Hypaque분리적단개핵세포적순도가체95％,기활세포솔＞95％,획득적림파세포점90％～ 95％,형태교균일.“2 100V、10 ms、1개맥충”전천공림파세포후가견산재형광,단수착시간추이형광축점감약,72 h기본간불도형광.만병독단차감염정식기림파세포48 h후무록색형광,류식세포술검측GFP양성세포＜1％.만병독단차감염증식기림파세포가견록색형광,차수착병독농도증대,형광축점증강,GFP양성세포재1％～5％지간.만병독중복감염증식기림파세포가견교강록색형광,GFP양성세포재5％～10％좌우,수착시간추이형광축점증강.결론 만병독중복감염증식기림파세포,능유효지장외원기인도입림파세포내은정표체.
Objective To explore the transfection efficiency of primary lymphocytes from human peripheral blood by different methods to acquire the method with higher transfection efficiency.Methods Mononuclear cells from human peripheral blood were isolated using Ficoll-Hypaque.Cell viability was detected by Trypan blue staining.Suspending lymphocytes were sucked out and were incubated in 24-well plate after cultured in 6-well plate for 2 h.Activated lymphocytes were transfected by electroporation with plasmid(PEGFP-N1).Resting or activated lymphocytes were transfected by lentivirus vector(LVGFP) single infection or repeated infection,respectively.Green fluorescence protein (GFP) was detected under the fluorescence microscopy and percentage of positive cells was checked by flow cytometry at different time points after infection.At the same time,the effectiveness of lentivirus infection was compared under different conditions.Results Purity of mononuclear cells isolated by Ficoll-Hypaque was 95 ％ and its viability was over 95 ％.The percentage of lymphocytes obtained with a uniform shape was 90 ％-95 ％.Scattered fluorescence was observed by electroporation under the conditions of voltage 2 100 V,pulse width 10 ms,pulse number 1 for lymphocyte,while fluorescent became weaker over time and no green fluorescent was observed after transfection for 72 h.After resting lymphocytes were infected once for 48 h by lentivirus vector,green fluorescent was not found and positive cells were less than 1％.1％-5 ％ of activated lymphocytes could express GFP after single lentivirus infection and the expression levels were enhanced with concentration increasing,while 5 ％-10 ％ of activated lymphocytes showed strong green fluorescent by repeated lentivirus infection.In contrast with electroporation,the fluorescent with lentivirus infection was stronger over time.Conclusion Repeated lentivirus infection could efficiently transfect exogenous genes into activated lymphocytes for stable expression.