阿霉素免疫脂质体对体外生长的人乳腺癌细胞的杀伤作用

Killing effect of immunoliposomal adriamycin on breast cancer cells in vitro

目的 构建Trastuzmab F（ab’）2修饰的阿霉素免疫脂质体并观察其对体外生长的人乳腺癌MCF-7细胞的杀伤作用.方法 抗体交联法制备Trastuzmab F（ab’）2段修饰的阿霉素免疫脂质体；透射电子显微镜观察免疫脂质体的形态及粒径分布；采用荧光标记法测定免疫脂质体活性；噻唑蓝（MTT）法检测MCF-7细胞在不同药物浓度下培养8、16、24、32 h后的增殖差异；流式细胞术检测200 mg/L药物浓度下培养32 h后的各组细胞凋亡.结果 成功构建了Trastuzmab F（ab’）2修饰的阿霉素免疫脂质体；所得免疫脂质体平均粒径为192 nm,粒径小于200 nm者占92.20％；投药10、30、50 min时摄入阿霉素免疫脂质体及阿霉素脂质体的细胞比例分别为（19.39±3.33）％、（57.36 ±4.12）％、（89.72±5.88）％和（10.17±4.05）％、（25.15±3.77）％、（38.86±6.24）％（P＜0.05）；阿霉素免疫脂质体对MCF-7细胞增殖的抑制作用强于阿霉素脂质体（F=1 256.225,P＜0.01）,且该抑制作用具有时间（F=2 145.325,P＜ 0.01）和浓度（F=1 287.472,P＜0.01）的依赖性；相同时间内,MCF-7细胞对阿霉素免疫脂质体的摄取要明显高于对照组（P＜0.05）；TrastuzmabF（ab,）2修饰的阿霉素免疫脂质体对MCF-7细胞增殖具有一定的抑制作用（P＜0.01）,且该抑制作用具有时间和浓度的依赖性（P＜0.01）；转染32 h后,阿霉素免疫脂质体组的细胞凋亡率[（35.22±3.52）％]高于对照组[（10.31±4.62）％,P＜ 0.05].结论 成功构建TrastuzmabF（ab,）2修饰的阿霉素免疫脂质体,其对体外生长的乳腺癌细胞具有较强的杀伤作用.

Objective To construct the immunoliposomal adriamycin tagged with trastuzumab F （ab＆#39;） 2 and to investigate its killing effect on human breast cancer cells in vitro.Methods The tastuzmab F（ab＆#39;）2 and the liposomal adriamycin were linked together by antibody crosslinking method.The morphology of immunoliposomes was observed by transmission electron microscopy （TEM） and their size distribution was measured by graphics software.The immune activity of immunoliposomes was evaluated using the fluorescence microscopy after the fluorescent immunoliposomes or lipoosomes were incubated together with MCF-7 cells.Inhibitory effect of the cell growth at different drug concentrations was measured using methyl thiazol tetrazolium （MTT） 8,16,24 and 32 h after cell culture.The apoptosis was examined by using flow cytometry 32 h after cell culture.Results The immunoliposomal adriamycin tagged with trastuzumab F（ab＆#39;）2 that was constructed in vitro has an average diameter of 192 nm,and that the diameters under 200 nm accounted for 92.20% of the total.The immunoliposome was superior to liposome at the identification capability to MCF-7 cells,the proportions of the cells which intake drug were （19.39 ±3.33）%,（57.36 ±4.12）%,（89.72 ±-5.88）% and （10.17 ±4.05）%,（25.15 ± 3.77）%,（38.86 ±6.24）% （P〈0.05） in two groups respectively after 10,30 and 50 min cell cultrue.The immunoliposome could inhibit the proliferation of MCF-7 cells in a dose （F =1 287.472,P 〈 0.01） and time （F =2 145.325,P〈 0.01）dependent manner; Apoptotic index of MCF-7 cells in immunoliposome group （35.22 ± 3.52） % was obviously higher than that in the control groups （10.31 ± 4.62） % 32 h after transfection.（P 〈 0.05）.Conclusion The immunoliposomal adriamycin tagged with trastuzumab F （ab＆#39;） 2 was constructed successfully and it can inhibit the proliferation of kill the MCF-7 cells effectively in vitro.