喜树碱负载外泌体抗结肠癌作用

Effect of camptothecin-loaded exosome on colon cancer

目的探讨喜树碱负载外泌体(exosome)的抗结肠癌作用。方法结肠癌colon-26细胞分为对照组、喜树碱组、喜树碱负载exosome组(负载组)和喜树碱与exosome混合组(混合组)。对照组、喜树碱组、负载组均经电转、过滤处理,混合组不经电转,仅过滤处理。对照组给予生理盐水,喜树碱组、负载组和混合组分别给予喜树碱、喜树碱负载exosome、喜树碱与exosome混合物孵育24、48h。各组采用MTT法检测细胞杀伤率,采用流式细胞术检测细胞凋亡情况,采用DAPI、DIC、Merge观察细胞中喜树碱荧光强度。将40只C57BL6小鼠分为对照2组、喜树碱2组、负载2组和混合2组,每组10只,接种结肠癌colon-26细胞制备结肠癌皮下瘤小鼠模型,于接种第8天分别给予相应药物,给药后第10天和第20天测量各组小鼠体质量和皮下瘤体积,并进行比较。结果 5、25和100μmol/L喜树碱孵育24h时和25、100μmol/L喜树碱孵育48h时,负载组细胞杀伤率均明显高于对照组、混合组和喜树碱组(P<0.05);5μmol/L喜树碱孵育48h时,负载组细胞杀伤率高于对照组和混合组(P<0.05),与喜树碱组比较差异无统计学意义(P>0.05);各浓度和各时间点时喜树碱组细胞杀伤率均高于对照组和混合组,混合组高于对照组(P<0.05);负载组结肠癌细胞凋亡率(14.2%)明显高于对照组(0.1%)、喜树碱组(1.1%)和混合组(1.6%)(P<0.05);负载组结肠癌细胞colon-26喜树碱荧光强度(952.40±28.72)明显高于对照组(72.23±9.65)、喜树碱组(345.26±14.32)和混合组(180.65±8.65)(P均<0.05);给药20d时负载2组瘤块体积[(280.5±3.4)mm3]明显低于对照2组[(350.8±6.7)mm3]、喜树碱2组[(300.8±3.3)mm3]和混合2组[(340.4±2.3)mm3](P均<0.05),喜树碱2组低于对照2组和混合2组(P<0.05),混合2组与对照2组比较差异无统计学意义(P>0.05);给药20d时负载2组体质量[(32.4±2.0)kg]明显高于喜树碱2组[(27.9±1.2)kg](P<0.01),与对照2组[(34.2±0.8)kg]、混合2组[(30.0±2.0)kg]比较差异无统计学意义(P>0.05),喜树碱2组和混合2组体质量低于对照2组(P<0.01)。结论喜树碱负载exosome具有较好抗结肠癌的作用。

Objective To study the effect of camptothecin loaded with exosome on colon cancer. Methods The colon-26 cells were divided into control group, camptothecin group, camptothecin-loaded exosome group （loaded group） and camptothecin mixed with exosome group （mixed group）. Control group, camptothecin group and loaded group were transfected and filtrated, and mixed group was only filtrated. Control group, camptothecin group, loaded group and mixed group were treated with normal saline, camptothecin, camptothecin-loaded exosome and the mixture of camptothecin and exosome respectively for 24 or 48 hours. The cell killing rate was detected by MTT. The apoptosis was detected by flow cytometry. The fluorescence intensity of camptothecin in cells was observed by DAPI, DIC and Merge. Forty C57BL6 mice were randomly divided into control group 2, camptothecin group 2, loaded group 2 and mixed group 2, with 10 mice in each group, and were vaccinated with colon-26 cells to establish mice models of subcutaneous tumor. The body mass of mice and the volume of tumor were recorded and compared by day 10 and 20 after administration.Results The killing rates for colon 26 ceils at 5, 25 and 100 μmol/L after 24-hour incubation or at 25 and 100 μmol/L after 48-hour incubation in loaded group were significantly higher than those in control group （P〈0.05）. The killing rate at 5 μmol/L after 48-hour incubation was obviously higher in loaded group than that in control group and mixed group （P〈0.05）, and showed no difference in comparison with that in camptothecin group （P〈0.05）. The killing rates for colon-26 cells at different concentrations and different time points were significantly higher in camptothecin group than those in control group and mixed group, and higher in mixed group than those in control group （P〈0.05）. The apoptosis rate of colon-26 cells was higher in loaded group （14. 2%） than that in control group （0. 1%）, camptothecin group （1.1 % ） and mixed group （1.6 % ） （P〈0.05）. The fluorescence intensity of camptothecin in colon-26 cells was obviously higher in loaded group （952.40 ± 28. 72） than that in control group （72. 2±9. 65）, camptothecin group （345. 26 ± 14.32） and mixed group （180.65±8.65） （P〈0.05）. The volume of tumor in loaded group 2 （（280.5±3.4） mm3） was significantly smaller than that in control group 2 （（350.8±6.7） mm3）, camptothecin group 2 （（300.8：k3.3） mm3） and mixed group 2 （（340.4 ± 2.3） mma） by day 20 after administration （P〈0.05）, and smaller in camptothecin group 2 than that in control group 2 and mixed group 2 （P〈0.05）, and there was no significant difference between control group 2 and mixed group 2 （P〉0.05）. The body mass in loaded group 2 （（32.4±2.0） kg） was significantly higher than that in camptothecin group 2 （（27.9±1.2） kg） by day 20 after administration （P〈0. 01）, and lower in camptothecin group 2 and mixed group 2 than that in control group 2 （P〈0. 01）, and there was no significant difference between control group 2 （（34.2±0.8） kg） and mixed group 2 （（30.0±2.0） kg） （P〈0.05）. Conclusion Camptothecin-loaded exosome is effective for suppressing colon cancer.