Mcl-1基因siRNA囊泡制备及其对人肝癌HepG2细胞的生长抑制作用观察

Preparation of Mcl-1 siRNA niosomes and its inhibitory effect on human hepatoma carcinoma HepG2 cells

目的制备包载髓样细胞白血病1(Mcl-1)基因的小干扰RNA(siRNA)囊泡(Mcl-1基因siRNA囊泡),并观察其对人肝癌HepG2细胞的生长抑制作用。方法 1Mcl-1基因siRNA囊泡制备及其功能鉴定:采用乙醇注入法制备空白囊泡,将其分别和FAM标记(荧光标记物,可在显微镜下被观察)的正常siRNA(FAM-siRNA)、Mcl-1基因siRNA溶液涡旋、静电复合,室温静置30 min,获得包载FAM-siRNA囊泡、Mcl-1基因siRNA囊泡。采用马尔文激光粒度仪测定空白囊泡、包载FAM-siRNA囊泡的平均粒径和zeta电位;采用超滤离心-荧光分光光度法测定包载FAM-siRNA囊泡的荧光强度并计算包封率;观察FAM-siRNA释放情况,计算FAM-siRNA释放率。取HepG2细胞分为A、B、C、D组,培养24 h时C组加入包载FAM-siRNA囊泡,D组加入包载FAM-siRNA囊泡和Lipofectamin转染试剂,B组加入游离FAM-siRNA,A组不做任何处理,继续培养6 h时观察各组细胞FAM-siRNA摄取情况。2转染Mcl-1基因siRNA囊泡对HepG2细胞的生长抑制作用观察:取HepG2细胞,分为1、2、3、4、5组,5×105/孔接种于6孔板,每组3个复孔。培养24 h时1、2、3、4组分别加入游离Mcl-1 siRNA、包载FAM-siRNA囊泡、Mcl-1基因siRNA囊泡、Mcl-1基因siRNA+Lipofectamin转染试剂,5组不做任何处理。培养78 h时检测各组细胞Mcl-1蛋白。取HepG2细胞分为甲、乙、丙、丁组及对照组,培养24 h时甲、乙、丙、丁组分别加入终浓度为1、10、50、100、200 nmol/L的游离Mcl-1基因siRNA、包载FAM-siRNA囊泡、Mcl-1基因siRNA囊泡、Mcl-1基因siRNA+Lipofectamin转染试剂,对照组不做任何处理。培养48 h时观察各组细胞生存情况,计算细胞存活率。结果空白囊泡、包载FAM-siRNA囊泡粒径、zeta电位相比,P均<0.01。包载FAM-siRNA囊泡包封率82.3%±2.1%。培养1、3、6、12、24、36、48、72、96 h时FAM-siRNA囊泡的FAM-siRNA释放率分别为13.5%±2.8%、26.1%±1.6%、38.0%±2.9%、50.6%±2.3%、56.8%±2.9%、59.9%±2.3%、63.3%±2.0%、65.0%±2.7%、67.2%±2.9%。C、D组对FAM-siRNA摄取情况优于B组。1、2、3、4、5组细胞Mcl-1蛋白相对表达量分别102.0±4.9、103.8±8.3、25.2±3.7、29.4±6.4、96.1±5.8,3组Mcl-1蛋白相对表达量明显低于1、2、5组,P均<0.01。当转染浓度为100nmol/L时,丙、丁组细胞生存率均高于甲、乙组,P均<0.01;丁组细胞生存率高于丙组,P<0.01。结论成功制备Mcl-1基因siRNA囊泡。HepG2细胞中Mcl-1蛋白低表达。转染Mcl-1基因siRNA囊泡的HepG2细胞存在生长抑制。

Objective To prepare myeloid cell leukemia-1（ Mcl-1） small interfering RNA（ siRNA） loaded niosomes and to observe its inhibitory effect on human hepatoma carcinoma HepG2 cells. Methods 1Preparation and functional identification of Mcl-1 siRNA loaded niosomes： Ethanol injection method was employed to prepare blank niosomes,which were then separately incubated with FAM-siRNA and Mcl-1 siRNA to form FAM-siRNA and Mcl-1 siRNA loaded niosomes through electrostatic interaction for 30 min. Malvern apparatus was used to determine the average particle size and zeta potential of blank niosomes and siRNA loaded niosomes. The encapsulation efficiency and in vitro release profile of siRNA were determined by centrifugal ultrafiltration-fluorescence spectrophotometry method. HepG2 cells were divided into groups A,B,C,and D for 24-hour cultivation,then groups C and D were treated with FAM-siRNA loaded niosomes and FAMsiRNA loaded Lipofectamin,respectively. Group B was exposed to equivalent free FAM-siRNA,while group A was subjected to no disposal. After 6-hour incubation,cellular uptake of FAM-siRNA in HepG2 cells was examined. 2The inhibitory effect of transfection of Mcl-1 siRNA loaded niosomes on HepG2 cells： Cells in logarithmic phase were divided into groups1,2,3,4,and 5 and were seeded at a density of 5 × 105 per well for cultivation. At 24 h,cells in the groups 1,2,3,and 4 were treated with free Mcl-1 siRNA,FAM-siRNA loaded niosomes,Mcl-1 siRNA loaded niosomes,and Mcl-1 siRNA loaded Lipofectamin for 6 h,respectively. After another 72 h,Mcl-1 protein of cells in each group was detected.HepG2 cells were divided into groups a,b,c,d and the control group,and were treated as previously mentioned. The final concentration of siRNA of each group was at a gradient of 1,10,50,100,and 200 nmol/L. The growth of cells was examined and cell viability was calculated. Results The particle size of blank niosomes was smaller while its zeta potential was larger as compared with FAM-siRNA loaded niosomes（ all P〈0. 01）. The encapsulation efficiency of FAM-siRNA was 82. 3% ± 2. 1%. The in vitro cumulative FAM-siRNA release rates of FAM-siRNA loaded noisomes at 1,3,6,12,24,36,48,72,and 96 h were 13. 5% ± 2. 8%,26. 1% ± 1. 6%,38. 0% ± 2. 9%,50. 6% ± 2. 3%,56. 8% ± 2. 9%,59. 9% ± 2. 3%,63. 3% ± 2. 0%,65. 0% ± 2. 7%,67. 2% ± 2. 9%,respectively. Compared with group B,cellular uptake of FAM-siRNA in groups C and D was better. The Mcl-1 protein expression levels of groups 1,2,3,4 and 5 were102. 0 ± 4. 9,103. 8 ± 8. 3,25. 2 ± 3. 7,29. 4 ± 6. 4,and 96. 1 ± 5. 8,respectively. Obviously,the level of group 3 was significantly less than that of groups 1,2,and 5（ all P〈0. 01）. Cell viability of groups c and d was significantly higher that that of groups a and b,meanwhile,the cell viability of group d was higher than that of group c（ all P〈0. 01）. Conclusion We successfully prepare Mcl-1 siRNA loaded niosomes and the expression of Mcl-1 protein is low expressed in HepG2 cells,and HepG2 cells are inhibited after transfection of Mcl-1 siRNA loaded niosomes.