腺病毒携带IL-12治疗肝癌细胞株HepG2的体外实验  

In vitro antitumor effect of an adenoviral vector expressing IL-12 in HepG2 cells

作　　者：崔清昱[1] 秦万民[1] 张炳远[1] 刘世海[2] 孙传东[1] 

机构地区：[1]青岛大学附属医院普外二科,山东省青岛市266071 [2]青岛大学中心实验室,山东省青岛市266071

出　　处：《世界华人消化杂志》2013年第30期3190-3197,共8页World Chinese Journal of Digestology

基　　金：山东省2012科学技术发展计划基金资助项目;No.2012G0021845;山东省自然科学基金资助项目;No.2011ZRB01588;青岛市科技计划基础研究基金资助项目;No.11-2-4-2(28)-jch~~

摘　　要：目的:探讨腺病毒介导的白介素12(interleukin-12,IL-12)基因(AdvIL-12)转染能否在体外诱导出更强的抗(hepatocellular carcinoma,HCC)免疫效应.方法:在体外,用AdvIL-12转染HepG2作为实验组,以Adv-绿色荧光蛋自(green fluorescent protein,GFP)转染的HepG2和未转染的HepG2作为对照组进行培养.用划痕试验检测载有IL-12的腺病毒对HepG2细胞的迁移能力的影响;结晶紫法检测对HepG2的生长能力的抑制和杀伤作用;MTT检测对HepG2细胞增殖能力的抑制作用的影响;TUNEL法检测对HepG2细胞的凋亡效应的影响.结果:正常生长的HepG2单层贴壁生长,生长旺盛,镜下观察为梭形,胞浆均质透明,折光性好,胞间紧密连接,核质均匀,核仁清晰可见.实验组AdvIL-12作用24 h后HepG2细胞迁移距离明显受到抑制,且随浓度增大,HepG2细胞向划痕区移动的距离越来越小,细胞数量越少,划痕越宽.实验组AdvIL-12结晶紫染色深度随浓度梯度减少而逐渐加深.取10 L AdvIL-12时对HepG2细胞杀伤效果最为明显.酶标仪检测AdvIL-12组、Adv-GFP组和HepG2空白对照组分别培养24、48、72 h后的A值(F1=95.7,F2=103.6,F3=165.8,P<0.01)与对照组相比有统计学意义.光镜下可见凋亡细胞明显固缩,体积变小,胞质致密,核浓缩甚至消失,形成凋亡小体.实验组AdvIL-12组凋亡细胞数量明显多于对照组Adv-GFP组.结论:转染AdvIL-12的HepG2细胞能有效抑制HepG2细胞的生长,增殖和迁移能力,效果随浓度增大而增强,并能够促进HepG2细胞的凋亡效应,从而在体外达到明显的肿瘤抑制作用.AIM： To explore whether an adenoviral vector expressing the interleukin-12 （IL-12） gene （AdvIL-12） can induce a strong anti-hepatic carcinoma effect in vitro. METHODS： HepG2 cells transfected with AdvIL-12 were cultured as the experimental group, and cells transfected with Adv-GFP or non-transfected cells were used as controls. The scratch test was used to detect the migration of AdvIL-12-infected HepG2 cells. The crystal violet staining assay was used to examine the survival of HepG2 cells. MTT assay was used to assess the proliferation of HepG2 cells. TUNEL assay was used to detect the apoptosis of HepG2 cells. RESULTS： AdvIL-12-infection inhibited the migration of HepG2 cells in a dose-dependent manner. In the experimental group, the dye depth of crystal violet staining deepened with the decrease in the concentration of AdvIL-12. The killing effect of 10 μL of AdvIL-12 had the most obvious killing effect on HepG2 cells. The absorbance values differed significantly between the AdvIL-12 group, Adv-GFP group and non-transfected HepG2 group （F1 = 95.7, F2 = 103.6, F3 = 165.8, P 〈 0.01） after culture for 24, 48 or 72 h. Under an optical microscope, typical apoptotic cells were visible. The number of apoptotic cells in the experimental group was significantly more than those in the two control groups. CONCLUSION： AdvIL-12 infection can effectively inhibit the ability of HepG2 cells to grow, proliferate and migrate and promote the apoptosis of HepG2 cells.

关 键 词：腺病毒 白介素-12 肝癌 TUNEL 

分 类 号：R735.7[医药卫生—肿瘤]