摘要目的 观察重组慢病毒Lv-hTERTp-TK与Lv-hTERTp-tumstatin在肝癌HepG2细胞中的靶向表达,探讨二者联合在体内外对HepG2细胞的抑制作用. 方法 用不同MOI重组慢病毒Lv-hTERTp-TK、 Lv-hTERTp-tumstatin转染肝癌细胞HepG2、正常肝细胞L02,72 h后荧光显微镜观察转染情况;逆转录PCR检测TK、 tumstatin mRNA在HepG2、 L02细胞中的表达;四甲基偶氮唑蓝法观察两种细胞增殖情况;流式细胞术检测两种细胞凋亡情况;RT-PCR、 Western blot检测HepG2细胞bcl-2、血管内皮生长因子(VEGF) mRNA和蛋白的表达;将HepG2细胞接种于30只BALB/c裸鼠皮下,构建动物移植瘤模型,随机分为5组,空白对照组、空载体组、tumstatm组、TK组及联合组,每组6只,瘤内分别注射磷酸盐缓冲溶液、空载体慢病毒Lv-hTERTp-eGFP、重组慢病毒Lv-hTERTp-tumstatin、重组慢病毒Lv-hTERTp-TK和重组慢病毒Lv-hTERTp-tumstatin+Lv-hTERTp-TK;4周后处死裸鼠,观察各组移植瘤体积及质量的变化;行HE染色进行各组肿瘤组织及主要脏器的形态学观察;免疫组织化学法检测肿瘤内微血管密度;TUNEL法检测肿瘤细胞凋亡率.数据采用方差分析. 结果 转染后TK、 tumstatin基因在HepG2细胞中特异表达,在L02细胞中无表达.TK组、tumstatin组及联合组均对HepG2细胞生长有抑制作用,联合组抑制率明显高于单基因作用组(F=731.679,P＜0.05),各组L02细胞抑制率差异无统计学意义(F=0.774,P＞0.05);联合组HepG2细胞总凋亡率达72.61％±3.29％,明显高于TK组(53.63％±3.06％)、tumstatin组(45.13％±2.15％)、空白对照组(13.29％±1.15％)(F=365.022,P＜0.05),各组L02细胞凋亡率差异无统计学意义(F=0.391,P＞0.05);与对照组相比,实验组HepG2细胞bcl-2及VEGF表达水平均下降(bcl-2 mRNA:F=322.780,P＜0.05;VEGF mRNA:F=561.136,P＜0.05;bcl-2蛋白:F=34.203,P＜0.05;VEGF蛋白:F=32.988,P＜0.05),其中联合组bcl-2及VEGF表达水平均低于单基因组(P值均＜0.05).tumstatin组、TK组和联合组的抑瘤率分别为40.68％、 44.96％和72.09％,联合组抑瘤效果最显著(P＜0.05);HE染色显示联合组肿瘤细胞凋亡最明显.与空白对照组相比,其他各组裸鼠体内肝、脾和肾等主要脏器组织形态学均无显著变化;空白对照组、空载体组、TK组、tumstatin组和联合组的微血管密度分别为28.4±4.28、29.2±2.59、20.8±3.35、 14.2±4.87、5.8±2.77,tumstatin组抗肿瘤血管生成作用强于TK组(P＜0.05),且联合组作用最明显(P＜0.05).联合组肿瘤细胞凋亡率为70.11％±5.67％,明显高于tumstatin组(23.75％±5.21％)、TK组(48.30％±4.99％)、空白对照组(3.89％±1.13％)(P＜0.05),其中TK组的促凋亡作用较tumstatin组显著(P＜0.05). 结论 hTERT启动子驱动TK、 tumstatin在HepG2细胞中靶向表达,两种基因联合在体内外显著抑制HepG2细胞增殖和促进HepG2细胞凋亡,作用明显强于单基因,其机制可能与bcl-2及VEGF表达下调有关.

abstractsObjective To observe targeted expression of recombinant lentivirus-mediated (Lv)-hTERTp-TK and Lv-hTERTp-tumstatin in HepG2 cells, and explore the inhibitory effect of their combination on HepG2 cells both in vitro and in vivo.Methods Lv-hTERTp-TK and Lv-hTERTptumstatin were used to infect HepG2 and L02 cells at different MOIs.Transfection efficiency was observed by fluorescence microscopy.Expression of TK and tumstatin mRNA was detected by reverse-transcriptase PCR.Proliferation and apoptosis were detected by MTT and flow cytometry, respectively.The HepG2 cells were examined by real time-PCR and western blotting to determine expression level of bcl-2 and VEGF mRNA and protein.A murine hepatocellular carcinoma model was established by injecting 1 × 107 HepG2 cells into 30 BALB/c nude mice.The modeled mice were randomly divided into a control group, mock group, Lv-hTERTp-tumstatin group, Lv-hTERTp-TK group, and combination group for four weeks of injections at regular intervals of PBS, Lv-hTERTp-null, Lv-hTERTp-tumstatin, Lv-hTERTp-TK, and Lv-hTERTp-tumstatin plus Lv-hTERTp-TK, respectively.Changes in tumor volume and weight, and cell morphology of tumor and major organs, were assessed by hematoxylin-eosin staining.Microvascular density of tumor tissue and cell apoptosis were assessed by immunohistochemical and TUNEL staining, respectively.Results The Lv-infected HepG2 cells, and not the Lv-infected L02 cells, expressed TK and tumstatin.Lv-hTERTp-TK and Lv-hTERTp-tumstatin, alone or in combination, inhibited proliferation and increased apoptosis of the HepG2 cells, but the combination was more effective than either alone (P ＜ 0.05).None of the treatments affected proliferation or apoptosis of the L02 cells (P ＞ 0.05).The combination also led to a greater reduction of bcl-2 and VEGF than either alone (all, P ＜ 0.05).Tumor growth was significantly inhibited by the combination (P ＜ 0.05).In vivo, the combination treatment induced the greatest amount of apoptosis of the HepG2 cells.Cell morphology of major organs such as liver, spleen and kidney were similar to the control group.The combination also produced the most significant effect on tumor microvascular density (P ＜ 0.05) and the highest apoptosis index (P ＜ 0.05).Conclusion The HTERT promoter can induce targeted expression of TK and tumstatin in HepG2 cells.Lv-hTERTp-TK combined with Lv-hTERTp-tumstatin can significantly inhibit proliferation and induce apoptosis of human HepG2 cells in vitro and in vivo, which may be related to down-regulation ofbcl-2 and VEGF.