包载反义RNA重组腺病毒微球的制备及逆转肝癌的体内外效果

Construction of microsphere encapusulating recombinant adenovirus with antisense multidrug resistance-associated protein gene and its effect on hepatocellular carcinoma

目的:构建携带反义多药耐药相关蛋白(MRP)基因的重组腺病毒微球,了解包载反义RNA重组腺病毒微球逆转肝细胞癌MRP的治疗效果. 方法:采用可降解的生物材料聚乳酸-聚乙烯醇(PELA) 包被携带反义MRP基因的重组腺病毒制成微球,体外测定微球的粒径、载病毒量、包封率及释放规律,并用其转染人肝癌耐药细胞株HepG2/ADM,48 h及120 h后检测转染细胞荧光强度.MTT法检测耐药细胞的阿霉素半数致死量(IC50);流式细胞仪检测细胞内红色荧光强度;以β-actin为内参照,以RT-PCR法观察转染后MRPmRNA表达量的高低.观察经肝动脉注射rAdV微球后肿瘤体积大小、生长率及平均生存时间. 结果:成功构建了携带反义MRP基因的重组腺病毒微球,直径约1.765 μm,包封率为52.4%,载病毒率为5.5×1011efu/g,在120 h内释放病毒量接近50%, 总的释放时间长于240 h.释放出的病毒保持活性, 10 mg微球48 h对HepG2/ADM耐药细胞株转导效率可达90%以上.HepG2/ADM细胞48 h及120 h阿霉素IC50为9.72,4.15 ug;以HepG2细胞内DNR的荧光强度为对照,rAdv微球组转染后120 h与HepG2/ ADM及rAdv组比较,细胞内DNR浓度有所升高(168.6±6.97 vs 98.39±6.17;168.6±6.97 vs 112.52±9.21,t=13.68及9.69,P=0.001及0.001<0.01).诱导后HepG 2/ADM MKP高表达,转染Adv微球后48 h及120 h,MRPmRNA的表达强度较转染前明显降低,以120 h表达最弱.转染Adv微球后120 h与48 h相比,MRPmRNA/β-actin的比值分别较转染前降低16.7% 及63.6%;120 h较转染AdV48 h表达降低26.25%. 治疗组vs对照组,生理盐水组,空白微球组及rAdv 组(n=4),肿瘤生长率显著降低(0.96±0.25 vs 8.79±0.34;4.82±0.30;4.67±0.67;2.97±0.29,t=36.10, 24.43.12.28及13.81,P=0.0001,0.0009, 0.001及0.001<0.05).平均生存时间显著延长(43.6±7.4 vs 23.4±3.2;25.3±3.7;26.5±4.1;33.7±2.9, t=5.521,4.599,4.522,2.796,P=0.005,0.007, 0.007及0.049<0.05). 结论:聚乳酸-聚乙烯醇共聚物(PELA)包载反义RNA 重组腺病毒制备的微球,可有效抑制MRP的表达提高耐药细胞对化疗药物的敏感性,这为高分子化学与基因治疗的结合提供了临床应用的理论基础.

AIM: to construct the microsphere, in which recombinant adenovirus (rAdV) was encapusulated, carrying antisense multidrug resistance-associated protein (MRP) gene (as-mrp) and to investigate its effect on reversing MRP-mediated mutidrug resistance of hepatocellular carcinoma. METHODS: The microsphere was constructed with biodegradable poly-DL-lactide-poly (ethylene-glycol) encapsulating as-mrp rAdV, and its diameter, the encapsulating rate, the virus loads and the releasing kinetics were determined in vitro. Human hepatocellular carcinoma HepG2/ADM cells were transfected with the microspheres, and the fluorescence intensity was assayed after 48 hours and 120 hours. IC50 of adriamycin on drug-resistant cells was determined. The level of MRP mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR), and the ratio of MRP mRNA to p-actin mRNA (MRP/p-actin) was calculated. Intracelluar rubidomycin (DNR) concentration was examined by flow cytometry. Wistar rats were implanted with Walker-256 tumor solid piece to establish a liver cancer model. After injected with rAdV, the tumor size, growth rate and the average survival time were determined. RESULTS: The microsphere, in which as-mrp rAdV was encapusulated, was constructed successfully. Its diameter, the encapsulating rate and the virus loads were 1.765 μm, 52.4% and 5.5×1011efu/g, respectively. Almost 50% of the viruses were released within 120 h, and the total releasing time lasted more than 240 h. The released viruses remained active. More than 90% HepG2/ADM cells could be transfected with 10 mg microspheres. IC50 of adriamycin on HepG2/ADM cells 48 and 120 h after transfection were 9.72 and 4.15 ug, respectively. Intracellular DNR intensity of the cells 120 h after transfection was significantly higher than that 48 h after transfection and the non-transfected cells (168.6±6.97 vs 98.39±6.17, f = 13.68, P< 0.01; 168.6±6.97 vs 112.52±9.21,t = 9.69, P<0.01). The values of MRP/p-actin 120 and 48 h after transfection were decreased by 16.7% and 63.6% respectively as compared with that before transfection, and the value after 120 h was decreased by 26.25% as compared with that after 48 h. Compared with control group, normal saline group, rAdv group and microsphere without rAdv group, the tumor growth rate was decreased significantly in rAdv microsphere group (0.96±0.25 vs 8.79±0.34, 4.82±0.30, 4.67±0.67, 2.97±0.29; P <0.05).The mean life time was prolonged significantly (43.6±7.4 vs 23.4±3.2, 25.3±3.7, 26.5±4.1, 33.7±2.9; P<0.05). CONCLUSION: The microsphere, in which rAdV carrying antisense MRP was encapusulated, can effectively inhibit MRP expression and improve the sensitivity of drug-resistant cells. This provides an experimental basis for the combination of macromolecular chemistry and gene therapy in the treatment of hepatocellular carcinoma.