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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.
Objective: To construct recombinant adenovirus microspheres carrying antisense multi-drug resistance associated protein (MRP) gene and to understand the therapeutic effect of recombinant adenovirus microspheres containing anti-sense RNA on reversing hepatocellular carcinoma (MRP) .Methods: The biodegradable biomaterial Materials Polylactic acid-polyvinylalcohol (PELA) was coated with recombinant adenovirus carrying antisense MRP gene to make microspheres. The microspheres size, viral load, entrapment efficiency and release pattern were determined in vitro and transfected into human The hepatocellular carcinoma cell line HepG2 / ADM was used to detect the fluorescence intensity of transfected cells 48 h and 120 h later.MTT assay was used to detect the doxorubicin (LD50) of drug-resistant cells, the intracellular fluorescence intensity was detected by flow cytometry, RT-PCR was used to observe the expression of MRP mRNA after transfection.β-actin was used as an internal reference to observe the expression of MRP mRNA after transfection.Rats volume, growth rate and mean survival time were observed after hepatic artery injection of rAdV microspheres.Results: The recombinant adenovirus microspheres with the MRP gene of about 1.765 μm in diameter, entrapment efficiency of 52.4%, viral load rate of 5.5 × 1011efu / g, released nearly 50% of the virus in 120 h and the total release time longer than 240 h The released virus remained active and 10 mg microspheres were resistant to HepG2 / ADM for 48 h The transduction efficiency of strain was up to over 90% .The IC50 of HepG2 / ADM cells for 48 h and 120 h were up to 9.72 and 4.15 μg respectively. The fluorescence intensity of DNR in HepG2 cells was the control, Compared with HepG2 / ADM and rAdv groups, intracellular DNR concentration increased (168.6 ± 6.97 vs 98.39 ± 6.17; 168.6 ± 6.97 vs 112.52 ± 9.21, t = 13.68 and 9.69, P = 0.001 and 0.001 <0.01) The expression of HepG 2 / ADM MKP at 48 h and 120 h after transfection of Adv microspheres significantly decreased the expression of MRP mRNA, The ratio of MRP mRNA / β-actin was decreased by 16.7% and 63.6% respectively compared with that before transfection, and decreased by 26.25% after 120 h compared with that of AdV transfected with AdV.Compared with control group, saline group, blank microsphere group and rAdv group (n = 4), the tumor growth rate was significantly lower (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 and 13.81, P = 0.0001, 0.0009, 0.001 <0.05). The mean survival time was significantly longer (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 and 0.049 <0.05) .Conclusion: Polylactic acid-polyvinyl alcohol copolymer (PELA) Adenovirus carrying antisense RNA prepared microspheres, can effectively inhibit the expression of MRP resistant cells to improve sensitivity to chemotherapeutic drugs, which provides the theoretical basis for clinical application in conjunction with gene therapy polymer chemistry.