包载人基质金属蛋白酶组织抑制因子1重组腺病毒微球的制备及其表征

Preparation and characterization of microspheres encapusulating recombinant adenovirus with human tissue inhibitors of matrix metalloproteinase-1 gene

背景:高分子载药微球可控释药物,易于实现靶向给药,是近年发展快速的的新剂型。目的:制备携带人基质金属蛋白酶组织抑制因子1的重组腺病毒微球,并进行表征分析。方法:采用可降解的生物材料聚乳酸-聚乙烯醇包被携带人基质金属蛋白酶组织抑制因子1基因的重组腺病毒制成微球,体外检测其形态大小、包封率、载病毒率及释放规律。重组腺病毒微球感染人肝癌细胞株HepG2,检测感染效率,明胶酶谱分析测定微球对HepG2分泌TIMP-1的影响,Western blot检测人基质金属蛋白酶组织抑制因子1蛋白的表达,MTT实验检测细胞增殖率,体外侵袭小室实验检测细胞侵袭力。结果与结论:构建的包载人基质金属蛋白酶组织抑制因子1重组腺病毒的PELA微球直径约1.965μm,包封率为60.0%,载病毒率为10.5×108/mg,在120h内释放病毒量接近60%,总释放时间长于240h。腺病毒微球感染HepG2细胞,当微球>10mg时,感染效率可达90%以上。微球感染HepG2细胞后经Western blot检测可见人基质金属蛋白酶组织抑制因子1蛋白的表达。MTT实验及体外侵袭实验结果均提示重组腺病毒微球对HepG2细胞的体外增殖和侵袭有抑制作用,腺病毒微球组细胞增殖率较低(P<0.05),腺病毒微球感染的HepG2细胞体外侵袭力明显降低(P<0.01)。结果证实,制备的人基质金属蛋白酶组织抑制因子1重组腺病毒微球,高效缓释,可明显抑制肝癌HepG2细胞体外增殖和侵袭。

BACKGROUND：Polymer vehicle microsphere is a new form of medicine developed rapidly in recent years,which can control drug release,prolong the biological half-life of drugs,lessen side effects,and achieve targeted delivery. OBJECTIVE：To construct the polymer microsphere encapusulating recombinant adenovirus with human tissue inhibitors of matrix metalloproteinase-1（TIMP-1） gene,and to discuss its characterization. METHODS：The microsphere was constructed by biodegradable poly-DL-lactide-poly（PELA） encapsulating rAdTIMP-1,the recombinant adenovirus carrying TIMP-1. The morphous,diameter,virus encapusulating and loading rate,and releasing kinetics of the microsphere were determined in vitro. HepG2 cells were infected with the microsphere,then,the efficiency of infection was checked by fluorescent microscope. The production and expression of TIMP-1 was identified by gelatin zymography and Western blotting analysis,and the proliferation and invasiveness were detected by MTT analysis and Boyden Chamber assay,respectively. RESULTS AND CONCLUSION：The microsphere encapsulating rAdTIMP-1 was successfully constructed and its diameter,encapsulating rate,and virus loads were 1.965 μm,60.0%,and 10.5×108/mg,respectively. Almost 60% of the viruses were released within 120 hours,and the total releasing time would last more than 240 hours. The resultant microsphere encapsulating rAdTIMP-1 can efficiently infected HepG2 cells with an efficiency of infection about 90%. As a result,the infected HepG2 cells significantly increased their TIMP-1 enzyme activity and the expression of TIMP-1 was detected by Western blot. And the tumor cells’ proliferative activity and invasive ability were significantly inhibited by the microsphere. The resultant medical microsphere,with high-performance and slow-release,can markedly inhibit the in vitro biological behaviors of HepG2 cells,which may pave the way for application in prospective in vitro experiment and further liver cancer comprehensive therapy.