Construction Of Microsphere Encapusulation Of Recombinant Adenovirus Carrying Antisense Multidrug Resistance-associated Protein Gene And The Study Of Reversing MRP With Hepatocellular Carcinoma

Objective: to construct the Microsphere encapusulated recombinant adenovirus carrying antisense multidrug resistance- associated protein and transfect human hepatoma resistant cells, moreover, going on hepatic arterial embolism to undersand the effect of reversing MRP with hepatocellular carcinoma.Mothods : Microsphere was made by using degradable biomaterial poly-DL-lactide-poly(ethylene-glycol) and encapulated the recombinant adenovirus carrying MRP,its diameter and the encapusulating rate were tested,the releasing test and the bioactivity of viruses incorporated were studied in vitro. The transfected cells were observed fluorescence intensity at 48 hours and 120 hours after transfecting HepG₂/ADM . In vitro drug sensitivity was measured by MTT assay, resistant index of andromycin resistant variants was determined by drawing cell dosage reaction curves.Levels of MRPmRNA expression were detected by reverse transcription polymerase chain reaction (RT-PCR) and The ratio of MRPmRNA/β -actin was detected. Intracelluar rubidomycin (DNR) concertration was examined by flow cytometry. The liver cancer model was made by inserting Walker-256 tumor solid piece into wistar rats.7 days after implantation on liver,the rats were injected through hepatic artery, the rats were divided randomly into 5 groups, group lxontrol; group 2:normal saline; group 3:microsphere carrying no viruses; group 4: recombinant adenovirus carrying antisense MRP; group 5: Microsphere encapusulation of recombinant adenovirus carrying antisense MRP.Hepatic arteries were ligated except control qroup,following by injecting ADM( 0.2ug/g,2 times every week)intraperitoneously for 14 days.The volume and weight changes of tumorand the life time were measured. The tissues including tumor and around tumor,kidney,lung were observed fluroscense intensity at 24 hours injecting ADM .Results: the Microsphere encapusulated recombinant adenovirus carrying antisense MRP was constructed successfully,its diameter was about 1.765um, the encapusulated rate was 52.4%,, carrying viral rate was 5.5 X 10 efu/mg, the release test shows that the adenovirus released more than 240 hours,50% were shed within first 120 hours,and their activity were retained ,and more than 90% HepG₂/ADM cells could be transfected when 10 mg microspheres.(the multiplicity of infection was 100).AdV microsphere inhabited the expression of mRNA in HepG₂/ADM cells,enhanced the sensitivity of HepG₂/ADM to chemotherapeutic drug and intracellular DNR concentration.The tumors growth inhabitation and the mean life length ingroup 5 were superior than other groups. The tumor tissue were observed fluorescense intensity but not other tissuesConclusions: Microsphere encapusulation of recombinant adenovirus carrying antisense MRP was effective vector retaining highly viral bioactivity and effectively reversed HepG2/ADM cells and tumor,therefore,the combination of high molecular material and gene therapy will be effective for hepatocellular carcinoma.