Construction Of Transgenic Human Toll-like Receptor 3 Tupaia Hepatocytes Model

Objective:we inserted the human Toll-like Receptor 3(hTLR3)gene into Tupaia hepatocytes following modified genetically,in order to establish transgenic hTLR3 Tupaia hepatocyte model.Laying the foundation for in-depth study of the role of TLR3 and its signal transduction pathway and antiviral mechanism.At the same time,providing cellular basis for the drug screening of anti hepatitis b virus.Methods:(1)We isolated primary Tupaia hepatocytes by the two-step collagenase perfusion method and optimized the top culture pattern by cocultivation with cells of feeder layer.The purity of primary Tupaia hepatocytes were comprehensively identified by the observation of cell morphology,PAS staining and anti-keratin18 immunocytochemistry staining.(2)The hTLR3 gene was amplied by PCR method.The amplification products were connected with p LV to construct the recombinant plasmids of p LV-hTLR3.At the same time,the recombinant plasmids would undergo colony PCR assay and sequencing identification.Then,We would make p LV-hTLR3 recombinant plasmid and packing coated plasmid to transfect 293 T cells by employing liposomal-mediated transfection method,in order to fulfil the packing of lentivirus.We would collect virus supernatant and continue infection in 293 Tcells,and after several rounds of amplification,we determined the titer of lentivirus by coubling dilution.(3)Transgenic hTLR3 Tupaia hepatocytes were constructed by using the condensed and packaged lentivirus solution to transfect primary hepatocyte.And it was comprehensively identified by the observation of expression of transgenic cell under a fluorescence microscope fluorescent and molecular biology method.Results:(1)Each Tupaia belangeri usually can get 3.0×10~7 hepatocytes,the cell survival rate was 85%±5%.After 4 hours hepatocytes began to stick wall,ovoid shape.After 24 hours,hepatocytes completely sticked wall,cell volume obviously become large,flat growth,we can clearly see double nucleus and three nuclei.After 48 hours the hepatocytes were interconnected and gradually formed islands.Observing cells morphology were uniform under the microscope,hepatocytes cytoplasms were dyed respectively amaranth and tan by PAS staining and anti-keratin18 immunocytochemistry staining and its purity up to 90%.(2)By exploring the dosage of lipidosome and ratio of four kinds of plasmids in orthogonal test in the packing of lentivirus,We established the optimal conditions about 2.75μL the dosage of lipidosome and the ratio of four plasmids system(vector plasmid,p Gag/Pol,p Rev,p VSV-G)for 10:7:3:3,and to conduct the package of virus,finally achieving the lentivirus particles of 1.2×10~7TU/m L titer.(3)The concentrated lentivirus successfully transfected primary Tupaia hepatocytes,with the transfection efficiency about 60%.Conclusions:(1)The obtained primary Tupaia hepatocytes were confirmed by isolation 、purification 、cultivation and identification,making substantial cellular basis for subsequent experiments.(2)To successfully construct lentivirus vector carrying the human TLR3 gene.Next,the virus subject to packing and purification was available to transfecting primary Tupaia hepatocytes,making foundations for the fabrication of transgenetic cell.(3)After the preliminary appraisal,building successfully transgenic hTLR3 Tupaia hepatocyte model.