The Experimental Study Of RNAi Targeting Atp6i Gene To Inhibit The Bone Absorption Function Of Osteoclasts

Objective To select high-efficient, high-specific siRNAs which can block the translantion of mRNAs transcripted from atp6i gene of rats osteoclasts, which are cultured in vitro by inducing, then observe the influence about bone absorption of osteoclasts after the target gene which has been transcripted is blocked.Methods To dissect The SD male rats of 3-4 weeks, take and wash out the femurs and shin bone marrows of two hind legs, with 1, 25- (OH)₂D₃ as inducing pharmaceutical, and further set up osteoclasts system in vitro, which is cultured with bone slices made from cattle femurs . The osteoclasts system is divided into 6 groups: control group , BLANK group , siRNA1 group , siRNA2 group , siRNA3 group and siRNA4 group . After the osteoclasts system has been induced and cultred in vitro for 6 days, above siRNAs are transfected into each group separately through transfecting reagent kit and marking reagent kit made in Allele Biotechnology Pharacology Inc Company (U.S.).72 hours after transfecting , bone slices culturesd with osteoclasts in each group are taken out, fixed and washed in supersonic wave machine for 1-2 minutes , stained with 1% toluidine blue directly. Bone absorption pits are observed with microscope and recorded by photo-taking system , then computer picture analyticl system is utilized to calculate the entire area of bone absorption pits on each bone slice, the result is expressesed by means of the area of bone absorption pits / slice (mm² / slice). The datas of every group...