Construction Of Ad-XBP1S And Effect On The Chondrocyte Diffferentiation

Objective To construct the recombinant adenoviral vector containinghuman X-box binding protein1spliced（XBP1S）,and to study the effectof the recombinant adenovirus on the differentiation of chondrocyte.Method By using AdEasyTM adenovirus vector system, to build andpack Ad-XBP1S adenovirus pellet.Determination of virus drops for thefollow-up test. To generate XBP1S small interfering RNA (siRNA)expression constructs, Adenovirus XBP1S siRNA was constructed usingmethods described in Part I and Part II. Micro mass cultures ofC3H10T1/2、BMSC and ATDC5cells infected with either Ad-GFP (CTR)or Ad-XBP1S (siXBP1S) were incubated with300ng/ml of BMP2forvarious time points, as indicated, and the level of the genes related withchondrocyte differentiation were measured by real-time PCR. Inaddition，determining whether XBP1S and Runx2combined with theformation of protein complexes, and then discussed XBP1S regulation themolecular mechanisms of chondrocyte differentiation andhypertrophy.Results The recombined adenovirus vectors ofAd-XBP1Sand Ad-siRNA had been successfully constructed;differentialexpression of XBP1S in the course of chondrogenesis both in vitriol and vivo;determining XBP1S functions as a positive regulator of hypertrophicchondrocyte differentiation by assaying the expression of the relatedmarker gene of chondrocyte differentiation; XBP1S associates with Runx2and enhances Runx2-induced hypertrophic chondrocyte differentiation.Over expressing XBP1S enhances the expression of IHH whereas itinhibits PTHrP. Conclusion the binding of XBP1S to Runx2and acting asits cofactor for hypertrophic chondrocyte formation and affectingIHH/PTHrP signaling which is known to be important in controllingchondrocyte hypertrophy,XBP1S is a new regular factor of regulatingchondrocyte differentiation.