The Effect Of Lentivirus-mediated Androgen Receptor Gene Overexpression On Articular Chondrocytes Chondrogenic Capacity Under Hypoxia In Vitro

Background:Hormones and their receptors are the important factors regulating the physiological function of articular chondrocytes,but the effect of androgen receptor expression on the physiological functions of chondrocytes is still unclear.This study explores the effect of androgen receptor overexpression on the chondrogenic capacity of articular chondrocytes under hypoxic culture,and to provide theoretical support for understanding the regulation of articular chondrocytes physiological functions.Materials and methods:Primary chondrocytes of knee joints of New Zealand white rabbits(6-8 weeks)were obtained by enzymatic digestion,and chondrocytes were identified by toluidine blue,cellular immunochemistry and immunofluorescence.Chondrocytes were infected with androgen receptor(Androgen Receptor,AR)overexpressing lentivirus(Lenti-AR-GFP)and green fluorescent empty vector lentivirus(Lenti-GFP),and the expression of green fluorescent protein was observed under an inverted fluoroscope at 4d,7d.The lentivirusinfected chondrocytes were cultured under normoxia(20% oxygen concentration)and hypoxia(5% oxygen concentration),and CCK-8 was used to detect the cell proliferation ability of each group at 1d,4d,and 7d after culture.The expression of cartilage-related moleculars was detected by q RT-PCR and Western-Blot at two oxygen concentrations at1 d,4d,7d,and 14 d.Results:The results of cellular immunochemistry and immunofluorescence showed that primary chondrocytes expresse specific chondrogenesis-related moleculars col2a1 and aggrecan,and the results of toluidine blue staining was blue.After the infection of lentivirus,the expression of green fluorescence was observed under an inverted fluorescence microscope,and it was positively correlated with time.Overexpression of AR can promote the proliferation of chondrocyte under hypoxic culture.The results of RealtimePCR and Western-Blot showed that overexpression of AR can promote the up-regulation of chondrogenesis-related factors col2a1,aggrecan and sox-9,and the down-regulation of chondrocyte extracellular matrix metalloproteinase enzyme mmp13 under normoxic culture.But overexpression of AR can promote the down-regulation of chondrogenesisrelated factors col2a1,aggrecan,and sox-9,and the up-regulation of chondrocyte extracellular matrix metalloproteinase enzyme mmp13 under hypoxic culture.At day 7of hypoxic culture,the expression of P-S6 protein was significantly increased after the overexpression of AR.Conclusions:After the overexpression of AR,the oxygen concentration has an effect on the physiological function of chondrocytes.Overexpression of AR can promote the downregulation of chondrogenic-specific moleculars and the degradation of chondrogenic extracellular matrix under hypoxic culture.