| Objective:1.Preliminarily investigate whether autophagy occurs in RASFs cultured in vitro and the autophagy in the pathogenesis of RA.2. Given different concentrations of MTX for 24 hours, then the protein and gene expression levels of autophagy marker microtubule-associated protein 1 light chain 3 and P62 were measured respectively. Through this we preliminarily investigate the role of autophagy in RA pathogenesis and the effects of MTX on autophagy.Methods: In this experiment, synovial tissues from 8 patients with RA and 5 patients with OA were collected during knee replacement. Synovial tissues were treated with enzyme digestion and then the cells were collected and cultured with different concentrations of MTX for 24 hours. The expression level of LC3 protein and p62 protein were detected by Western-blotting; the LC3 m RNA and p62 m RNA were detected by RT-q PCR; autophagosomes were detected by transmission electron microscopy.Results:1.The result of WB:The protein of LC3 and p62 were detected in RASFs and OASFs. With the concentration of Methotrexate increasing, the expression of LC3 decreased and the expression of P62 increased2.The result of RT-q PCR : LC3 m RNA and P62 m RNA were expressed both in RASFs and OASFs. Compared with the blank group of OASFs, the LC3 m RNA expression of RASFs blank group increased while P62 m RNA decreased. There is no significant difference between blank group anddrug-treated groups in RASFs.3. The result of TEM: In RASFs and OASFs autophagy formation can be detected.Conclusions:1.The level of autophagy in RASFs is higher than control group;2. Starvation-induced autophagy dramatically declined after 24 hours3.Methotrexate can inhibit autophagy of RASFs,in addition, with the increase of drug concentration, inhibition enhanced. Autophagy inhibitor treat synovial hyperplasia is expected to become possible.4.The expression of LC3 m RNA and P62 m RNA showed no change before and after MTX treatment. |
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