The Study On Mechanism Of Proliferation Of Fibroblast-like Synoviocytes Induced By TNF-α In Rheumatoid Arthritis

ObjectiveRheumatoid arthritis (RA) is a common chronic inflammatory and autoimmune disease which mainly affects synovial joint. The main pathological features of RA include abnormal hyperplasia of synovial tissues, infiltration by inflammatory cells , obvious increase of neonatal microvessel count and pannus formation , which lead to the subsequent erosion of bone and cartilage , as well as articular destruction. At present study manifesting , The main cause of synovial membrane expanding is abnormal hyperplasia of fibroblast-like synoviocytes(FLS) . The study suggested that hyperplastic fibroblast-like synoviocytes which occupy abnormal active state on function can secrete multi pro-inflammatory cytokines,grow factor,chemotatic factor,matrix protease and so on, and possess invert property , posing the erosion of bone and cartilage and acceleration of joint destruction. therefore inhibiting hyperplasia and activation of fibroblast-like synoviocy, there is significant sense for controling morbidity of RA and progression of pathogenetic condition .TNF-α(Tumor necrosis factor-alpha) , a multifulctional cytokine is produced by activated monocytes and macrophages. Because of different tissue-specific, TNF-αpromote apoptosis of majority tumor cells, on the contrary it also may promote proliferation of T lymphocyte,B lymphocyte,thymocyte. TNF-αmay promote proliferation of FLS, however, the concrete mechanism remains to be fully understood.TNF-αeduces biological role by binding to TNF receptors on different cell surface, TNF-αhas two receptors which can be divided into TNFRI and TNFRII, and blongs to I type membrane protein, tumor necrosis factor receptor family, their ectodomain have 28% homology, however, their internal structural domain are completely different, TNFRI has death domain, TNFRII hasn't death domain, TNFRI is almost expressed on all cells, but TNFRII is expressed on immune cell and endothelial cell, both of receptors mediate alonely distinct signals transduce pathway independently or together.Because TNF-αproduce effect through its receptors, in our empirical study, we suppose to observe proportional change of TNFRI and TNFRII expressed in RA FLS through TNF-αof invariable concentration affecting in RA FLS, accordingly approaching possible mechanism that TNF-αinduces proliferation of RA FLS.Methods1. Isolation and culture of RA FLS: synovial cells were isolated from the sterile fresh ynovial tissues by collagenase digestion, cells between passage 3 and 9 were used for experiments.2. After RA FLS was treated with TNF-α, HE stain, observing morphological change of FLS.3. Proliferative response to distinct concentration in RA FLS was observed by Cell growth inhibiting experiment (MTT).4. After RA FLS was treated with invariable concentration TNF-α, observing proportional change of TNFRI and TNFRII expressed in RA FLS by RT-PCR.Results1. Observing morphological changes of RA FLS: After RA FLS was stimulated 48h with 20ng/ml TNF-α, HE stain, compared with control group(0ng/ml), observing cell morphology under Microscope. density of FLS stimulated with 20ng/ml TNF-αenhanced, but cell morphology hadn't obvious change, cell growing fascl , cell morphology was main fusiform , two-pole spine was slender, majority karyomegaly, present round and ellipse under high power lens, nuclei were manifest, the number were 1-3.2. Proliferative response to distinct concentration TNF-αin RA FLS: After FLS were stimulated 72h with distinct concentration TNF-α, MTT results displayed that growth rate of FLS in lng/ml concentration group compared with control group had not difference, other concentration groups compared with control group had significant difference.(P<0.01), the proliferative response of RA synovial cells was increased in a dose-dependent manner and reached a plateau at 10ng/ml concentration.3. The result of RT-PCR analysis: When RA FLS were unstimulated by TNF-α, expression ofTNFRImRNA was obvious higher TNFRIImRNA, their ratios was 2.449, upon TNF-αstimulation, their ratios was 1.461 in 6h, their ratios was 0.951 in 12h, their ratios was 0.512 in 24h, the ratios decreased reaching at the minimal level, both ratios was 0.648, following time change, expression rations of TNFRI and TNFRII took place change, expression of TNFRI was downregulated, expression of TNFRII was upregulated and reached a plateau in 24h, it took on time-dependent manner.Conclusion1. TNF-αhad the function to inhibit the apoptosis of RA FLS and promote the proliferation of FLS in vitro.2. TNF-αcould upregulate the expression of TNFRII mRNA and downregulate expression of TNFRI mRNA, decreasing the apoptosis of RA FLS , thereby promoting the proliferation of RA synovial tissues.