ABSTRACT
Aim To compare high-content cell imaging system and other methods in detecting cell proliferation, including the traditional thymidine ( 3H-TdR) incorporation method, methyl thiazolyl tetrazolium ( MTT ) method and cell counting kit-8 (CCK-8) method. Methods The fibroblast-like synovial cells (FLS) were used as the study object to observe the sensitivity and stability of FLS proliferation in different methods by using the usual proliferative stimulant tumor necrosis factor-α ( TNF-α) and the known proliferation inhibitor methotrexate at different concentrations. Results The 3H-TdR method and high-content cell imaging could detect a significant inhibitory effect of 1 nmol ·L-1 MTX on FLS cell proliferation, MTT assay and CCK-8 method could detect the significant inhibitory effect of 10 nmol· L-1 MTX on FLS cell proliferation. 3H-TdR method was found to have a large degree of discretization in the data set, with a standard deviation of 32.61% ~61.36% , and the MTT method was 11.9% ~ 17.8% , the CCK-8 method was 17.15% ~32.88% , and the high-content cell imaging system method was 12.66% ~26.54%. Conclusion The method of high-content cell imaging system is more accurate and stable for detecting cell proliferation.
ABSTRACT
<p><b>BACKGROUND</b>Signaling pathways that regulate the production of cytokines and destructive enzymes have been implicated in rheumatoid arthritis (RA) pathogenesis. There are co-relations between signaling pathways. The aim of this study was to investigate interactions and cross-talks between MEK1/2-extracellular signal-related kinase (ERK1/2) signaling and G protein-couple signaling in synoviocytes of collagen-induced arthritis (CIA) rats by the stimulation of interleukin-1 (IL-1), U0126, isoprenaline hydrochloride and aminophylline respectively.</p><p><b>METHODS</b>Twenty Sprague-Dawley (SD) rats were induced by chicken type II collagen. Synoviocytes of CIA rats were isolated and cultured. The expressions of Gi, phosphorylated MEK1/2 (p-MEK1/2) and phosphorylated ERK1/2 (p-ERK1/2) were detected by Western blotting. cAMP level and protein kinase A (PKA) activity were measured by radioimmunoassay and kinase-glo luminescent kinase assay respectively.</p><p><b>RESULTS</b>There was remarkable inflammation in CIA rats accompanied by swelling paws, hyperplastic synovium, pannus and cartilage erosion. cAMP level and PKA activity of synoviocytes decreased. Gi, p-ERK1/2 and p-MEK1/2 increased. rIL-1alpha improved the expression of Gi, p-ERK1/2 and p-MEK1/2. cAMP and PKA increased with stimulation of rIL-1alpha. U0126 inhibited Gi, cAMP and PKA of synoviocytes stimulated by rIL-1alpha. Isoprenaline hydrochloride enhanced Gi, cAMP and PKA, but had no effects on p-MEK1/2 and p-ERK1/2. Aminophylline increased cAMP and PKA, but inhibited p-MEK1/2 and p-ERK1/2.</p><p><b>CONCLUSIONS</b>Mitogen-activated protein kinases (MAPKs) and G protein-couple signaling are associated with synovitis. There are cross talks between MAPKs and G protein-couple signaling. The two signaling pathways represent potential therapeutic targets for RA.</p>