The Experimental Study Of The Influence By MTX And Alkylating Agent Upon SS Patients' PBMC Cultivated In Vitro

Object To investigate the effects of alkylating agent(4-hydroperoxycyclophosphamide, 4HC) and methotrexate(MTX) on apoptosis, cell cycle and the secretion level of IL-1βand TNF-αof the peripheral blood mononuclearcell (PBMC) in the patients who get Sjogren syndrome(SS). Besides, it should be eveluated that weather they have synergistic effect, how the mechanisms work and what kind of scientific evidence can be provided for the clinical practice.Method SS patients'PBMC was cultivated with the effect of MTX and 4HC of different levels(0μg/ml, 1μg/ml, 10μg/ml, 100μg/ml) alone or combined in vitro for 48 hours. Both the cell cycle and the apoptosis were analyzed by flow cytometry (FCM) with PI staining method. The level of IL-1βand TNF-αwas detected by ELISA. Factorial design was adopted at three concentration levels(0μg/ml, 1μg/ml, 10μg/ml)of MTX and 4HC. Statistical analysis was performed by the software SPSS 13.Result1. In MTX-treated group, the percentage of G0/G1 phase increased while the percentage of the S phase and G2/M phase decreased, plus, the PT% decreased either. The percentage of apoptosis increased with the increasing of MTX concentration.2. When PBMC was affected by 4HC, each phrase of cell cycle of it did not vary obviously, however, the lethal influence of 4HC on G0/G1 phase seem a litter stronger than the other phases. The percentage of apoptosis increased with the increasing of 4HC concentration.3. Under the combined treatement of MTX and 4HC, the percentage of G0/G1 phase decreased, conrespondently, the percentage of the S phase, G2/M phase and PT% imcreased. With the increasing of concentration, the percentage of apoptosis of the combination group increased. However, compared with single-drug-treated groups at the same level, the percentage of apoptosis of the combination group increased more significantly.4. The secretion level of IL-1βand TNF-αdescended gradually with the raising concentration of drugs in all three groups, especially in the combination group.5. The interaction between MTX and 4HC (P<0.05) on the apoptosis, cell cycle and the secretion level of IL-1βand TNF-αexisted which displayed as synergistic effect. Conclusion In vitro, the cell cycle-specific inhibiter MTX has synergistic effect with the cell cycle-nonspecific inhibiter 4HC. The combined treatement of MTX and 4HC is more effective to increase the apoptosis of the SS patients'PBMC which is suppressed in G0/G1 phase. Besides, the secretion level of IL-1βand TNF-α, two kinds of proinflammatory factor, are descend more significantly.