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Objective:To preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis(EAU).Methods:RPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73 -/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/Ladenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4 + T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73 -/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data. Results:When the stimulation mode was AMP, the proliferation of CD4 + T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the nonintervention group ( F=13.28, P<0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4 + T cells between the two groups ( F=7.78, 6.58; P>0.05). There was no statistically significant difference in the proliferation capacity of CD4 + T cells between the CD73 -/- MMP-9 inhibitor intervention group and the non-intervention group ( F=5.24, 6.12, 7.04; P>0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group( F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group ( F=15.24, P<0.01). Conclusion:MMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.
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The P2Y12 receptor antagonist is used widely in prevention and treatment of cardiovascular and cerebrovascular disease. Monitoring changes of platelet function after treatment can improve the prognosis of patients. The platelet function test is the important way to evaluate high residual platelet reactivity after antiplatelet treatment, including light transmission aggregometry (LTA), whole blood impedance aggregometry assay (WBIA), vasodilator- stimulated phosphoprotein (VASP), thrombelastogram (TEG), platelet function analyzer- 100 (PFA-100) and VerifyNow system (VerifyNow). It is very different for the reflecting ability with residual reactivity of platelets among these tests after anti-platelet therapy, and also significant difference for assessment effect. Among them, LTA is a classic method for the curative effect evaluation of anti-platelet agents, which is convenient and cheap, but it is susceptible to the operating and environment interference. The clinical application of WBIA is less, and which lacks threshold value for assessment. VASP is sensitive for the changes of platelet function, but the test is complex and expensive. TEG can monitor the inhibition ratio of drugs on anti-platelets, but it needs to verify the safety of treatment. It is not clear for sensitivity and specificity with monitoring anti-platelet agent by PFA-100. VerifyNow is effective and reliable, but the cost is high. The evidence of clinical study shows that LTA, VASP and VerifyNow can reflect the effect of platelet inhibition of P2Y12 receptor antagonists sensitively, and is associated with the risk of major adverse cardiac events (MACE) in patients with cadiovascular diseases.
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Objective To study how CD73 is shed from the retinal pigment epithelium (RPE) surface. Methods CD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared. Results LPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73. Conclusion MMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
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Objective To study how CD73 is shed from the retinal pigment epithelium (RPE) surface. Methods CD73 shedding was induced by treating RPE with lipopolysaccharides (LPS) and TNF-α. After Phospholipase C (PLC) or pan matrix metalloproteinase (MMP) inhibitors were added, surface amount of CD73 was evaluated by flow cytometry (FACS). Then selective inhibitors or their corresponding siRNAs of MMP-2 and MMP-9 were applied to the treatments of RPE; and their effects on induced CD73 shedding were evaluated by FACS. By site directed mutagenesis, mutations were introduced to Lys547-Phe548 coding sites of CD73 cDNA, which was cloned in a pcDNA mammalian expression vector. Both wt-CD73 and mutated-CD73 were over expressed in CD73-/- RPE and their induced shedding was compared. Results LPS and TNF-α induced CD73 shedding from RPE was completely blocked by the addition of pan MMP inhibitor but not PLC inhibitor. Selective MPP-9, but not MMP-2, inhibitor or its siRNA blocked CD73 shedding. In CD73-/- RPE induced CD73 shedding was happened to overexpressed wt-CD73 but not Lys547-Phe548 sites mutant CD73. Conclusion MMP-9 is responsible for shedding CD73 from RPE through hydrolyzing its Lys547 -Phe548 sites.
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Objective To investigate the expression of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor receptor-4 ( FGFR-4 ) in the papillary thyroid carcinomas ( PTC ) and clinical significance . Methods Immunohistochemistry and Western blotting for the expression of FGF-2 and FGFR-4 were performed in 89 cases of PTC and 30 cases of normal thyroid tissues ( NTT) adjacent to the tumors .Results Immunohistochemistry results showed that , FGF-2 and FGFR-4 expressions were high in thyroid carcinoma (P0.05).Analyzed by Western blotting technique ,FGF-2 and FGFR-4 expressions in thyroid carcinoma were significantly higher than that in normal tissue ,with decrease of cancer degree of tissue differentiation and significantly up regulated expression (P<0.05).Expressions of FGF-2 and FGFR-4 were in a positive linear correlation in the disease (rs=0.434,P<0.01).Conclusion The expressions of FGF-2 and FGFR-4 are correlated with papillary thyroid cancer and they participated in the process of invasion and metastasis , both of which have a positive synergistic effect .The degree of malignancy and biological behavior are meaningful and comprehensive indicators ,which provide a theoretical basis for the subsequent experimental studies of cellular and molecular biology .
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Objective To elucidate the expression diversity of IL-2R subunits in T helper(Th) cells under different activation condition and clarify its relationship with the response to IL-2 induced proliferation.Methods In vitro cultured Th1 cells were activated by plate bound anti-mouse CD3 McAb.In both activated and inactivated Th1 cells the expression of different IL-2R subunit was evaluated by real-time PCR and fluorescence staining,3H incorporation assay was adopt to measure the proliferation of the cells in response to IL-2 treatment,IL-2 affinity was determined via I125 labeled IL-2.IL-2Rα siRNA transfection was applied to knockdown CD25 expression in activated Th1 cells and its effect was confirmed by Western blot,IL-2 induced proliferation in IL-2Rα siRNA transfected Th1 cells were subsequently detected.CD4 cells were isolated from na(i)ve BALB/c mice and were also stimulated by plate bound anti-CD3 McAb.Cells were harvested at different days posterior to the stimulation,CD25 content and IL-2 induced proliferation were determined by flow cytometry and 3H incorporation assay respectively.Results In comparison with inactivated cells,only the expression of CD25but not other IL-2R subunits was significantly increased in activated Th1 cells.In accordance with elevated CD25,IL-2 affinity was also increased in activated Th1 cells,however,which resulted in decreased cell proliferation in response to IL-2 treatment.In activated Th1 cells,when CD25 expression was differently knockdown by different-dosed IL-2Rα siRNA transfection,the highest IL-2 response was observed in the cells with partially CD25 depression.Forin vitro activated na(i)ve CD4 cells the elevated CD25 expression gradually decreased and the highest proliferation response was detected at day 8 post stimulation upon IL-2 treatment.Conclusion Although CD25 is necessary for IL-2 induced proliferation,however,the excessively expressed CD25 lead to lower proliferation response.Not fully activated Thl or CD4 cells,but partially activated cells with lightly increased CD25 content possessed highest proliferation rate in response to IL-2 treatment.