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Objective To prepare recombinant adenovirus pAd-gal-9 containing murine galectin-9 and explore galectin-9's pro-apoptotic effect on T lymphocytes. Methods The recombinant adenovirus plas-mid pAd/CMV/V5-DEST-gal-9 was prepared by conventional molecular cloning and LR reaction. The pAd/ CMV/V5-DEST-gal-9 linearlized by Pac I was transfected into 293A cells with Lipofectin 2000. Eight days after transfection, the 293A cells were subjected to freeze/thraw circle for three times and the supernatant was collected after centrifugation. Higer titer pAd-gal-9 was produced by large-scale infection of 293A cells with the supernatant containing pAd-gal-9. The supernatant was condensed to get purified pAd-gal-9 by CsCl density gradient centrifugation. After titer determination with gradient dilution of harvested pAd-gal-9 infec-tion in 293A-seeded 96-wells, pAd-gal-9 was used to infect the CHO cell line. Immunohistological assay, Western blot and flow cytometry were employed to ascertain the subcellular location expression of galectin-9. We added solid-phase transgenic CHO cells or freshly-cultured supernatant to medium containing activated T cells to detect the pro-apoptotic effect of galectin-9. Results The pAd-gal-9 was prepared successful. Im-munohistochemical staining of CHO infected with pAd-gal-9 confirmed that galectin-9 was expressed in the cytosol. Intercellular staining indicated that mean fluorescence intensity of galectin-9 was significantly higher in pAd-gal-9-infected CHO group than control group. Supernatant from pAd-gal-9-infected CHO promoted the apoptosis of T cells. The percent of apoptotic T cells was higher than the Tim-3 positive T cells. Conclu-sion CHO infected with pAd-gal-9 can secret galectin-9 to promote the apoptosis of activated T cells via Tim-3-independent mechanisms.
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0.05 ). Conclusion FTY720 is an effective specific and reversible immunosuppressant.
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Objective To investigate the influence of FTY720 on the lymphocytes and their recovery after withdrawal of FTY720,and on the changes in T cell subsets of inbred mice so as to direct the administration of FTY720. Methods Male inbred C57/bl mice were given FTY720 at the dose of 3 mg/kg per day by oral gavage once a day for 14 days. The PBLs were counted and T cell subsets were analyzed by flow cytometry at the selected time points. Results When FTY720 was administrated, the PBLs showed a sharp decrease and reached the minimum 4 hours later.Then the PBLs remained stable during the whole pe- riod of administration.The PBLs didn’t restore immediately when the reagent was withdrawn,but increased slowly 2 weeks later and completely recovered 6 weeks later.The changes of T cells were similar to PBLs.CD - 8 subsets showed relatively higher sensitivity to FTY720. Conclusions FTY720 is a safe reagent which has rapid and specific effects on PBLs,and the changes in PBLs are reversible.The effect of FTY720 on T cell subsets is similar to that on PBLs.CD - 8 T cells are relatively more sensitive to FTY720.