Apoptosis of Tim-3~- T cells induced by secretion of galectin-9 in mice
10.3760/cma.j.issn.0254-5101.2010.02.010
- VernacularTitle:小鼠galectin-9可通过分泌形式诱导Tim-3~-T细胞凋亡
- Author:
Wentao HE
;
Jin YUAN
;
Yi XU
;
Hongmin ZHOU
;
Lanjun CAI
;
Hui GUO
;
Chao LI
;
Liqun ZUO
;
Nianqiao GONG
;
Zhonghua CHEN
- Publication Type:Journal Article
- Keywords:
Galectin-9;
Intracellular staining;
Apoptosis;
T cell
- From:
Chinese Journal of Microbiology and Immunology
2010;30(2):144-149
- CountryChina
- Language:Chinese
-
Abstract:
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.
