角膜内皮细胞高表达TGF-β_1的免疫学作用

Immunological effect of TGF-β_1 in transferred corneal endothelial cells

目的探讨TGF-β1基因转染角膜内皮细胞后表达产物的免疫学活性。方法分别用RT-PCR、Western blot、免疫组织化学法检测pSecTag2-TGF-β1MP质粒转染角膜内皮细胞48h后TGF-β1基因的表达,ELISA法检测TGF-β1浓度;MTT法检测其对ConA诱导的淋巴细胞转化反应的影响;用流式细胞仪法检测其对淋巴细胞凋亡以及淋巴细胞CD3+、CD4+、CD8+、CD25+、CD69+、CD71+亚群分化的影响。结果从mRNA转录和蛋白质水平均证实TGF-β1基因在角膜内皮细胞获得高效表达。瞬时转染48h角膜内皮细胞培养上清中TGF-β1的表达为(98±3)pg/ml,高于对照组。在其作用下淋巴细胞活化增生受抑制;出现明显的亚二倍体峰;CD25+、CD69+、CD71+阳性细胞比率下降。结论基因转染角膜内皮细胞高表达TGF-β1,可抑制淋巴细胞的活化增生,促使淋巴细胞凋亡。

Objective Transforming growth factor-β1 （ TGF-β1 ） is a muhifunctional peptide, and it has been used to antagonize allograft rejection as one of the most effective immunosuppressive eytokines recently, This study aimed to investigate the immunological property of expressive production from transferred corneal endothelial cells with TGF-β1 gene. Methods The expression of TGF-β1 in the corneal endothelial cells was detected by RT-PCR, Western blotting and immunohistoehemistry. The concentration of TGF-β1 protein secreted in the corneal endothelial cell supernatant was assayed by ELISA, The effect on the spleen lymphoeytes activated by ConA was measured by 3-（4,5-dimethyhhiazol-2-yl） 2,5-diphenyl tetrazoliym bromide （MTT） eolorimetrie assay in vitro, The effect of spleen lymphocyte on the apoptosis, effect of lymphocyte subsets（ CD3^＋、CD4^＋、CD8^＋、CD25^＋、CD69^＋、CD71^＋）on the transformation were detected with flow eytometry. Results TGF-β1 MP gene was expressed in corneal endothelial cells of transferred gene group at mRNA and protein level, The concentration of TGF-β1 in the transferred cell supernatant was （98±3）pg/ml after cells were euhured for 48 hours in vitro, OD value of transferred group was the lowest in MTT test. An obvious subdiploid peak was detected in the transferred gene group with flow eytometry. Compared with control group,CD69^＋ ,CD25^＋ ,CD71^＋ lymphoeytes rate in transferred gene group was significantly decreased, Conclusion The high expression of recombinant TGF-β1 in the transferred corneal endothelial cells possesses negative immunological activity, which may be benefit for suppressing immune rejection induced by corneal transplantation.