过表达TAP1上调人胶质瘤细胞株U251 HLA-I的表达

摘要

AIM: To investigate the relationship between the overexpression of transporter associated with antigen processing 1 (TAP1) and the human leukocyte antigen I (HLA-I). METHODS: The full length of TAP1 gene was obtained from the cDNA library. The lentiviral vector pSIN-EF2-IRES-GFP-puro was digested by BamH I and EcoR I, and the full length of TAP1 gene was inserted into the vector by T4 DNA ligase. Subsequently, the recombinant plasmid was transformed into Escheriehia coli DH5α cells and the correct transformant was selected. The recombinant plasmid and the Lenti-X HTX packaging mixture were co-transfected into 293T cells, and the virus particle was acquired. Human glioma U251 cells were transfected with the lentivirus. The expression of TAP1 and HLA-I was determined by real-time fluorescence quantitative PCR, Western blotting and flow cytometric analysis. RESULTS; TAP1 gene was successfully transfected into the U251 cells and stably expressed in the cell line. The expression of TAP1 in U251 cells at mRNA and protein levels increased by ( 8. 73 ± 1. 07) and (11. 71 ± 0. 83 ) folds, respectively. As a result, the mRNA expression of HLA-A, HLA-B, HLA-C (heavy chain) and β2-microglobulin (light chain) was up-regulated by (3. 51 ±0. 36) , (4. 78 ±0. 85) , (2.94 ±0.28) and (3. 23 ±0. 24) folds, respectively. The protein expression of HLA-I also increased to (3.14 ±0. 53) fold. The surface expression of HLA-I on the U251 cells transfected with TAP1 gene was largely enhanced as well. CONCLUSION : Overexpression of TAP1 up-regulates the expression of HLA-I. TAP1 plays an important role in HLA-I pro-cessing pathway.%目的:探讨抗原提呈相关转运蛋白1(TAPl)过表达对人胶质瘤细胞株U251人类白细胞抗原I(HLA-I)表达的影响.方法:培养U251细胞,构建慢病毒载体并转染U251细胞,获得稳定高表达TAP1的细胞株并设立转染空载体对照组,分别收集U251细胞组、空载体对照组和TAPl基因转染细胞组细胞的蛋白和RNA,行real-time PCR和Western blotting分析过表达的TAP1对U251细胞HLA-I表达的影响,并用流式细胞术分析U251细胞HLA-I表面呈现的变化.结果:成功建立TAP1高表达U251细胞株,TAP1 mRNA和蛋白分别升高(8.73±1.07)倍和(11.71±0.83)倍.高表达的TAP1促进U251细胞HLA-A、HLA-B、HLA-C(重链)和β2微球蛋白(轻链)mRNA表达上调,分别升高(3.51±0.36)倍、(4.78±0.85)倍、(2.94±0.28)倍和(3.23±0.24)倍,HLA-I蛋白表达升高(3.14±0.53)倍,同时U251细胞HLA-I的表面呈现明显增多,差异有统计学意义(P＜0.05).结论:过表达TAP1能促进U251细胞HLA-I表达及其在细胞表面呈现的提高.