b7h4靶向shrna干扰小鼠间充质干细胞c3h10t12的构建及其生物学功能的初步分析word格式论文.docxVIP

优秀毕业论文 精品参考文献资料 B7-H4 靶向 shRNA 干扰小鼠间充质干细胞系 C3H10 T1/2 的构建及其生物学功能的初步研究 英文摘要 The construction and primary study for the biological character of the mouse mesenchymal stem cell line C3-H10T1/2 with B7-H4 knocked down by shRNA interference Abstract Objective To construct B7-H4 targeted shRNA stable transfected mouse mesenchymal stem cell line C3H10T1/2 (C3H10), and study the role and mechanism of the immune inhibition of C3H10 cells mediated by B7-H4. Methods The expression of B7-H4 on C3H10 was detected by flow cytometry, Western blot; the lentiviral vectors with mouse B7-H4 target shRNA were transfected into the C3-H10 cells (C3H10-shRNA-B7-H4). The shRNA transfection efficiencies were measured by detecting the green fluorescence on C3-H10 cells using the fluorescence microscope and flow cytometry (FCM). The transfected cells were sorted by puromycin. The knocked down effect of the different sequence of specific shRNAs were measured by detecting the protein by flow cytometry and Western blot, or mRNA by RT-PCR. The C3H10 cells with highest B7-H4 knocked down efficiency was defined as C3-H10-shRNA-B7-H4 cell line, which could be passaged for many times. These cells were co-cultured with the PHA activated mice spleen lymphocytes, and the activation , proliferation, cell cycle and apoptosis of the spleen lymphocytes were detected by flow cytometer analysis. Experimental allergic encephalitis (EAE) mice models were established, and the in vivo experiment group were set as: sham operated group, EAE model group, C3H10 cells implanted group, C3H10-shRNA-NC cells implanted group, and C3H10-shRNA-B7-H4 cells implanted group. In the C3H10 cells implanted group, C3H10-shRNA-NC cells implanted group, and C3H10-shRNA-B7-H4 cells implanted group, 8 days after the immunization, C3H10 cells, C3H10 shRNA - NC cells and C3H10 - shRNA -B7-H4 cells were injection to EAE mice respectively, and the neurological impairment function score and weight were measured everyday. 英文摘要 B7-H4 靶向 sh