(siRNA?IL?17和IL?17基因逆转录病毒载体的构建及其转染致糖尿病性T细胞中IL?17的表达)Construction of the retrovirus vectors carrying the IL?17 or siRNA?IL?17 genes and expression of IL?17 gene in the transgenic diabetogenic T cells
WANG Hai?Dong，SUN Jiao，XIA Shi?Jin.
Department of Endocrinology, Institute of Geriatrics, Huadong Hospital, Fudan University, Shanghai 200040, China
(siRNA?IL?17和IL?17基因逆转录病毒载体的构建及其转染致糖尿病性T细胞中IL?17的表达)【Abstract】 Objective To construct retrovirus vectors carrying IL?17 or siRNA?IL?17 genes with Thy1.1 gene and determine the infected ability of the retrovirus vectors to diabetogenic BDC2.5 T cells. Methods The IL?17 cDNA and Thy1.1 full?length cDNA were subcloned into MIT(MSCV?IRES?Thy1.1) retrovirus vector, and the siRNA?IL?17, U6 promoter and Thy1.1 full?length cDNA were also inserted into retrovirus vector of pMND?BANSHEE.The recombined vectors were transfected 293 packaging cells by DNA calcium phosphate coprecipitation. Virus supernatant which infected pre?activated spleen cells from NOD/BDC mice was collected. After incubation, the IL?17 expression in diabetogenic T cells was detected. Results By flow cytometry, retrovirus vectors carrying IL?17 or siRNA?IL?17 genes were constructed successfully. After infection of IL?17 or siRNA?IL?17 retrovirus to pre?activated primary NOD/BDC spleen T cells, the percentages of Thy1.1+/Thy1.2+ double positive cells were (0.6±0.3)%, (7.2±2.4)%, (6.8±2.6)%, (6.4±2.4)% and (4.6±1.8)% in Mock control, MIT empty vector, Bcl?2 positive control, IL?17 and siRNA?IL?17 vector groups, respectively. After the retrovirus with IL?17 infecting BDC2.5 T cells in vitro, the expression of IL?17 was significantly higher than that of siRNA?IL?17 group and control (both of P＜0.01). Infected IL?17 retrovirus to either non?activated or activated T cells respectively in vitro, the expressions of IL?17 were distinctly higher than those of siRNA?IL?17 groups and control (both of P＜0.01). Moreover, the expression of IL?17 in the T cells activated groups were higher than those of non?activated T cells groups(P<0.01). On the other hand, the expression of IL?17 was markedly reduced in the groups of infection siRNA?IL?17, and had no difference to the control group(both of P＜0.01). Conclusions The IL?17 expression of IL?17 diabetogenic BDC2.5 T cells in vitro is higher than that of the transgenic cells of siRNA?IL?17(P<0.01). The retrovirus vectors can be used as an important tool to transfer a foreign gene into T cells efficiently.
【Key words】 Retrovirus vector； IL?17 gene；T cells；NOD mouse(siRNA?IL?17和IL?17基因逆转录病毒载体的构建及其转染致糖尿病性T细胞中IL?17的表达)
白细胞介素?17（IL?17）是初始CD4 T淋巴细胞（naive CD4 T cells）分化而来的效应性Th?17细胞产生的一种组织性促炎性细胞因子〔1〕。目前研究表明，IL?17可调节约60种编码炎症分子的基因，是多种组织细胞的促炎性介质和CD4 T细胞参与组织炎症的重要途径之一〔2〕。为明确IL?17在1型糖尿病中的作用，本研究利用Thy1.1基因作标志，分别构建了携带IL?17和siRNA?IL?17基因的逆转录病毒载体，观察其对致糖尿病性T细胞的感染能力，为进一步研究IL?17在老年糖尿病中的致病机制奠定实验基础。
1 材料与方法(siRNA?IL?17和IL?17基因逆转录病毒载体的构建及其转染致糖尿病性T细胞中IL?17的表达)
1.1 主要试剂和仪器 藻红素(PE)、异硫氰基荧光素(FITC)或Cychrome标记的抗CD4、Vβ4 TCR、CD69、CD62L、Thy1.1、Thy1.2、BDC2.5 TCR?Biotin的单抗均购自美国BD公司，细胞因子IL?17 ELISA试剂盒购自Quantikine公司，各种限制性内切酶、牛小肠碱性磷酸酶(CIAP)、T4 DNA连接酶（MBI）及核酸纯化试剂盒、小、大量质粒提取试剂盒、pGEM T Vector试剂盒均为美国Promega公司产品，MACS MultiSort Kit为德国Miltenyi Biotec产品，其他试剂均由Cincinnati Diabetes Center提供。BD FACS Calibur流式细胞仪。
1.2 引物 根据开放阅读框（ORF）序列设计IL?17的一对引物，正义：5′?AGATCTGGACGCGCAAACATGAG?3′，反义：5′?GGGTCGTCGACGGGTCTCTGTTTAG?3′；在IL?17的cDNA序列的转录起始点AUG开始，选择下游第123个碱基位点作为siRNA 的作用位点，借助Design Support System软件设计IL?17的siRNA：正义：5′?TGGACTTCCTCCAGAATGTGTTCAAGAGACAC?ATTCTGGAGGAAGTCCTTTTTTC?3′，反义5′?TCGAGAAAAAAG?GACTTCCTCCAGAATGTGTCTCTTGAACACATTCTGGAGGAAG?TCCA?3′。通过与www.ncbi.nlm.nih.gov/BLAST比对，确保了所选择的RNAi分子对作用的基因具有特异性，上述基因产品均订购于Integrated DNA Technologies公司。(责任编辑：南粤论文中心)
