IL-17和siRNA-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

目的构建含有Thy1.1细胞表面抗原(Thy-1.1cell surface antigen,Thy1.1)基因的携带IL-17或siRNA-IL-17基因的逆转录病毒载体,并观察逆转录病毒对致糖尿病性BDC2.5T细胞的转染能力。方法利用基因工程和细胞克隆技术分别将IL-17的cDNA插入MSCV-IRES-Thy1.1(MIT)逆转录病毒载体,将Thy1.1、U6增强子(U6promoter)基因和IL-17的siRNAcDNA插入逆转录病毒载体pMND-BANSHEE,采用磷酸钙沉淀法将重组载体转染293包装细胞;收集病毒上清,转染预先活化的NOD/BDC小鼠脾细胞,测定致糖尿病性T细胞的IL-17和siRNA-IL-17的表达。结果流式细胞术检测显示,成功构建携带IL-17和siRNA-IL-17的逆转录病毒载体。携带IL-17或siRNA-IL-17逆转录病毒分别感染原代培养并活化的NOD/BDC脾细胞,在空白对照组、空载体组、阳性对照组、IL-17组和siRNA-IL-17组,Thy1.1+/Thy1.2+细胞比例分别为(0.6±0.3)%,(7.2±2.4)%,(6.8±2.6)%,(6.4±2.4)%和(4.6±1.8)%;体外实验携带IL-17基因逆转录病毒转染BDC2.5T淋巴细胞后,IL-17的表达显著高于siRNA-IL-17转染组和未转染对照组(均P<0.01)。体外培养活化实验显示,携带IL-17逆转录病毒转染非活化组和活化组T淋巴细胞,转染后IL-17的表达均显著高于siRNA-IL-17转染组和未转染对照组(均P<0.01),其中IL-17转染活化组的IL-17的表达较非活化组更高(P<0.01);在siRNA-IL-17转染的非活化组和活化组,IL-17的表达显著降低,与未转染对照组相比无显著性差异(均P>0.05)。结论逆转录病毒载体能够快速、稳定地将外源基因IL-17或siRNA-IL-17转移至BDC/NODT细胞,可作为介导T细胞基因转移的重要工具。

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.