α-胞衬蛋白-小干扰RNA真核表达载体的构建及其对原发性干燥综合征模型小鼠治疗作用的研究

Construction of eukaryotic expression vector for α-Fodrin siRNA and evaluation of its therapeutic effects on NOD mice

目的针对α胞衬蛋白（α-Fodrin）构建2段小干扰RNA（siRNA）真核表达载体，观察其对原发性干燥综合征模型小鼠（NOD小鼠）的治疗作用。方法16只8周龄的NOD小鼠采用单纯随机抽样法分为4组，对照组、空载体组、α-胞衬蛋白．siRNA1组及α-胞衬蛋白-siRNA2组，每组4只。化学合成siRNA的模板DNA4条，退火形成2条双链DNA（dsDNA），BamH Ⅰ和HindⅢ双酶切后插入线性化质粒载体pGFP—V—RS的启动子下游，重组体采用双酶切和测序鉴定。将测序鉴定成功的0．5mg／kg siRNA真核表达载体分别尾静脉注射NOD小鼠，1次／周，共2次，对照组注射等体积的磷酸盐缓冲液（PBS），空载体组注射等剂量的空载体。组织切片在荧光显微镜下观察其是否靶向到泪腺，反转录-聚合酶链反应（RT-PCR）检测NOD小鼠肺脏α-胞衬蛋白（mRNA）的表达，免疫组织化学法检测NOD小鼠泪腺和肺脏仪．胞衬蛋白的表达水平。酶联免疫吸附（ELISA）法检测NOD小鼠血清中干扰素叫和白细胞介素（IL）-17的表达水平；苏木精-伊红（HE）染色法观察各组小鼠泪腺和脏器的病理变化。统计学方法采用重复测量的方差分析。结果①成功构建2段siRNA真核表达载体；②α-胞衬蛋白-siRNA质粒载体靶向到泪腺；③α-胞衬蛋白-siRNA组与对照组和空载体组相比，α-胞衬蛋白mRNA和蛋白的表达明显降低；④α-胞衬蛋白-siRNA组[1组（4．38±1．02）pg／ml，2组（4．55±0．06）pg／ml]与对照组[（11．73±2．73）pg/ml]和空载体组[（15．40±1．99）pg／ml]相比，2次注射后小鼠血清中IL-17水平显著下降（P〈0．05），干扰素-γ水平无明显下降（P〉0．05）；⑤α-胞衬蛋白-siRNA组与对照组和空载体组相比，小鼠泪腺淋巴细胞浸润减少，肺间质炎细胞浸润明显减少。结论α-胞衬蛋白siRNA能抑制炎症反应，减轻NOD小鼠的泪腺和肺脏炎细胞浸润程度，从而抑制疾病的进展。

Objective To construct two vectors of small interfering RNA （siRNA） expressing α-Fodrin and investigate its therapeutic effects on mice model with primary Sjoegren＇s syndrome （non-obese diabetic mice, NOD mice）. Methods Sixteen 8-week-old NOD mice were randomly divided into four groups： the control group, the vector group, the α-Fodrin-siRNA1 group and α-Fodrin-siRNA2 group, 4 mice in each group. Four template DNA of α-Fodrin siRNA were chemically synthesized and annealed to two double stranded （dsDNA）, then digested by BamH Ⅰ and Hind Ⅲ. The digested double strands oligos were inserted into the downstream of .U6 promoter of linearized pGFP-V-RS vector. Recombinant were confirmed by restrictive enzyme digestion and sequencing. Then the vectors were injected throughtail veil once a week, two times in total, while mice in the control group were injected with the same dose of phosphate buffer saline （PBS）and the vector group were injected with the same dose of vector vehicle, pGFP-V-RS was labeled by green fluorescent protein（GFP） and lacriminal glands underwent pathological examination. In addition, Ihe expression of α-Fudrin mRNA in lung were detected by reverse transcription-polymerase cilain reaction （RTPCR）, and α-Fodrin protein in lacriminal glands and lung were detected by immuno-histochemistry. Sennn inte, rferon （IFN-γ）, interleukin-17 （1L-17） concentrations in each group were deteeted by enzyme linked immunosorbent assay （EI,ISA） in order to observe changes in cytokine levels. At the same time, the pathological changes of tire lacriminal glamts and organs with hema/oxylin-eosin （HE） staimng were observed. The repeat ANOVA was used for slatistical analysis. Results （1） We constructed two siRNA eukaryotie expression vector successfully; （2） α-Fodrin-siRNA couht target to the lacriminal glands. （3） Compared with the contnA gnmp aud vector vehicle group, the expression of α-Fodrin mRNA and protein were. significantly decreased in the trealment grolJps. （4） Compand with the control group [（11.73±2.73） pg/ml] and vector whicle group [ （ 15.40±1.99 ） pg/ml ], serum IL-17 levels in the treatment groups were [ α-Fodrin-siRNA 1 group （4.38 ± 1.02） pg/ml; α-Fodrin-siRNA 2 group （4.55±0.06） pghrd] significantly decreased （P〈0.05）, but IFN-γ levels in the α-Fodrin-siRNA group were not decreased significantly （P〉0.05）. （5） Comparc, d with the control group and vector vehicle group, lymphocyte infiltration of lacriminal gland and inflammatory cell infihration of alveolar and interstitial were significantly reduced in α-Fodrin-siRNA groups. Conclusion Speeific α-Fodrin siRNA can inhibi the imflammafion, and suppress the inflmnmato irffiltration of lacriminal glands and lung in mice with primary Sjsgren＇s syndrome. So tire constnlcted vectors may slow tire progression of pSS.