CAJ | 학술논문

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DNA 4条,退火形成2条双链DNA(dsDNA),BamH Ⅰ和Hind Ⅲ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0.5 mg/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α-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-γ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.
목적 침대α포츤단백(α-Fodrin)구건2단소간우RNA(siRNA)진핵표체재체,관찰기대원발성간조종합정모형소서(NOD소서)적치료작용.방법 16지8주령적NOD소서채용단순수궤추양법분위4조,대조조、공재체조、α-포츤단백-siRNA1조급α-포츤단백-siRNA2조,매조4지.화학합성siRNA적모판DNA 4조,퇴화형성2조쌍련DNA(dsDNA),BamH Ⅰ화Hind Ⅲ쌍매절후삽입선성화질립재체pGFP-V-RS적계동자하유,중조체채용쌍매절화측서감정.장측서감정성공적0.5 mg/kg siRNA진핵표체재체분별미정맥주사NOD소서,1차/주,공2차,대조조주사등체적적린산염완충액(PBS),공재체조주사등제량적공재체.조직절편재형광현미경하관찰기시부파향도루선,반전록-취합매련반응(RT-PCR)검측NOD소서폐장α-포츤단백(mRNA)적표체,면역조직화학법검측NOD소서루선화폐장α-포츤단백적표체수평.매련면역흡부(ELISA)법검측NOD소서혈청중간우소-γ화백세포개소(IL)-17적표체수평；소목정-이홍(HE)염색법관찰각조소서루선화장기적병리변화.통계학방법채용중복측량적방차분석.결과 ①성공구건2단siRNA진핵표체재체；②α-포츤단백-siRNA질립재체파향도루선；③α-포츤단백-siRNA조여대조조화공재체조상비,α-포츤단백mRNA화단백적표체명현강저；④α-포츤단백-siRNA조[1조(4.38±1.02) pg/ml,2조(4.55±0.06) pg/ml]여대조조[(11.73±2.73) pg/ml]화공재체조[(15.40±1.99) pg/ml]상비,2차주사후소서혈청중IL-17수평현저하강(P＜0.05),간우소-γ수평무명현하강(P＞0.05)；⑤α-포츤단백-siRNA조여대조조화공재체조상비,소서루선림파세포침윤감소,폐간질염세포침윤명현감소.결론 α-포츤단백siRNA능억제염증반응,감경NOD소서적루선화폐장염세포침윤정도,종이억제질병적진전.
Objective To construct two vectors of small interfering RNA (siRNA) expressing α-Fodrin and investigate its therapeutic effects on mice model with primary Sj(o)gren'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,the expression of α-Fodrin mRNA in lung were detected by reverse transcription-polymerase chain reaction (RTPCR),and α-Fodrin protein in lacriminal glands and lung were detected by immuno-histochemistry.Serum interferon (IFN-γ),interleukin-17 (IL-17) concentrations in each group were detected by enzyme linked immunosorbent assay (ELISA) in order to observe changes in cytokine levels.At the same time,the pathological changes of the lacriminal glands and organs with hematoxylin-eosin (HE) staining were observed.The repeat ANOVA was used for statistical analysis.Results ① We constructed two siRNA eukaryotic expression vector successfully; ② α-Fodrin-siRNA could target to the lacriminal glands.③ Compared with the control group and vector vehicle group,the expression of α-Fodrin mRNA and protein were significantly decreased in the treatment groups.④ Compared with the control group [(11.73±2.73) pg/ml] and vector vehicle 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) pg/ml] significantly decreased (P＜0.05),but IFN-γ levels in the αt-Fodrin-siRNA group were not decreased significantly (P＞0.05).⑤ Compared with the control group and vector vehicle group,lymphocyte infiltration of lacriminal gland and inflammatory cell infiltration of alveolar and interstitial were significantly reduced in α-Fodrin-siRNA groups.Conclusion Specific α-Fodrin siRNA can inhibit the inflammation,and suppress the inflammatory infiltration of lacriminal glands and lung in mice with primary Sj(o)gren's syndrome.So the constructed vectors may slow the progression of pSS.