CAJ | 학술논문

目的：研究STAT6圈套寡核苷酸（ODN）对支气管哮喘（简称哮喘）小鼠脾淋巴细胞转录IL‐4 mRNA的影响作用。方法实验细胞分组：空白组（A组）、哮喘OVA组（B组）、治疗哮喘OVA组（C组）、干扰哮喘OVA组（D组）、脂质体哮喘OVA组（E组）。设计并人工合成STAT6圈套ODN及无序ODN ，全硫代修饰的ODN。用OVA和氢氧化铝复制哮喘模型，用淋巴细胞分离液分离小鼠脾淋巴细胞，体外培养后，导入由阳离子脂质体2000转染剂携带的圈套ODN进入淋巴细胞培养，抽提RNA进行反转录，建立实时荧光聚合酶链反应（PCR）反应条件，通过比较ct进行基因表达的相对定量分析。观察圈套ODN的转染对脾淋巴细胞转录IL‐4 mRNA的影响。结果 A、B、C、D、E组的IL‐4 mRNA分别表达为：0．02545±0．00433，0．06742±0．00128，0．03151±0．00530，0．06504±0．00775，0．05952±0．00561。A组和C组低于B组、D组和E组，其差异有统计学意义（ t＝6．204，P＜0．01），C组和A组间差异无统计学意义（t ＝1．310，P ＞0．05），B组和D组、E组之间的差异无统计学意义（P ＞0．05）。结论熔解曲线分析结果证明了PCR反应的特异性，应用SYBRG reen I实时荧光PCR可以特异、准确地分析STAT6圈套ODN能下调STAT6活性，降低转录IL‐4 mRNA的表达。
목적：연구STAT6권투과핵감산（ODN）대지기관효천（간칭효천）소서비림파세포전록IL‐4 mRNA적영향작용。방법실험세포분조：공백조（A조）、효천OVA조（B조）、치료효천OVA조（C조）、간우효천OVA조（D조）、지질체효천OVA조（E조）。설계병인공합성STAT6권투ODN급무서ODN ，전류대수식적ODN。용OVA화경양화려복제효천모형，용림파세포분리액분리소서비림파세포，체외배양후，도입유양리자지질체2000전염제휴대적권투ODN진입림파세포배양，추제RNA진행반전록，건립실시형광취합매련반응（PCR）반응조건，통과비교ct진행기인표체적상대정량분석。관찰권투ODN적전염대비림파세포전록IL‐4 mRNA적영향。결과 A、B、C、D、E조적IL‐4 mRNA분별표체위：0．02545±0．00433，0．06742±0．00128，0．03151±0．00530，0．06504±0．00775，0．05952±0．00561。A조화C조저우B조、D조화E조，기차이유통계학의의（ t＝6．204，P＜0．01），C조화A조간차이무통계학의의（t ＝1．310，P ＞0．05），B조화D조、E조지간적차이무통계학의의（P ＞0．05）。결론용해곡선분석결과증명료PCR반응적특이성，응용SYBRG reen I실시형광PCR가이특이、준학지분석STAT6권투ODN능하조STAT6활성，강저전록IL‐4 mRNA적표체。
Objective To explore the effect of STAT6 decoy oligonucleotides (ODN ) on transcription IL‐4 mRNA by spleen lymphocyocytes of mice by bronchial asthma (asthma) .Methods Mouse spleen lymphocytes were divided into 5 groups:a unstimulation group (group A ) ,a ovalbumin (OVA) group (group B) ,a treated OVA group (group C) ,a intervention OVA group (group D) ,a liposome OVA group (group E) .Double strand of decoy oligonucleotide and scrambled oligonucleotide was designed and synthesized ,and fluorescein conjugated PS‐ODN .The asthma model was duplicated with OVA absorbed to aluminum hydroxide ,spleen lymphocytes of mice were separated ,then the STAT6 decoy ODN was introduced into the lymphocytes with cationic liposome transfection and cultured in vitro . Then collected for RNA extraction and reverse transcription ,real‐time PCR was carried out after optimization of PCR parameter .The relative quantification analysis was performed with comparative threshold cycle method .The effect of STAT6 decoy ODN on IL‐4 mRNA transcribed by spleen lymphocytes was observed .Results The level of IL‐4 mRNA expression in group A ,group B ,group C , group D and group E was 0 .025 45 ± 0 .004 33 ,0 .067 42 ± 0 .001 28 ,0 .031 51 ± 0 .005 30 ,0 .065 04 ± 0 .007 75 ,0 .059 52 ± 0 .005 61 .The expression of IL‐4 mRNA was lower in group A and group C than group B ,group D and group E ( t = 6 .204 ,P < 0 .01) .There was no evident change of IL‐4 mRNA expression among group C and group A ,group B and group D ,group E ( P>0 .05) .Conclusions Melting curve analysis confirmed the specificity of the amplification products .The developed real time PCR assay for detecting the expression of genes related to IL‐4 mRNA proliferation with high specificity and good quality ,STAT6 decoy ODN can downregulate activeness of STAT6 and lower transcription of IL‐4 mRNA .