荧光实时定量PCR检测STAT6圈套寡核苷酸对支气管哮喘小鼠脾淋巴细胞转录IL-4mRNA的影响  

Detecting the expression of genes in STAT6 decoy oligonucleotides on transcription IL-4 mRNA by spleen iymphocyocytes of mice with bronchial asthma by SYBR Green real-time PCR

作　　者：谢鹏[1] 熊瑛 赵学飞 刘春风 王文强[1] 黄超群[3] 

机构地区：[1]川北医学院第二临床医院南充市中心医院重症医学科,637000 [2]四川医科大学附属第一医院呼吸内科,泸州646000 [3]遂宁市中医院呼吸内科,629000

出　　处：《国际呼吸杂志》2015年第17期1292-1298,共7页International Journal of Respiration

摘　　要：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-4mRNA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-4mRNA的影响。结果A、B、C、D、E组的IL-4mRNA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SYBRGreenI实时荧光PCR可以特异、准确地分析STAT6圈套0DN能下调STAT6活性，降低转录IL-4mRNA的表达。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 0.0Z5 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 Mehing 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.

关 键 词：SYBR Green I 实时定量荧光PCR 信号转导和转录激活因子 IL-4 mRNA 淋巴细胞 哮喘 

分 类 号：R562.25[医药卫生—呼吸系统]