【作者】 纳斯尔阿吉·买买提艾力；

【导师】 库热西·玉努斯； 帕尔哈提·阿布都热依木；

【作者基本信息】 新疆医科大学 ， 生物化学暨分子生物学， 2007， 硕士

【摘要】 目的:研究溃结安对溃疡性结肠炎（Ulcerative colitis,UC）大鼠白细胞介素-6（interleukin 6,IL-6）基因mRNA水平的影响及构建具有特异性阻断大鼠IL-6基因的小干扰RNA（small intefferening RNA,siRNA）表达体系,为确定溃疡性结肠炎发病基因及研究维药西帕依溃结安治疗溃疡性结肠炎作用机理提供新的方法。方法:Wistar大鼠分为正常组,溃疡性结肠炎模型组,生理盐水阴性对照组组,5-氨基水杨酸阳性对照组,溃结安干预组共5组,用2,4-二硝基氯苯（DNCB）复合乙酸方法造成溃疡性结肠炎大鼠模型,从结肠中提取总RNA经逆转录-聚合酶链反应（RT-PCR）得到目的基因的cDNA,经PCR得到目的基因的扩增片段。应用siRNA设计软件在大鼠IL-6的mRNA上寻找特异性的短核苷酸序列并设计合成两条互补的小发夹结构的DNA序列,经退火后形成双链DNA片段,采用基因克隆技术,将其克隆到pSilencer^TM1.0-U6的载体中,转化DH5α大肠杆菌,提取质粒,酶切方法和测序法对重组体进行鉴定。结果:溃结安降低了大鼠IL-6基因mRNA水平。酶切和序列测定表明,成功构建了大鼠IL-6基因的siRNA表达体系。结论:大鼠IL-6基因表达的变化可能与溃疡性结肠炎的发生发展相关联;溃结安治疗作用可能与其下调了IL-6基因表达有关;成功构建siRNA表达体系,为确定溃疡性结肠炎发病基因及研究维药溃结安治疗溃疡性结肠炎的靶点打下了基础。更多还原

【Abstract】 Objective:To investigate the expression of intedeukin 6（IL-6）in intestinal tissue of rat with Ulcerative Colitis（UC）and further studies the pathogenesis of UC.To construct the specific small interfereing RNA（siRNA）expression vector that can block the rat IL-6 gene by RNA interference（RNAi）technique,which provides a new approach to determine the onset gene of Ulcerative Colitis and investigate the target of the Uygur medicine Kuijie’an（KJA）that can treat the UC.Methods:Wistar rats were derided into 5 groups:control group,UC groups,NS group,5-ASA group,UC rats induced by DNCB and acetic acid.Observe and assay the expression of IL-6 gene by RT-PCR.According to the encoding sequence of mRNA of rat IL-6 provided by Genbank,two pieces of specific hairpin structure short chain oligonucleotide sequences were designed and synthesized by using the siRNA software.The annealed oligonucleotide fragment were subcloned into pSilencer ^TM1.0-U6 expression vector by gene clone technique and transformed into DH5αE.coli.Finally the plasmid evaluated by using enzyme cutting and sequencing. Results:Expression of IL-6 gene remarkably increased in intastinal tissue of rats with UC as compared with control group;but it remarkably decreased after theraphy of KJA. Identification using enzyme cutting and sequencing showed that the IL-6 siRNA expression vector was constructed successfully.Conclusion:It demonstrated tentavtively that KJA had an effect on UC.Successful construction of rat IL 6 siRNA expression vector will facilitate further studies of determine the onset gene of Ulcerative Colitis and investigate the target of the Uygur medicine KJA that can treat.the Ulcerative Colitis.更多还原