溃结安对溃疡性结肠炎模型中iNOS的影响及相关RNAi技术研究
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摘要
目的:1)构建大鼠溃疡性结肠炎动物模型并给予药物干预,检测大鼠结肠组织中iNOS mRNA表达的变化,从mRNA水平上探讨维药西帕依溃结安对iNOS表达的影响;2)建立RNAi技术平台,为借助RNAi技术进一步研究溃疡性结肠炎发病敏感点和维药西帕依溃结安治疗溃疡性结肠炎的作用靶点奠定基础。方法:采用DNCB(2,4-二硝基氯苯)复合乙酸灌肠的方法制备大鼠溃疡性结肠炎模型,将动物分为正常组、溃疡性结肠炎模型组、生理盐水(NS)阴性对照组、5-氨基水杨酸(5-ASA)阳性对照组、溃结安干预组,采用半定量RT-PCR法检测各组大鼠结肠组织中iNOS mRNA的表达,分析其表达差异。同时构建大鼠iNOS基因的真核表达GFP载体和RNA干扰载体,共转染COS-7细胞,应用Western blot方法检测抑制效率。结果:1)模型组大鼠体征、症状、结肠粘膜损伤情况、病理切片结果均提示溃疡形成;2)半定量RT-PCR结果显示大鼠结肠组织中iNOS mRNA的表达在模型组与其余各组之间差异有统计学意义(P＜0.05),而在溃结安干预组与生理盐水阴性对照组之间差异无统计学意义(P＞0.05);3)酶切和测序报告显示大鼠iNOS基因的真核表达GFP载体和RNA干扰载体构建成功,转染结果的镜下及Westernblot结果均提示iNOS-GFP融合蛋白表达下降。结论:1)大鼠溃疡性结肠炎动物模型构建成功;2)初步确定iNOS可能不是维药西帕依溃结安治疗溃疡性结肠炎的直接作用靶点;3)在COS-7细胞中应用RNAi技术成功调节了大鼠iNOS基因的表达。
Objective: 1) To construct acute ulcerative colitis models of rats and interfere with Kui Jie'an, detected the difference of iNOS mRNA expression to study the mechanism of Kui Jie'an; 2) To establish a platform of RNA interference technique, which play the basic role in research on the occurrence of ulcerative colitis and the mechanism of Kui Jie'an. Methods: Using 2, 4-dinitrochlorobenzene(DNCB) and acetic acid to induce ulcerative colitis model,rats were divided randomly into 5 groups: normal group, model group, normal salt(NS) negative control group, 5-ASA positive control group, Kui Jie'an intervention group. Using semi-quantitative RT-PCR to detect the difference of iNOS mRNA expression in rats' colons. Construct an eukaryotic expression recombonant containing the rat iNOS gene and a recombinant plasmid carrying siRNA to rat iNOS gene. After transfecting the two plasmids into COS-7 cells together, detected the inhibitive effect by Western blot. Results: 1) Pathological section of colon showed that there were ulcerations in model group; 2) The expression of iNOS mRNA showed the statistical significance between model group and the other groups (P<0.05). But the expression between Kui Jie'an intervention group and normal salt group didn't show the statistical significance (P>0.05). 3) Identifcation by enzyme cutting and sequencing showed that the two plasmids were constructed successfully. Cotransfection showed that the expression of iNOS-GFP fusion protein was decreased in COS-7 cells. Conclusion: 1) The construction of model was successful; 2) Concluded that the effect of Kui Jie'an on ulcerative colitis didn't relate to iNOS expression directly; 3) Regulated the expression of rat iNOS gene in COS-7 cells using RNA interference (RNAi) technique successfully.
Objective: 1) To construct acute ulcerative colitis models of rats and interfere with Kui Jie'an, detected the difference of iNOS mRNA expression to study the mechanism of Kui Jie'an; 2) To establish a platform of RNA interference technique, which play the basic role in research on the occurrence of ulcerative colitis and the mechanism of Kui Jie'an. Methods: Using 2, 4-dinitrochlorobenzene(DNCB) and acetic acid to induce ulcerative colitis model,rats were divided randomly into 5 groups: normal group, model group, normal salt(NS) negative control group, 5-ASA positive control group, Kui Jie'an intervention group. Using semi-quantitative RT-PCR to detect the difference of iNOS mRNA expression in rats' colons. Construct an eukaryotic expression recombonant containing the rat iNOS gene and a recombinant plasmid carrying siRNA to rat iNOS gene. After transfecting the two plasmids into COS-7 cells together, detected the inhibitive effect by Western blot. Results: 1) Pathological section of colon showed that there were ulcerations in model group; 2) The expression of iNOS mRNA showed the statistical significance between model group and the other groups (P<0.05). But the expression between Kui Jie'an intervention group and normal salt group didn't show the statistical significance (P>0.05). 3) Identifcation by enzyme cutting and sequencing showed that the two plasmids were constructed successfully. Cotransfection showed that the expression of iNOS-GFP fusion protein was decreased in COS-7 cells. Conclusion: 1) The construction of model was successful; 2) Concluded that the effect of Kui Jie'an on ulcerative colitis didn't relate to iNOS expression directly; 3) Regulated the expression of rat iNOS gene in COS-7 cells using RNA interference (RNAi) technique successfully.
引文
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