目的 探讨miR-199a在2,4,6-三硝基苯磺酸(TNBS)/乙醇诱导的溃疡性结肠炎(UC)大鼠中的表达情况及雷公藤多苷(TWP)对其的作用。方法 采用2,4,6-三硝基苯磺酸(TNBS)/乙醇灌肠法建立溃疡性结肠炎动物模型。对大鼠结肠组织进行大体形态损伤评分和镜下结肠损伤评分;采用芯片分析、Real-time PCR技术评估各组结肠黏膜组织中miR-199a的表达水平;针对 milwalk数据库中预测的下游靶基因mRNA用表达谱进一步筛选分析靶基因mRNA,并用Real-time PCR技术验证靶基因mRNA在各组中的表达, 最后在DAVID数据库进行pathway分析,以分析miR-199a参与UC炎症活动时所调节的靶基因mRNA。结果 TWP高剂量组结肠黏膜大体形态损伤评分、组织病理损伤评分与模型对照组间差异有统计学意义(P＜0.01)。芯片分析显示,模型对照组中miR-199a较正常对照组上调(P＜0.01),在AZA组表达较模型对照组下调(P＜0.01,P＜0.05)。miR-199a-3p在TWP中剂量组,miR-199a-5p 在TWP高剂量组中较模型对照组均下调(P＜0.05)。miR-199a 的 Real-time PCR结果显示, 模型对照组miR-199a的表达水平较正常对照组明显上调(P＜0.01);TWP的中、高剂量组和AZA组中miR-199a-3p的表达水平较模型对照组均下调(P＜0.05);TWP中剂量组miR-199a-5p的表达水平较模型对照组下调(P＜0.05)。表达谱分析显示,FASL为miR-199a的靶基因。模型对照组中靶基因FASL表达水平较正常对照组上调(P＜0.05),TWP组、AZA组中靶基因FASL表达水平较模型对照组下调,其中以AZA组的下调为显著(P＜0.01)。靶基因FASL 的Real-time PCR结果显示,模型对照组中靶基因FASL表达水平较正常对照组明显上调(P＜0.01);TWP中剂量组、高剂量组和AZA组中靶基因FASL表达水平较模型对照组下调(P＜0.01)。结论 miR-199a在TNBS/乙醇 UC大鼠中有上调表达,FASL是miR-199a的下游靶基因。TWP能改善UC的炎症活动,对UC活动时上调的miR-199a具有下调作用;FASL在UC活动时呈上调表达,TWP对miR-199a下游靶基因FASL具有下调作用。
Abstract
OBJECTIVE To explore the expression of miR-199a in ulcerative colitis (UC) rats induced by 2,4,6-trinitrobenzene sulfonic (TNBS) / ethanol and the study on the effect of TWP on them. METHODS Through injecting TNBS/ethyl alcohol acid liquid into the anus of the rats to establish the UC rat model . The colitics commom morphous damage and grade the histopathological score (CMDI) of colon mucosa injury were evaluated. Chip analysis and Real-time PCR were used to verify the expression of miR-199a in each colon mucosa tissue. Based on the expression profile, the downstream target genes mRNA in milwalk database was selected, then the expression of target genes mRNA by Real-time PCR in each group was veritied, at last the relevant signal pathway in the DAVID database was analyzed. Doing these to analyse the target gene mRNA regulated by the miR-199a in the inflammatory activity of UC. RESULTS Compared with the model group, TWP high dose group was significantly lower on gross morphological damage score and histopathological injury score(P＜0.01). Chip analysis showed that in model group, the expression of miR-199a was significantly higher than the normal group(P＜0.01), and expression of the AZA group was significantly lower than the model group(P＜0.01,P＜0.05). The expression of miR-199a-3p in medium dose group and the expression of miR-199a-5p in high dose group were significantly lower than the model group(P＜0.05).The RESULTS of Real-time PCR showed that expression of miR-199a in the model group was significantly increased than that in the normal group(P＜0.01). The expression of miR-199a-3p in TWP medium dose group, high dose group and AZA group were decreased than that in model group(P＜0.05). Meanwhile, the expression of miR-199a-5p in TWP medium dose group was decreased than that in model group(P＜0.05). The gene expression profile showed that FASL was the target gene of miR-199a. In the model group, the expression of FASL was higher than that in the normal group. The expression of FASL in AZA group was significantly decreased than that in the model group(P＜0.01). The RESULTS by the Real-time PCR of the target gene FASL showed that in the model group, the expression of FASL was higher than that in the normal group (P< 0.01). The expression of FASL in medium dose group, high dose group and AZA group were significantly decreased than that in the model group (P<0.01). CONCLUSION miR-199a is up-regulated in TNBS/Ethanol UC rats, and FASL is the downstream target gene of miR-199a. TWP can reduce the UC's inflammatory effectively and decrease the up-regulated miR-199a in UC. FASL is up-regulated in UC's inflammatory activity. TWP can reduce downstream target gene FASL of miR-199a.
关键词
溃疡性结肠炎 /
雷公藤多苷 /
miR-199a /
表达谱分析 /
FASL
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Key words
ulcerative colitis /
Tripteryginum wilfordii polyglycoside /
miR-199a /
gene expression profile /
FASL
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中图分类号：
R965
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脚注
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基金
国家自然科学基金资助项目(81273903)
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