炎症相关miRNAs在LPS诱导的小鼠乳腺上皮细胞炎性反应中的表达及miR-223真核表达载体的构建

发布时间：2018-03-25 05:21

本文选题：炎症相关miRNAs　切入点：乳腺上皮细胞　出处：《畜牧兽医学报》2016年01期

【摘要】：小分子RNA(miRNA)是近年来发现的一类内源性、非编码单链小RNA,其在乳腺组织免疫防御过程中发挥的调控作用逐渐引起了人们的关注。作者拟用qRT-PCR检测炎症相关miRNAs在LPS诱导的小鼠乳腺上皮细胞炎症反应中的表达变化;构建3种miR-223真核表达载体并检验重组载体的转染效率。用不同浓度LPS处理小鼠乳腺上皮细胞(EpH4-Ev)48h,qRT-PCR检测miR-223、miR-146a、miR-181a和miR-155的表达变化。以EpH4-Ev细胞基因组DNA为模板,PCR扩增pre-miR-223序列,以质粒Minicircle、pcDNA3.1(+)和转座子piggyBac为母本构建3种miR-223真核表达载体,将其转染EpH4-Ev细胞,通过观察荧光、qRT-PCR检测miR-223的表达,评价重组质粒转染效率。结果:qRT-PCR检测结果表明,LPS诱导EpH4-Ev细胞炎症反应时,4种炎症相关miRNAs的表达均显著(P0.05或P0.01)增加,并呈现不同程度的剂量依赖性;重组质粒转染EpH4-Ev细胞后,qRT-PCR检测结果显示,Mini-miR-223质粒转染组的miR-223表达水平无统计学差异(P0.05);PBmiR-223和pcDNA-miR-223转染EpH4-Ev细胞后,miR-223的表达均极显著(P0.001)增加。本研究表明炎症相关miRNAs参与LPS诱导小鼠EpH4-Ev细胞的炎性反应过程。成功构建3种miR-223真核表达载体,为后续miRNAs在乳腺炎症反应中的功能研究奠定基础。
[Abstract]:Small molecule RNAs miRNAs are a kind of endogenous RNAs that have been discovered in recent years. The role of non-coding single-stranded small RNAs in the immune defense of mammary gland has attracted more and more attention. The authors intend to use qRT-PCR to detect the expression of inflammatory miRNAs in mouse mammary epithelial cells induced by LPS. Three miR-223 eukaryotic expression vectors were constructed and the transfection efficiency of the recombinant vectors was tested. The expression changes of miR-2223 miR-146a miR-181a and miR-155 in mouse mammary epithelial cells treated with different concentrations of LPS were detected by qRT-PCR. Pre-miR-223 sequences were amplified by PCR using genomic DNA of EpH4-Ev cells as template. Three kinds of miR-223 eukaryotic expression vectors were constructed from plasmid Minicircleus pcDNA3.1 () and transposon piggyBac as female parent, and were transfected into EpH4-Ev cells. The expression of miR-223 was detected by fluorescence qRT-PCR. Results the results showed that the expression of four kinds of inflammatory miRNAs was significantly increased in EpH4-Ev cells induced by LPS-induced inflammation (P0.05 or P0.01), and showed a dose-dependent manner. After transfection of the recombinant plasmid into EpH4-Ev cells, the results of qRT-PCR showed that there was no significant difference in the expression level of miR-223 between the two groups. The expression of miRNAs in EpH4-Ev cells transfected with pcDNA-miR-223 was significantly higher than that in the control group (P0.001). This study indicated that the inflammation related miRNAs was involved in LPS inducement. Three kinds of miR-223 eukaryotic expression vectors were successfully constructed by guiding the inflammatory reaction of mouse EpH4-Ev cells. To lay a foundation for the study of the function of miRNAs in the inflammation of mammary gland.
【作者单位】：河南农业大学农业部动物生化与营养重点开放实验室;河南农业大学牧医工程学院;
【基金】：国家自然科学基金(31470120)
【分类号】：S852.3

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