甘草miRNA对人免疫细胞基因表达谱的影响研究
发布时间：2018-12-30 11:50
【摘要】：背景及目的:前期高通量测序结果显示,miRNA156、miRNA1511、miRNA8155在甘草水煎剂miRNA中丰度较高。本研究通过细胞形态学、细胞的增殖情况及免疫相关基因表达方面探究甘草的miRNA156、miRNA1511、miRNA8155对人免疫细胞的影响。通过表达谱测序发现差异基因并加以验证,从而为甘草药效新机制的相关研究开拓新的思路。方法:1、根据高通量测序结果中的miRNA156、miRNA1511、miRNA8155序列,设计合成相应的反转录及检测引物,通过RT-PCR确认甘草组织miRNA中确实存在前期从其水煎剂中通过高通量测序技术鉴定出来的几种microRNA分子:miRNA156、miRNA1511、miRNA8155。2、使用FAM标记的小分子DNA来验证脂质体Lip2000的转染效率以建立可靠的转染方法;设计合成miRNA156、miRNA1511和miRNA8155相对应的mimic;将其分别通过Lip2000转染人的免疫细胞(PBMC、CIK、Jurkat细胞系),并以无关的miRNA NC mimic作为阴性对照组;使用显微镜观察并进行细胞计数来比较各组间细胞形态和细胞增殖情况;使用RT-PCR检测各组细胞间部分免疫相关基因的表达变化情况。3、将各miRNA mimic转染健康人PBMC细胞,培养24小时后,提取细胞的总RNA,进行表达谱测序,分析各组细胞基因表达的差异,并通过GO及KEGG富集分析对差异基因进行分类和注释。4、使用Real-time PCR及Western-blotting技术分别从基因水平和蛋白水平来验证表达谱测序结果的可靠性。结果:1、通过RT-PCR检测目标miRNA,发现甘草组织总RNA中确实能检测到miRNA156、miRNA1511、miRNA8155,验证了前期高通量测序结果,为后续研究奠定了可靠基础。2、通过用Lip2000转染FAM标记的小分子DNA,倒置荧光显微镜下观察发现Lip2000有较高的转染效率。制备合成了miRNA156、miRNA1511、miRNA8155相对应的miRNA mimic,并成功通过Lip2000转染进人的免疫细胞(PBMC、CIK、JURKAT细胞);实验组与对照组间细胞形态有明显差异;甘草的三个miRNA对PBMC细胞和CIK细胞生长有增殖作用,而对急性T细胞白血病细胞系JURKAT的生长存在明显抑制作用。RT-PCR结果显示,甘草miRNA使PBMC、CIK、JURKAT细胞中部分免疫相关细胞因子、信号分子和转录因子的表达水平发生了显著的变化。3、表达谱测序分析结果显示实验组与对照组比较,存在较多表达差异基因,GO及KEGG富集分析结果提示这些差异基因参与重要的免疫过程,与人的免疫功能密切相关。4、Real-time PCR的验证结果显示,差异基因的表达变化情况与表达谱测序结果基本一致。而Western-blotting结果显示,部分差异基因在PBMC和CIK细胞中未检测到明显的信号。结论:结合本研究前期实验结果,提示甘草的miRNA对人免疫细胞有明显的免疫调节作用,这是对甘草药效作用机制研究领域新的探索。本研究结果对后期计划进行的动物实验研究奠定了基础。对后期进一步研究甘草的作用机制、临床应用和甘草新剂型的开发以及新药的研发,也带来了新的研究思路。
[Abstract]:Background and objective: the results of high throughput sequencing showed that miRNA156,miRNA1511,miRNA8155 was more abundant in Glycyrrhiza uralensis decoction miRNA. In this study, the effects of miRNA156,miRNA1511,miRNA8155 of Glycyrrhiza uralensis on human immune cells were investigated in terms of cell morphology, cell proliferation and immune-related gene expression. The differentially expressed genes were identified and verified by sequencing of expression profiles, which opened up a new idea for the study of the new mechanism of licorice pharmacodynamics. Methods: 1. According to the miRNA156,miRNA1511,miRNA8155 sequence of high throughput sequencing, the corresponding reverse transcription and detection primers were designed and synthesized. Identification of several microRNA molecules in Glycyrrhiza uralensis miRNA by RT-PCR: miRNA156,miRNA1511,miRNA8155.2, The transfection efficiency of liposome Lip2000 was verified by using FAM labeled small molecule DNA to establish a reliable transfection method. Mimic; corresponding to miRNA156,miRNA1511 and miRNA8155 were designed and synthesized to transfect Lip2000 into human immune cells (PBMC,CIK,Jurkat cell line) respectively, and the unrelated miRNA NC mimic was used as negative control group. Cell morphology and cell proliferation were compared by microscope and cell count. RT-PCR was used to detect the expression of partial immune-related genes in each group. 3. The miRNA mimic was transfected into healthy PBMC cells and cultured for 24 hours. The total RNA, expression profile of the cells was sequenced. The difference of gene expression in each group was analyzed, and the differentially expressed genes were classified and annotated by GO and KEGG enrichment analysis. Real-time PCR and Western-blotting techniques were used to verify the reliability of expression profile sequencing at gene level and protein level, respectively. Results: 1. Through RT-PCR detection of target miRNA, it was found that miRNA156,miRNA1511,miRNA8155, could be detected in total RNA of Glycyrrhiza uralensis, which confirmed the pre-high throughput sequencing results, which laid a reliable foundation for further study. 2. The transfection efficiency of Lip2000 was found to be higher by using Lip2000 transfection of FAM labeled small molecule DNA, under inverted fluorescence microscope. MiRNA mimic, corresponding to miRNA156,miRNA1511,miRNA8155 was prepared and successfully transfected into human immune cells (PBMC,CIK,JURKAT cells) by Lip2000. There were significant differences in cell morphology between the experimental group and the control group. Three miRNA of Glycyrrhiza uralensis had proliferative effects on PBMC cells and CIK cells, but on the growth of acute T cell leukemia cell line JURKAT. RT-PCR results showed that miRNA of Glycyrrhiza uralensis could induce PBMC,CIK,. The expression levels of immune-associated cytokines, signal molecules and transcription factors in JURKAT cells changed significantly. 3. The results of expression profiling showed that there were more differentially expressed genes in the experimental group than in the control group. The results of GO and KEGG enrichment analysis suggested that these differentially expressed genes were involved in the important immune process and were closely related to human immune function. 4 the results of Real-time PCR showed that the expression changes of the differentially expressed genes were basically consistent with the sequencing results of the expression profiles. Western-blotting results showed that some differentially expressed genes were not detected in PBMC and CIK cells. Conclusion: combined with the experimental results in the early stage of this study, it is suggested that miRNA of Glycyrrhiza uralensis has obvious immunomodulatory effect on human immune cells, which is a new exploration in the research field of the mechanism of action of Glycyrrhiza uralensis. The results of this study lay a foundation for the later planned animal experiments. Further research on the mechanism of licorice, clinical application, the development of new dosage forms of liquorice and the development of new drugs also bring new research ideas.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285
[Abstract]:Background and objective: the results of high throughput sequencing showed that miRNA156,miRNA1511,miRNA8155 was more abundant in Glycyrrhiza uralensis decoction miRNA. In this study, the effects of miRNA156,miRNA1511,miRNA8155 of Glycyrrhiza uralensis on human immune cells were investigated in terms of cell morphology, cell proliferation and immune-related gene expression. The differentially expressed genes were identified and verified by sequencing of expression profiles, which opened up a new idea for the study of the new mechanism of licorice pharmacodynamics. Methods: 1. According to the miRNA156,miRNA1511,miRNA8155 sequence of high throughput sequencing, the corresponding reverse transcription and detection primers were designed and synthesized. Identification of several microRNA molecules in Glycyrrhiza uralensis miRNA by RT-PCR: miRNA156,miRNA1511,miRNA8155.2, The transfection efficiency of liposome Lip2000 was verified by using FAM labeled small molecule DNA to establish a reliable transfection method. Mimic; corresponding to miRNA156,miRNA1511 and miRNA8155 were designed and synthesized to transfect Lip2000 into human immune cells (PBMC,CIK,Jurkat cell line) respectively, and the unrelated miRNA NC mimic was used as negative control group. Cell morphology and cell proliferation were compared by microscope and cell count. RT-PCR was used to detect the expression of partial immune-related genes in each group. 3. The miRNA mimic was transfected into healthy PBMC cells and cultured for 24 hours. The total RNA, expression profile of the cells was sequenced. The difference of gene expression in each group was analyzed, and the differentially expressed genes were classified and annotated by GO and KEGG enrichment analysis. Real-time PCR and Western-blotting techniques were used to verify the reliability of expression profile sequencing at gene level and protein level, respectively. Results: 1. Through RT-PCR detection of target miRNA, it was found that miRNA156,miRNA1511,miRNA8155, could be detected in total RNA of Glycyrrhiza uralensis, which confirmed the pre-high throughput sequencing results, which laid a reliable foundation for further study. 2. The transfection efficiency of Lip2000 was found to be higher by using Lip2000 transfection of FAM labeled small molecule DNA, under inverted fluorescence microscope. MiRNA mimic, corresponding to miRNA156,miRNA1511,miRNA8155 was prepared and successfully transfected into human immune cells (PBMC,CIK,JURKAT cells) by Lip2000. There were significant differences in cell morphology between the experimental group and the control group. Three miRNA of Glycyrrhiza uralensis had proliferative effects on PBMC cells and CIK cells, but on the growth of acute T cell leukemia cell line JURKAT. RT-PCR results showed that miRNA of Glycyrrhiza uralensis could induce PBMC,CIK,. The expression levels of immune-associated cytokines, signal molecules and transcription factors in JURKAT cells changed significantly. 3. The results of expression profiling showed that there were more differentially expressed genes in the experimental group than in the control group. The results of GO and KEGG enrichment analysis suggested that these differentially expressed genes were involved in the important immune process and were closely related to human immune function. 4 the results of Real-time PCR showed that the expression changes of the differentially expressed genes were basically consistent with the sequencing results of the expression profiles. Western-blotting results showed that some differentially expressed genes were not detected in PBMC and CIK cells. Conclusion: combined with the experimental results in the early stage of this study, it is suggested that miRNA of Glycyrrhiza uralensis has obvious immunomodulatory effect on human immune cells, which is a new exploration in the research field of the mechanism of action of Glycyrrhiza uralensis. The results of this study lay a foundation for the later planned animal experiments. Further research on the mechanism of licorice, clinical application, the development of new dosage forms of liquorice and the development of new drugs also bring new research ideas.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285
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