甘草miRNA對人免疫細(xì)胞基因表達譜的影響研究
發(fā)布時間：2018-12-30 11:50
【摘要】：背景及目的:前期高通量測序結(jié)果顯示,miRNA156、miRNA1511、miRNA8155在甘草水煎劑miRNA中豐度較高。本研究通過細(xì)胞形態(tài)學(xué)、細(xì)胞的增殖情況及免疫相關(guān)基因表達方面探究甘草的miRNA156、miRNA1511、miRNA8155對人免疫細(xì)胞的影響。通過表達譜測序發(fā)現(xiàn)差異基因并加以驗證,從而為甘草藥效新機制的相關(guān)研究開拓新的思路。方法:1、根據(jù)高通量測序結(jié)果中的miRNA156、miRNA1511、miRNA8155序列,設(shè)計合成相應(yīng)的反轉(zhuǎn)錄及檢測引物,通過RT-PCR確認(rèn)甘草組織miRNA中確實存在前期從其水煎劑中通過高通量測序技術(shù)鑒定出來的幾種microRNA分子:miRNA156、miRNA1511、miRNA8155。2、使用FAM標(biāo)記的小分子DNA來驗證脂質(zhì)體Lip2000的轉(zhuǎn)染效率以建立可靠的轉(zhuǎn)染方法;設(shè)計合成miRNA156、miRNA1511和miRNA8155相對應(yīng)的mimic;將其分別通過Lip2000轉(zhuǎn)染人的免疫細(xì)胞(PBMC、CIK、Jurkat細(xì)胞系),并以無關(guān)的miRNA NC mimic作為陰性對照組;使用顯微鏡觀察并進行細(xì)胞計數(shù)來比較各組間細(xì)胞形態(tài)和細(xì)胞增殖情況;使用RT-PCR檢測各組細(xì)胞間部分免疫相關(guān)基因的表達變化情況。3、將各miRNA mimic轉(zhuǎn)染健康人PBMC細(xì)胞,培養(yǎng)24小時后,提取細(xì)胞的總RNA,進行表達譜測序,分析各組細(xì)胞基因表達的差異,并通過GO及KEGG富集分析對差異基因進行分類和注釋。4、使用Real-time PCR及Western-blotting技術(shù)分別從基因水平和蛋白水平來驗證表達譜測序結(jié)果的可靠性。結(jié)果:1、通過RT-PCR檢測目標(biāo)miRNA,發(fā)現(xiàn)甘草組織總RNA中確實能檢測到miRNA156、miRNA1511、miRNA8155,驗證了前期高通量測序結(jié)果,為后續(xù)研究奠定了可靠基礎(chǔ)。2、通過用Lip2000轉(zhuǎn)染FAM標(biāo)記的小分子DNA,倒置熒光顯微鏡下觀察發(fā)現(xiàn)Lip2000有較高的轉(zhuǎn)染效率。制備合成了miRNA156、miRNA1511、miRNA8155相對應(yīng)的miRNA mimic,并成功通過Lip2000轉(zhuǎn)染進人的免疫細(xì)胞(PBMC、CIK、JURKAT細(xì)胞);實驗組與對照組間細(xì)胞形態(tài)有明顯差異;甘草的三個miRNA對PBMC細(xì)胞和CIK細(xì)胞生長有增殖作用,而對急性T細(xì)胞白血病細(xì)胞系JURKAT的生長存在明顯抑制作用。RT-PCR結(jié)果顯示,甘草miRNA使PBMC、CIK、JURKAT細(xì)胞中部分免疫相關(guān)細(xì)胞因子、信號分子和轉(zhuǎn)錄因子的表達水平發(fā)生了顯著的變化。3、表達譜測序分析結(jié)果顯示實驗組與對照組比較,存在較多表達差異基因,GO及KEGG富集分析結(jié)果提示這些差異基因參與重要的免疫過程,與人的免疫功能密切相關(guān)。4、Real-time PCR的驗證結(jié)果顯示,差異基因的表達變化情況與表達譜測序結(jié)果基本一致。而Western-blotting結(jié)果顯示,部分差異基因在PBMC和CIK細(xì)胞中未檢測到明顯的信號。結(jié)論:結(jié)合本研究前期實驗結(jié)果,提示甘草的miRNA對人免疫細(xì)胞有明顯的免疫調(diào)節(jié)作用,這是對甘草藥效作用機制研究領(lǐng)域新的探索。本研究結(jié)果對后期計劃進行的動物實驗研究奠定了基礎(chǔ)。對后期進一步研究甘草的作用機制、臨床應(yīng)用和甘草新劑型的開發(fā)以及新藥的研發(fā),也帶來了新的研究思路。
[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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285
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