雷公藤甲素、雷公藤红素对外周血单个核细胞干扰素刺激基因表达的影响
发布时间：2018-09-17 18:22
【摘要】：研究目的： 通过检测人外周血单个核细胞(peripheral blood mononuclear cell, PBMC)接受LPS刺激前后干扰素刺激基因(IFN-stimulated gene, ISG)表达水平的改变,探究雷公藤甲素、雷公藤红素两种药物对HIV/AIDS患者免疫激活状态的抑制作用。 研究方法： 从北京协和医院艾滋病诊疗中心的就诊患者中选出长期接受cART治疗的HIV/AIDS患者13例、未接受抗病毒治疗的HIV/AIDS患者13例,并招募健康人对照8例,收集临床资料并留取外周血标本,分离PBMC,加入足量LPS刺激后在不同药物浓度下进行细胞培养,提取细胞总mRNA,进行逆转录,运用实时荧光定量相对定量方法检测目的基因MxA、MxB、SG15及内参基因GAPDH表达水平。观察药物及LPS刺激前后目的基因表达水平的变化,比较各组之间的差异。主要统计方法包括Kolmogorov-smirnov检验、Shapiro-Wilk检验、Levene检验、方差分析、配对t检验等,定义P0.05具有统计学显著性。 研究结果： 1、入组患者临床特征： 共入组患者及健康人34例,其中抗病毒治疗组13例,cART治疗时间中位数21个月,CD4+T细胞计数494.8±164.1/μl,均实现病毒的完全抑制；未抗病毒治疗组13例,CD4+T细胞计数278.1±191.1/μl；健康人对照组8例,年龄25-26岁,各项感染指标均为正常。 2、各分组未受LPS刺激外周血单个核细胞ISG表达水平存在差异： 在未接受LPS刺激的健康人对照组、抗病毒治疗组及未抗病毒治疗组PBMC中,ISG15基因相对表达量分别为0.23±0.09,0.28±0.11,0.45±0.26,各分组间存在一定差异(P0.05)； 3、足量LPS刺激影响外周血单个核细胞ISG表达水平： 在体外加入足量LPS (1ug/ml)刺激后,健康人对照组、治疗患者组、未治疗患者组ISG15、MxA基因的表达水平均有明显提高(P0.05),而MxB基因表达水平则无显著变化。 4、雷公藤甲素、雷公藤红素显著影响LPS刺激后外周血单个核细胞ISG基因表达水平： 各组PBMC在足量LPS刺激基础上加入不同浓度的雷公藤甲素后,ISG15、MxA、 MxB基因的相对表达量均出现了不同程度的下降,其中以ISG15最为显著,在浓度为10ng/ml的雷公藤甲素作用下,ISG15基因的相对表达水平在健康人组中由9.64±1.40下降至1.12±0.35(P=0.000),在浓度其40ng/ml的雷公藤红素作用下,ISG15基因的相对表达水平在健康人组中由9.64±1.40下降至2.35±0.51(P=0.000),抑制程度高于另外两组,且抗病毒治疗组抑制水平明显高于抗病毒治疗组。 研究结论： 1、ISGs表达水平可以体现HIV/AIDS患者免疫激活状态； 2、足量LPS刺激可明显提升ISG15、MxA基因表达水平,且上升程度与免疫激活水平相关,对MxB基因的表达水平则无明显影响； 3、雷公藤甲素、雷公藤红素可明显抑制ISG15、MxA基因表达水平,对MxB基因也有一定抑制作用,抑制程度与免疫激活水平相关,在正常人及抗病毒治疗患者中效果好。
[Abstract]:Objective: to investigate the changes of Interferon stimulating gene (IFN-stimulated gene, ISG) expression in human peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) before and after LPS stimulation, and to investigate the expression of triptolide in human peripheral blood mononuclear cells (PBMC). Inhibitory effect of tripterine on immune activation in patients with HIV/AIDS. Methods: 13 patients with cART, 13 patients with HIV/AIDS without antiviral therapy, and 8 healthy controls were selected from the AIDS center of Peking Union Hospital. Clinical data were collected and peripheral blood samples were collected. PBMC, was isolated and cultured in different concentrations after LPS stimulation. Total mRNA, was extracted for reverse transcription. The expression levels of target gene MxA,MxB,SG15 and internal reference gene GAPDH were detected by real-time fluorescence quantitative relative quantitative method. The changes of target gene expression before and after drug and LPS stimulation were observed. The main statistical methods included Kolmogorov-smirnov test Shapiro-Wilk test Levene test ANOVA paired t-test and so on. The definition of P05 was statistically significant. Results: 1. Clinical features of the patients: 34 patients and 34 healthy persons were enrolled. The median time of antiviral therapy was 21 months (median 21 months), CD4 T cell count was 494.8 卤164.1 / 渭 l, all of them could completely inhibit the virus. The count of CD4 T cells was 278.1 卤191.1 / 渭 l in the untreated group and 8 cases in the control group, aged 25-26 years. The expression of ISG in peripheral blood mononuclear cells (PBMC) without LPS stimulation was different in all groups: in the control group without LPS stimulation, the expression of ISG in peripheral blood mononuclear cells (PBMC) was significantly higher than that in control group (P < 0.05). The relative expression of ISG15 gene in PBMC of antiviral treatment group and non-antiviral treatment group was 0.23 卤0.09 卤0.28 卤0.110.45 卤0.26, respectively, and there were some differences among groups (P0.05). 3. The level of ISG expression in peripheral blood mononuclear cells (PBMC) was affected by sufficient LPS stimulation: after sufficient LPS (1ug/ml) was added in vitro, the healthy control group, the treatment group, the control group, the treatment group, The expression level of ISG15,MxA gene in untreated group was significantly increased (P0.05), while the expression level of MxB gene had no significant change. 4, triptolide, triptolide, Tripterin significantly affected the expression of ISG gene in peripheral blood mononuclear cells stimulated by LPS: the relative expression levels of MxB gene and ISG15 MxA were all significantly increased in each group after the addition of different concentrations of tripterygium wilfordii on the basis of sufficient LPS stimulation. There has been a decline of varying degrees, Among them, ISG15 is the most significant. The relative expression level of ISG15 gene decreased from 9.64 卤1.40 to 1.12 卤0.35 in healthy controls under the action of triptolide (10ng/ml). The relative expression level of ISG15 gene decreased from 9.64 卤1.40 to 9.64 卤1.40 in healthy controls under the action of tripterine of 40ng/ml. To 2.35 卤0.51 (P0. 000), the degree of inhibition was higher than that of the other two groups. The inhibition level of antiviral therapy group was significantly higher than that of antiviral treatment group. Conclusion: 1 the expression level of ISGs can reflect the immune activation state of HIV/AIDS patients, 2, sufficient LPS stimulation can significantly enhance the expression of ISG15,MxA gene, and the degree of increase is related to the level of immune activation. 3, triptolide and tripterygium wilfordii can significantly inhibit the expression of ISG15,MxA gene, and inhibit the expression of MxB gene, and the inhibition degree is related to the level of immune activation, and the expression level of MxB gene is not affected by triptolide, tripterygium wilfordii and tripterygium wilfordii. It works well in normal people and in patients with antiviral therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.91
本文编号：2246740
[Abstract]:Objective: to investigate the changes of Interferon stimulating gene (IFN-stimulated gene, ISG) expression in human peripheral blood mononuclear cells (peripheral blood mononuclear cell, PBMC) before and after LPS stimulation, and to investigate the expression of triptolide in human peripheral blood mononuclear cells (PBMC). Inhibitory effect of tripterine on immune activation in patients with HIV/AIDS. Methods: 13 patients with cART, 13 patients with HIV/AIDS without antiviral therapy, and 8 healthy controls were selected from the AIDS center of Peking Union Hospital. Clinical data were collected and peripheral blood samples were collected. PBMC, was isolated and cultured in different concentrations after LPS stimulation. Total mRNA, was extracted for reverse transcription. The expression levels of target gene MxA,MxB,SG15 and internal reference gene GAPDH were detected by real-time fluorescence quantitative relative quantitative method. The changes of target gene expression before and after drug and LPS stimulation were observed. The main statistical methods included Kolmogorov-smirnov test Shapiro-Wilk test Levene test ANOVA paired t-test and so on. The definition of P05 was statistically significant. Results: 1. Clinical features of the patients: 34 patients and 34 healthy persons were enrolled. The median time of antiviral therapy was 21 months (median 21 months), CD4 T cell count was 494.8 卤164.1 / 渭 l, all of them could completely inhibit the virus. The count of CD4 T cells was 278.1 卤191.1 / 渭 l in the untreated group and 8 cases in the control group, aged 25-26 years. The expression of ISG in peripheral blood mononuclear cells (PBMC) without LPS stimulation was different in all groups: in the control group without LPS stimulation, the expression of ISG in peripheral blood mononuclear cells (PBMC) was significantly higher than that in control group (P < 0.05). The relative expression of ISG15 gene in PBMC of antiviral treatment group and non-antiviral treatment group was 0.23 卤0.09 卤0.28 卤0.110.45 卤0.26, respectively, and there were some differences among groups (P0.05). 3. The level of ISG expression in peripheral blood mononuclear cells (PBMC) was affected by sufficient LPS stimulation: after sufficient LPS (1ug/ml) was added in vitro, the healthy control group, the treatment group, the control group, the treatment group, The expression level of ISG15,MxA gene in untreated group was significantly increased (P0.05), while the expression level of MxB gene had no significant change. 4, triptolide, triptolide, Tripterin significantly affected the expression of ISG gene in peripheral blood mononuclear cells stimulated by LPS: the relative expression levels of MxB gene and ISG15 MxA were all significantly increased in each group after the addition of different concentrations of tripterygium wilfordii on the basis of sufficient LPS stimulation. There has been a decline of varying degrees, Among them, ISG15 is the most significant. The relative expression level of ISG15 gene decreased from 9.64 卤1.40 to 1.12 卤0.35 in healthy controls under the action of triptolide (10ng/ml). The relative expression level of ISG15 gene decreased from 9.64 卤1.40 to 9.64 卤1.40 in healthy controls under the action of tripterine of 40ng/ml. To 2.35 卤0.51 (P0. 000), the degree of inhibition was higher than that of the other two groups. The inhibition level of antiviral therapy group was significantly higher than that of antiviral treatment group. Conclusion: 1 the expression level of ISGs can reflect the immune activation state of HIV/AIDS patients, 2, sufficient LPS stimulation can significantly enhance the expression of ISG15,MxA gene, and the degree of increase is related to the level of immune activation. 3, triptolide and tripterygium wilfordii can significantly inhibit the expression of ISG15,MxA gene, and inhibit the expression of MxB gene, and the inhibition degree is related to the level of immune activation, and the expression level of MxB gene is not affected by triptolide, tripterygium wilfordii and tripterygium wilfordii. It works well in normal people and in patients with antiviral therapy.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.91
【参考文献】
相关博士学位论文 前1条
1 谢静;异常免疫激活与HIV/AIDS疾病进展和抗病毒治疗疗效的相关性及临床干预研究[D];北京协和医学院;2013年
，本文编号：2246740
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