雷公藤甲素、雷公藤紅素對(duì)外周血單個(gè)核細(xì)胞干擾素刺激基因表達(dá)的影響
發(fā)布時(shí)間：2018-09-17 18:22
【摘要】：研究目的： 通過檢測(cè)人外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell, PBMC)接受LPS刺激前后干擾素刺激基因(IFN-stimulated gene, ISG)表達(dá)水平的改變,探究雷公藤甲素、雷公藤紅素兩種藥物對(duì)HIV/AIDS患者免疫激活狀態(tài)的抑制作用。 研究方法： 從北京協(xié)和醫(yī)院艾滋病診療中心的就診患者中選出長期接受cART治療的HIV/AIDS患者13例、未接受抗病毒治療的HIV/AIDS患者13例,并招募健康人對(duì)照8例,收集臨床資料并留取外周血標(biāo)本,分離PBMC,加入足量LPS刺激后在不同藥物濃度下進(jìn)行細(xì)胞培養(yǎng),提取細(xì)胞總mRNA,進(jìn)行逆轉(zhuǎn)錄,運(yùn)用實(shí)時(shí)熒光定量相對(duì)定量方法檢測(cè)目的基因MxA、MxB、SG15及內(nèi)參基因GAPDH表達(dá)水平。觀察藥物及LPS刺激前后目的基因表達(dá)水平的變化,比較各組之間的差異。主要統(tǒng)計(jì)方法包括Kolmogorov-smirnov檢驗(yàn)、Shapiro-Wilk檢驗(yàn)、Levene檢驗(yàn)、方差分析、配對(duì)t檢驗(yàn)等,定義P0.05具有統(tǒng)計(jì)學(xué)顯著性。 研究結(jié)果： 1、入組患者臨床特征： 共入組患者及健康人34例,其中抗病毒治療組13例,cART治療時(shí)間中位數(shù)21個(gè)月,CD4+T細(xì)胞計(jì)數(shù)494.8±164.1/μl,均實(shí)現(xiàn)病毒的完全抑制；未抗病毒治療組13例,CD4+T細(xì)胞計(jì)數(shù)278.1±191.1/μl；健康人對(duì)照組8例,年齡25-26歲,各項(xiàng)感染指標(biāo)均為正常。 2、各分組未受LPS刺激外周血單個(gè)核細(xì)胞ISG表達(dá)水平存在差異： 在未接受LPS刺激的健康人對(duì)照組、抗病毒治療組及未抗病毒治療組PBMC中,ISG15基因相對(duì)表達(dá)量分別為0.23±0.09,0.28±0.11,0.45±0.26,各分組間存在一定差異(P0.05)； 3、足量LPS刺激影響外周血單個(gè)核細(xì)胞ISG表達(dá)水平： 在體外加入足量LPS (1ug/ml)刺激后,健康人對(duì)照組、治療患者組、未治療患者組ISG15、MxA基因的表達(dá)水平均有明顯提高(P0.05),而MxB基因表達(dá)水平則無顯著變化。 4、雷公藤甲素、雷公藤紅素顯著影響LPS刺激后外周血單個(gè)核細(xì)胞ISG基因表達(dá)水平： 各組PBMC在足量LPS刺激基礎(chǔ)上加入不同濃度的雷公藤甲素后,ISG15、MxA、 MxB基因的相對(duì)表達(dá)量均出現(xiàn)了不同程度的下降,其中以ISG15最為顯著,在濃度為10ng/ml的雷公藤甲素作用下,ISG15基因的相對(duì)表達(dá)水平在健康人組中由9.64±1.40下降至1.12±0.35(P=0.000),在濃度其40ng/ml的雷公藤紅素作用下,ISG15基因的相對(duì)表達(dá)水平在健康人組中由9.64±1.40下降至2.35±0.51(P=0.000),抑制程度高于另外兩組,且抗病毒治療組抑制水平明顯高于抗病毒治療組。 研究結(jié)論： 1、ISGs表達(dá)水平可以體現(xiàn)HIV/AIDS患者免疫激活狀態(tài)； 2、足量LPS刺激可明顯提升ISG15、MxA基因表達(dá)水平,且上升程度與免疫激活水平相關(guān),對(duì)MxB基因的表達(dá)水平則無明顯影響； 3、雷公藤甲素、雷公藤紅素可明顯抑制ISG15、MxA基因表達(dá)水平,對(duì)MxB基因也有一定抑制作用,抑制程度與免疫激活水平相關(guān),在正常人及抗病毒治療患者中效果好。
[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 鹵164.1 / 渭 l, all of them could completely inhibit the virus. The count of CD4 T cells was 278.1 鹵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 鹵0.09 鹵0.28 鹵0.110.45 鹵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 鹵1.40 to 1.12 鹵0.35 in healthy controls under the action of triptolide (10ng/ml). The relative expression level of ISG15 gene decreased from 9.64 鹵1.40 to 9.64 鹵1.40 in healthy controls under the action of tripterine of 40ng/ml. To 2.35 鹵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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.91
本文編號(hào)：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 鹵164.1 / 渭 l, all of them could completely inhibit the virus. The count of CD4 T cells was 278.1 鹵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 鹵0.09 鹵0.28 鹵0.110.45 鹵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 鹵1.40 to 1.12 鹵0.35 in healthy controls under the action of triptolide (10ng/ml). The relative expression level of ISG15 gene decreased from 9.64 鹵1.40 to 9.64 鹵1.40 in healthy controls under the action of tripterine of 40ng/ml. To 2.35 鹵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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R512.91
【參考文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 謝靜;異常免疫激活與HIV/AIDS疾病進(jìn)展和抗病毒治療療效的相關(guān)性及臨床干預(yù)研究[D];北京協(xié)和醫(yī)學(xué)院;2013年
，本文編號(hào)：2246740
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