巨细胞病毒潜伏感染-再激活在溃疡性结肠炎患者病程中的作用机制的初步探究

发布时间：2018-05-29 17:02

本文选题：溃疡性结肠炎 + 巨细胞病毒感染　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景与目的：巨细胞病毒(cytomegalovirus, CMV)属于人群普遍易感的病原体之一,免疫力低下的患者,CMV感染或再激活可增加患者死亡率。因此长期以来其广受微生物学和感染领域专家关注。近几年来,消化科临床医生对这个跨学科领域也高度重视,因研究显示CMV感染与溃疡性结肠炎(Ulcerative colitis, UC)疾病复发和治疗激素抵抗有关。而UC在我国发病呈上升的趋势,且中-重度UC患者常需要应用糖皮质激素(简称激素)治疗,这为患者体内CMV感染或再激活提供了有利条件。临床研究提示：UC患者激素治疗过程中感染CMV会导致激素疗效下降或无效,患者病情加重[1],抗CMV治疗后患者激素抵抗可逆转。但目前CMV对UC病程的影响,治疗时机的选择等方面存在很多的临床问题,鉴于这个临床问题,本研究通过科学研究期望提供临床思路。因此本研究的目的是：1)建立CMV潜伏期及再激活的细胞模型；2)探究CMV潜伏期及再激活模型激素抵抗的发生机制及CMV对UC炎症的影响,CMV灭活后对激素抵抗和细胞因子的影响；3)研究CMV潜伏期及再激活模型以及感染灭活病毒的模拟潜伏和模拟再激活组、UC患者感染CMV及抗病毒治疗后,细胞模型和UC合并CMV感染的患者全血细胞miRNA基因的改变及相关的信号通路。研究分为三部分：第一部分巨细胞病毒潜伏期-再激活细胞模型的建立方法：1)模型建立初步阶段：应用MRC-5细胞培养CMV病毒；应用PMA诱导分化THP-1为巨噬细胞；2)潜伏期细胞模型：CMV感染未分化的单核细胞不同时间；3)再激活模型的建立：比较两个模型：感染潜伏期CMV的单核细胞分化为巨噬细胞模型,与CMV感染分化的巨噬细胞,这两种再激活模型。采用观察细胞形态、qRT-PCR检测单个细胞内病毒载量以及RT-PCR检测病毒早期、晚期相关基因的表达来验证模型的建立。最终选择了CMV感染分化的巨噬细胞模型。结果：1)CMV感染THP-1源性单核细胞系形态学：悬浮生长状态；2)CMV病毒载量在感染3天后稳定,病毒即刻早期基因72(immediate early 72, IE72)不表达,病毒晚期相关基因UL82表达,表明病毒感染THP-1后进入潜伏期；3)将感染潜伏期CMV的单核细胞分化为巨噬细胞后,细胞贴壁,但病毒载量不增加,IE72也不表达；4)将CMV感染分化的巨噬细胞后,细胞内病毒载量升高,在感染第3天时,IE72表达量升高最显著。第二部分CMV感染导致UC患者激素抵抗的机制及对炎症的影响方法：1)细胞分组：在已建立的潜伏期和再激活细胞模型的基础上,将THP-1细胞系分为4组：(Ⅰ)对照组；(Ⅱ) PMA诱导分化为巨噬细胞组(简称PMA组)；(Ⅲ) CMV潜伏期细胞模型(简称CMV组)；(Ⅳ) CMV再激活细胞模型(简称PMA+CMV组)。2)采用qRT-PCR方法检测4组细胞中GRβ、GRa及两者比值的变化,Western Blot方法检测GRp、GRa及磷酸化的GRa蛋白水平变化,多因子ELISA方法检测各组细胞上清液促炎因子和抑炎因子的变化。3)紫外线灭活CMV病毒，，检测病毒灭活后对激素受体和细胞因子水平变化。结果：1)CMV潜伏感染时GRβ、GRα mRNA和蛋白轻度升高,但GR β/α无显著改变；2)CMV再激活时GRβ、GTα显著升高,GRβ/升高(P0.05),且无功能的Ser226位磷酸化的GR α也升高,与激素抵抗相关；3)与对照组相比,CMV潜伏期感染IL-10轻度升高(0.72±0.12 pg/ml vs 0.87±0.21pg/ml, P=0.036),抗炎因子IL-5轻度下降(MFI:48.7±3.2 vs 43.6±2.6, P=0.02),其他炎性细胞因子无显著变化；4)再激活模型中促炎因子IL-6 (1.22±0.02pg/ml vs 4877.13±31.51 pg/ml, P0.001)、 TNF-α (1.35±0.15pg/ml vs 1479.88±29.8pg/ml, P＜0.001)显著升高,抗炎因子IL-5下降(MFI:48.7±3.2 vs 35.6±3.7, P=0.016),与UC炎症加重有关；CMV潜伏期及再激活,IL-10均升高；5)感染灭活病毒的模拟潜伏期和PMA+灭活CMV的模拟再激活模型,其激素受体和包括IL-10在内的各种细胞因子与对照相比均无变化。第三部分细胞模型和UC患者标本差异miRNA表达谱的变化方法：1)细胞模型分组：(Ⅰ)CMV潜伏感染模型；(Ⅱ)CMV再激活细胞模型；(Ⅲ)感染灭活病毒的潜伏期细胞模型；(Ⅳ)感染灭活病毒的再激活细胞模型；2)UC患者分组：选取3例合并CMV感染的激素抵抗UC患者抗CMV治疗前后的全血细胞。Trizol法提取各组细胞RNA,检测RNA的完整度和纯度,采用miRNA表达谱芯片检测细胞模型及患者标本中miRNA表达谱的变化,通过生物信息学软件预测靶基因,并进行基因富集分析。结果：1)获得miRNA动态表达谱；2)细胞模型分组中,潜伏期与再激活组有536个显著差异的基因；模拟潜伏期感染与模拟再激活感染模型有509种基因差异表达；再激活感染模型与模拟再激活感染模型有96种基因改变。生物信息学分析显示差异表达的基因主要与炎症、细胞粘附、分化、细胞核蛋白相关基因有关；3)UC患者miRNA表达谱显示,抗病毒治疗前后相比,hsa-miR-199a-5p、 hsa-miR-4419b和hsa-miR-642a-3p升高,这些miRNA与PMA诱导蛋白、DNA结合蛋白、G蛋白偶联受体、转化生长因子相关；4)细胞模型中CMV激活期、潜伏期及灭活病毒感染的差异表达的miRNA,与UC患者CMV感染治疗前后比较,hsa-miR-4419b均升高,研究hsa-miR-4419b的功能可能有助于揭示CMV感染UC患者病情和激素抵抗的机制。结论：1.成功建立模拟CMV潜伏感染和再激活细胞模型；2.CMV再激活感染时GRp绝对或相对升高,以及GRa磷酸化无功能,可能是患者激素抵抗的机制之一；3.CMV再激活感染使促炎因子(IL-10等)增加和抑炎因子(IL-5)下降,其失衡可能是激素疗效下降或抵抗的另一机制；并可解释UC患者粘膜损伤加重的现象；4. hsa-miR-4419b是细胞模型和CMV感染UC患者共同改变的miRNA,研究它的功能作用可能有助于揭示CMV感染对UC病情影响和激素抵抗的机制。
[Abstract]:Background and purpose: cytomegalovirus (CMV) is one of the most susceptible pathogens in the population. The patient with low immunity, CMV infection or reactivation can increase the patient's mortality. Therefore, it has long been concerned by experts in the field of Microbiology and infection. In recent years, clinicians in the digestive department have also been in this interdisciplinary field. Highly valued, CMV infection is associated with the recurrence of ulcerative colitis (Ulcerative colitis, UC) and the treatment of hormone resistance. But the incidence of UC is on the rise in our country, and moderate to severe UC patients often need to be treated with glucocorticoids (steroids), which provides favorable conditions for CMV infection or reactivation in the patients. The bed study suggests that the infection of CMV in UC patients can lead to the decrease or ineffectiveness of the hormone curative effect, the patient's condition aggravates [1], and the hormone resistance can be reversed after the anti CMV treatment. However, there are many clinical problems in the effect of CMV on the course of UC and the choice of the time of treatment. In view of this clinical problem, the study through scientific research The purpose of this study is to provide clinical ideas. 1) the purpose of this study is to establish CMV incubation period and reactivation cell model; 2) to explore the mechanism of CMV latency and reactivation of the model hormone resistance, the effect of CMV on UC inflammation, the effect of CMV on hormone resistance and cytokines, and 3) to study the latency and reactivation model of CMV and the model of reactivation and reactivation. The simulated latent and simulated reactivation group of inactivated virus infection, UC patients infected with CMV and antiviral therapy, the cell model and the changes of miRNA gene in the whole blood cells of the patients with UC combined with CMV infection and related signal pathways. The study is divided into three parts: the first part of the latent period of the giant cytomegalovirus - reactivation cell model: 1) model Preliminary stage: MRC-5 cells were used to cultivate CMV virus; PMA was used to induce THP-1 to be macrophage; 2) incubation period cell model: CMV infected undifferentiated mononuclear cells at different time; 3) the reactivation model was established: two models were compared: the mononuclear cells of the incubation period CMV were differentiated into macrophage model, and CMV sense Dyed macrophages, these two reactivation models. Using qRT-PCR to observe cell morphology, detection of viral load in single cells and RT-PCR detection of the virus early, and the expression of late related genes to verify the establishment of the model. Finally, the model of macrophage in CMV infection differentiation was selected. Results: 1) CMV infection of THP-1 derived mononuclear cells. State of state: suspension growth state; 2) the load of CMV virus is stable after 3 days of infection, and the immediate early gene 72 (immediate early 72, IE72) is not expressed, and the late related gene UL82 expression of the virus indicates that the virus infect THP-1 after the incubation period; 3) the monocyte that infected the latent period CMV is divided into macrophage, the cell is adhered to the virus, but the virus carrier is loaded. The amount of IE72 did not increase; 4) after the CMV infected macrophages, the viral load in the cells increased and the expression of IE72 increased most significantly at third days of infection. Second part of CMV infection led to the mechanism of hormone resistance in UC patients and the method of influence on inflammation: 1) cell grouping: the incubation period and reactivation of the cell model were established. The THP-1 cell line was divided into 4 groups: (I) the control group; (II) PMA induced differentiation into macrophage group (PMA group); (III) CMV latent period cell model (CMV group); (IV) CMV reactivation cell model (PMA+CMV group).2) using qRT-PCR method to detect GR beta, GRa and the ratio of the two groups in the qRT-PCR method. Methods the changes of the level of GRp, GRa and phosphorylation of GRa protein were detected, and the changes of.3 in the supernatant and anti inflammatory factors were detected by multifactor ELISA method.) CMV virus was inactivated by ultraviolet radiation, and the changes of the hormone receptor and cytokine level after the inactivation of the virus were detected. Results: 1) GR beta, GR alpha mRNA and protein slightly increased in CMV latent infection. There was no significant change in GR beta / alpha; 2) when CMV reactivates, GR beta, GT alpha significantly increased, GR beta / elevated (P0.05), and non functional Ser226 phosphorylated GR alpha increased, and was associated with hormone resistance; 3) IL-10 slightly increased in the CMV latency infection (0.72 + 0.12 pg/ml vs 0.87 +) compared with the control group. 3.2 vs 43.6 + 2.6, P=0.02), no significant changes in other inflammatory cytokines, 4) IL-6 (1.22 + 0.02pg/ml vs 4877.13 + 31.51 pg/ml, P0.001) in the reactivation model, TNF- alpha (1.35 + 0.15pg/ml vs 1479.88 + 29.8pg/ml, P < 0.001). CMV incubation period and reactivation, IL-10 increased; 5) the simulated incubation period of the inactivated virus and the simulated reactivation model of PMA+ inactivated CMV, the hormone receptor and all kinds of cytokines including IL-10 were not changed to the ratio of the photographic ratio. The changes of the miRNA expression profiles in the third part of the cell model and the UC patient were: 1) cell model grouping: (I) CMV latent infection model; (II) CMV reactivation cell model; (III) latent period cell model of inactivated virus infection; (IV) reactivation cell model of inactivated virus infection; 2) group of UC patients: select 3 cases of UC patients with CMV infection against CMV treatment before and after the anti CMV treatment of whole blood cell.Trizol method RNA was taken to detect the integrity and purity of RNA. The miRNA expression chip was used to detect the changes of the miRNA expression profiles in the cell model and the patient's specimen. The target gene was predicted by the bioinformatics software and the gene enrichment was analyzed. Results: 1) the dynamic expression spectrum of miRNA was obtained. 2) the incubation period and the reactivation group were 5. 36 genes with significant differences, 509 differentially expressed genes in the simulated incubation period and the simulated reactivation infection model, and 96 kinds of gene changes in the reactivated infection model and the simulated reactivation infection model. The bioinformatics analysis showed that the differentially expressed genes were mainly related to inflammation, cell adhesion, differentiation, and nuclear protein related genes. 3) miRNA expression profiles in UC patients showed that hsa-miR-199a-5p, hsa-miR-4419b and hsa-miR-642a-3p were increased before and after antiviral therapy, these miRNA and PMA induced proteins, DNA binding proteins, G protein coupled receptors, TGF related; 4) CMV activation period, incubation period and inactivated virus infection differential expression of miRNA, and UC Hsa-miR-4419b increased in patients with CMV infection before and after treatment. The function of hsa-miR-4419b may help to reveal the mechanism of CMV infection in patients with UC and the mechanism of hormone resistance. Conclusion: 1. a successful establishment of simulated CMV latent infection and reactivation cell model, 2.CMV reactivation of infection and GRp absolute or relative increase, and GRa phosphorylation reactive power. Yes, it may be one of the mechanisms of the patient's hormone resistance; 3.CMV reactivation of infection causes the increase of proinflammatory factor (IL-10) and the decrease of anti inflammatory factor (IL-5), and its imbalance may be another mechanism for decreasing or resisting hormone efficacy, and can explain the aggravation of mucosal damage in UC patients; 4. hsa-miR-4419b is the cell model and CMV infected UC patients. The altered miRNA may help to reveal the mechanism of CMV infection on UC and hormone resistance.
【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】：2016
【分类号】：R574.62;R511

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