發(fā)布時間：2018-05-29 17:02

  本文選題：潰瘍性結(jié)腸炎 + 巨細(xì)胞病毒感染　； 參考：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文

【摘要】：背景與目的：巨細(xì)胞病毒(cytomegalovirus, CMV)屬于人群普遍易感的病原體之一,免疫力低下的患者,CMV感染或再激活可增加患者死亡率。因此長期以來其廣受微生物學(xué)和感染領(lǐng)域?qū)＜谊P(guān)注。近幾年來,消化科臨床醫(yī)生對這個跨學(xué)科領(lǐng)域也高度重視,因研究顯示CMV感染與潰瘍性結(jié)腸炎(Ulcerative colitis, UC)疾病復(fù)發(fā)和治療激素抵抗有關(guān)。而UC在我國發(fā)病呈上升的趨勢,且中-重度UC患者常需要應(yīng)用糖皮質(zhì)激素(簡稱激素)治療,這為患者體內(nèi)CMV感染或再激活提供了有利條件。臨床研究提示：UC患者激素治療過程中感染CMV會導(dǎo)致激素療效下降或無效,患者病情加重[1],抗CMV治療后患者激素抵抗可逆轉(zhuǎn)。但目前CMV對UC病程的影響,治療時機(jī)的選擇等方面存在很多的臨床問題,鑒于這個臨床問題,本研究通過科學(xué)研究期望提供臨床思路。因此本研究的目的是：1)建立CMV潛伏期及再激活的細(xì)胞模型；2)探究CMV潛伏期及再激活模型激素抵抗的發(fā)生機(jī)制及CMV對UC炎癥的影響,CMV滅活后對激素抵抗和細(xì)胞因子的影響；3)研究CMV潛伏期及再激活模型以及感染滅活病毒的模擬潛伏和模擬再激活組、UC患者感染CMV及抗病毒治療后,細(xì)胞模型和UC合并CMV感染的患者全血細(xì)胞miRNA基因的改變及相關(guān)的信號通路。研究分為三部分：第一部分巨細(xì)胞病毒潛伏期-再激活細(xì)胞模型的建立方法：1)模型建立初步階段：應(yīng)用MRC-5細(xì)胞培養(yǎng)CMV病毒；應(yīng)用PMA誘導(dǎo)分化THP-1為巨噬細(xì)胞；2)潛伏期細(xì)胞模型：CMV感染未分化的單核細(xì)胞不同時間；3)再激活模型的建立：比較兩個模型：感染潛伏期CMV的單核細(xì)胞分化為巨噬細(xì)胞模型,與CMV感染分化的巨噬細(xì)胞,這兩種再激活模型。采用觀察細(xì)胞形態(tài)、qRT-PCR檢測單個細(xì)胞內(nèi)病毒載量以及RT-PCR檢測病毒早期、晚期相關(guān)基因的表達(dá)來驗證模型的建立。最終選擇了CMV感染分化的巨噬細(xì)胞模型。結(jié)果：1)CMV感染THP-1源性單核細(xì)胞系形態(tài)學(xué)：懸浮生長狀態(tài)；2)CMV病毒載量在感染3天后穩(wěn)定,病毒即刻早期基因72(immediate early 72, IE72)不表達(dá),病毒晚期相關(guān)基因UL82表達(dá),表明病毒感染THP-1后進(jìn)入潛伏期；3)將感染潛伏期CMV的單核細(xì)胞分化為巨噬細(xì)胞后,細(xì)胞貼壁,但病毒載量不增加,IE72也不表達(dá)；4)將CMV感染分化的巨噬細(xì)胞后,細(xì)胞內(nèi)病毒載量升高,在感染第3天時,IE72表達(dá)量升高最顯著。第二部分CMV感染導(dǎo)致UC患者激素抵抗的機(jī)制及對炎癥的影響方法：1)細(xì)胞分組：在已建立的潛伏期和再激活細(xì)胞模型的基礎(chǔ)上,將THP-1細(xì)胞系分為4組：(Ⅰ)對照組；(Ⅱ) PMA誘導(dǎo)分化為巨噬細(xì)胞組(簡稱PMA組)；(Ⅲ) CMV潛伏期細(xì)胞模型(簡稱CMV組)；(Ⅳ) CMV再激活細(xì)胞模型(簡稱PMA+CMV組)。2)采用qRT-PCR方法檢測4組細(xì)胞中GRβ、GRa及兩者比值的變化,Western Blot方法檢測GRp、GRa及磷酸化的GRa蛋白水平變化,多因子ELISA方法檢測各組細(xì)胞上清液促炎因子和抑炎因子的變化。3)紫外線滅活CMV病毒，，檢測病毒滅活后對激素受體和細(xì)胞因子水平變化。結(jié)果：1)CMV潛伏感染時GRβ、GRα mRNA和蛋白輕度升高,但GR β/α無顯著改變；2)CMV再激活時GRβ、GTα顯著升高,GRβ/升高(P0.05),且無功能的Ser226位磷酸化的GR α也升高,與激素抵抗相關(guān)；3)與對照組相比,CMV潛伏期感染IL-10輕度升高(0.72±0.12 pg/ml vs 0.87±0.21pg/ml, P=0.036),抗炎因子IL-5輕度下降(MFI:48.7±3.2 vs 43.6±2.6, P=0.02),其他炎性細(xì)胞因子無顯著變化；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)顯著升高,抗炎因子IL-5下降(MFI:48.7±3.2 vs 35.6±3.7, P=0.016),與UC炎癥加重有關(guān)；CMV潛伏期及再激活,IL-10均升高；5)感染滅活病毒的模擬潛伏期和PMA+滅活CMV的模擬再激活模型,其激素受體和包括IL-10在內(nèi)的各種細(xì)胞因子與對照相比均無變化。第三部分細(xì)胞模型和UC患者標(biāo)本差異miRNA表達(dá)譜的變化方法：1)細(xì)胞模型分組：(Ⅰ)CMV潛伏感染模型；(Ⅱ)CMV再激活細(xì)胞模型；(Ⅲ)感染滅活病毒的潛伏期細(xì)胞模型；(Ⅳ)感染滅活病毒的再激活細(xì)胞模型；2)UC患者分組：選取3例合并CMV感染的激素抵抗UC患者抗CMV治療前后的全血細(xì)胞。Trizol法提取各組細(xì)胞RNA,檢測RNA的完整度和純度,采用miRNA表達(dá)譜芯片檢測細(xì)胞模型及患者標(biāo)本中miRNA表達(dá)譜的變化,通過生物信息學(xué)軟件預(yù)測靶基因,并進(jìn)行基因富集分析。結(jié)果：1)獲得miRNA動態(tài)表達(dá)譜；2)細(xì)胞模型分組中,潛伏期與再激活組有536個顯著差異的基因；模擬潛伏期感染與模擬再激活感染模型有509種基因差異表達(dá)；再激活感染模型與模擬再激活感染模型有96種基因改變。生物信息學(xué)分析顯示差異表達(dá)的基因主要與炎癥、細(xì)胞粘附、分化、細(xì)胞核蛋白相關(guān)基因有關(guān)；3)UC患者miRNA表達(dá)譜顯示,抗病毒治療前后相比,hsa-miR-199a-5p、 hsa-miR-4419b和hsa-miR-642a-3p升高,這些miRNA與PMA誘導(dǎo)蛋白、DNA結(jié)合蛋白、G蛋白偶聯(lián)受體、轉(zhuǎn)化生長因子相關(guān)；4)細(xì)胞模型中CMV激活期、潛伏期及滅活病毒感染的差異表達(dá)的miRNA,與UC患者CMV感染治療前后比較,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機(jī)制。結(jié)論：1.成功建立模擬CMV潛伏感染和再激活細(xì)胞模型；2.CMV再激活感染時GRp絕對或相對升高,以及GRa磷酸化無功能,可能是患者激素抵抗的機(jī)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,其失衡可能是激素療效下降或抵抗的另一機(jī)制；并可解釋UC患者粘膜損傷加重的現(xiàn)象；4. hsa-miR-4419b是細(xì)胞模型和CMV感染UC患者共同改變的miRNA,研究它的功能作用可能有助于揭示CMV感染對UC病情影響和激素抵抗的機(jī)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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R574.62;R511

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相關(guān)碩士學(xué)位論文 前1條

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