發(fā)布時(shí)間：2018-07-08 13:22

  本文選題：乙型肝炎病毒 + 慢性乙型肝炎��； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：乙型肝炎病毒（HBV）感染已經(jīng)成為全球公共健康問(wèn)題，每年約有100萬(wàn)人死于HBV感染相關(guān)的肝臟疾病。活動(dòng)性HBV復(fù)制是導(dǎo)致肝臟損傷和疾病進(jìn)展的關(guān)鍵因素，因此最大限度地長(zhǎng)期抑制或清除HBV成為慢性乙型肝炎治療的總體目標(biāo)。目前用于慢性乙型肝炎抗病毒治療的藥物主要有兩大類：1、核苷（酸）類似物；2、干擾素（interferon, IFN）。干擾素具有抗病毒和免疫調(diào)節(jié)雙重作用，治療療程短而確定、耐藥少見(jiàn)，故逐漸成為臨床治療慢性乙型肝炎的主要選擇。然而，目前干擾素治療只能達(dá)到30%-40%的HBeAg血清學(xué)轉(zhuǎn)換，約有60%的患者盡管接受干擾素抗病毒治療，但仍然遭受乙肝病毒的侵害，因此短期內(nèi)預(yù)測(cè)干擾素治療療效顯得尤為重要，最新研究熱點(diǎn)是干擾素抗病毒作用信號(hào)通路中的干擾素刺激基因，泛素特異性蛋白酶18（Ubiquitin-specific protease18, USPl8）是其中重要的一個(gè)干擾素刺激基因。本研究旨在探討USPl8在干擾素-α治療慢乙肝過(guò)程中的預(yù)測(cè)意義及作用機(jī)制。 方法：選取慢性HBV感染者70例，均為免疫清除期患者，所有患者均進(jìn)行肝組織活檢，無(wú)明顯干擾素抗病毒治療禁忌癥。留取受試者治療前新鮮外周血提取PBMCs進(jìn)行體外干擾素-α孵育刺激試驗(yàn)，并對(duì)受試者應(yīng)用干擾素-α抗病毒治療，進(jìn)行隨訪觀察24周，其中無(wú)病毒學(xué)應(yīng)答患者（nonvirological response, NVR）18例，完全病毒學(xué)應(yīng)答患者（complete virologicalresponse,CVR）32例，部分病毒學(xué)應(yīng)答患者(partial virological response,PVR)20例，健康對(duì)照10例。 1體外干擾素-α孵育PBMCs：留取70例受試者治療前新鮮外周靜脈血，，密度梯度離心法分離PBMCs，無(wú)血清培養(yǎng)基懸浮沉淀細(xì)胞，分為兩組：第一組空白對(duì)照組（不加任何因子）；第二組加入干擾素-α因子刺激孵育PBMCs,4h后收集細(xì)胞。 2對(duì)所有受試者應(yīng)用干擾素-α抗病毒治療隨訪觀察，留取治療0周、1周、12周、24周時(shí)新鮮外周血，密度梯度離心法分離收集PBMCs。 3Trizol法抽提外周血單個(gè)核細(xì)胞總RNA，應(yīng)用Real-time PCR法檢測(cè)USP18、STAT1mRNA的表達(dá)水平。 4RIPA法提取細(xì)胞總蛋白應(yīng)用western blot法檢測(cè)USP18、STAT1、P-STAT1蛋白的表達(dá)水平。 結(jié)果： 1健康對(duì)照組外周血PBMCs中USP18的表達(dá)為陰性 2干擾素-α治療過(guò)程外周血PBMCs中USP18mRNA水平變化 70例CHB患者在干擾素-α治療過(guò)程中接受mRNA水平檢測(cè)，將PBMCs中內(nèi)參GAPDH相對(duì)表達(dá)量設(shè)定為1.00，與GAPDH表達(dá)量相比較，干擾素-α治療前NVR組PBMCs中USP18的表達(dá)水平(0.073±0.026)較CVR組（0.043±0.023）及PVR組（0.044±0.022）高，差異有統(tǒng)計(jì)學(xué)意義（F=10.665，P0.01）；治療1周時(shí)，各組PBMCs中USP18的表達(dá)水平明顯升高，NVR組（0.098±0.023）、CVR組（0.071±0.027）、PVR組（0.074±0.027），NVR組表達(dá)水平最高，差異有統(tǒng)計(jì)學(xué)意義（F=6.781，P=0.002）；治療12周及24周時(shí)，各組表達(dá)水平為NVR組（0.097±0.024）(0.096±0.023)、CVR組（0.069±0.025）（0.066±0.025）、PVR組（0.075±0.026）（0.074±0.025），NVR組表達(dá)水平仍最高，差異有統(tǒng)計(jì)學(xué)意義（F1=7.140，p=0.002；F2=8.654, P0.01）。 3干擾素-α治療過(guò)程外周血PBMCs中USP18蛋白水平變化 將PBMCs中內(nèi)參β-actin相對(duì)表達(dá)量設(shè)定為1.00，與β-actin表達(dá)量相比較，干擾素-α治療前NVR組外周血PBMCs中USP18的表達(dá)水平(0.308±0.046)較CVR組（0.250±0.044）及PVR組（0.248±0.048）高，差異有統(tǒng)計(jì)學(xué)意義（F=11.151，P0.01）；治療1周后，各組USP18的表達(dá)水平明顯升高，NVR組（0.563±0.050）、CVR組（0.431±0.047）、PVR組（0.428±0.047），NVR組表達(dá)水平最高，差異有統(tǒng)計(jì)學(xué)意義（F=51.722，P0.01）；治療12周及24周時(shí)，各組表達(dá)水平為NVR組（0.568±0.051）(0.566±0.049)、CVR組（0.430±0.047）（0.429±0.045）、PVR組（0.426±0.047）（0.430±0.049），NVR組表達(dá)水平仍最高，差異有統(tǒng)計(jì)學(xué)意義（F1=55.958，P0.01；F2=55.841, P0.01）。 4體外干擾素-α孵育刺激PBMCs后形態(tài)學(xué)觀察 PBMCs經(jīng)2h貼壁后，可見(jiàn)部分細(xì)胞懸浮，多數(shù)細(xì)胞為B細(xì)胞和外周血DC，干擾素-α孵育刺激4h后未見(jiàn)形態(tài)學(xué)變化，細(xì)胞數(shù)目無(wú)明顯變化。 5體外干擾素-α刺激PBMCs后USP18、STAT-1Mrna/蛋白水平變化 體外干擾素-α刺激治療前外周血PBMCs后，干擾素-α組PBMCs中USP18在mRNA/蛋白上的表達(dá)水平高于空白對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.01），而NVR組、CVR組及PVR組外周血PBMCs中STAT-1mRNA及其蛋白水平無(wú)顯著性差異。 6體外干擾素-α刺激PBMCs后USP18表達(dá)水平與P-STAT-1表達(dá)水平的相關(guān)性 空白對(duì)照組及干擾素-α組PBMCs中P-STAT-1表達(dá)水平分別為：NVR組（0.25±0.02）、（0.58±0.05），CVR組（0.26±0.02）、（0.66±0.05），PVR組（0.26±0.01）、（0.65±0.05），干擾素-α組PBMCs中P-STAT-1表達(dá)水平高于空白對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.01），回歸分析結(jié)果顯示：干擾素-α刺激后PBMCs中USP18的表達(dá)水平與P-STAT-1的表達(dá)水平成負(fù)相關(guān)性，相關(guān)系數(shù)為：-0.753（P0.01）。 7多種影響因素與干擾素-α治療療效的回歸分析 將受試者的年齡、治療前HBV-DNA基線水平、肝組織炎癥分級(jí)、治療前ALT水平、及治療前PBMCs中USP18的表達(dá)水平多種因素與干擾素-α治療療效進(jìn)行l(wèi)ogisitic回歸分析，通過(guò)逐步排除法，病毒載量、肝組織炎癥分級(jí)及治療前PBMCs中USP18的表達(dá)水平進(jìn)入回歸方程，y=0.588+20.552x1-39.299x2+19.641x3，回歸方程有統(tǒng)計(jì)學(xué)意義（χ~2=35.682，P0.01）。 結(jié)論： 1USP18的表達(dá)受干擾素刺激機(jī)體強(qiáng)烈誘導(dǎo)。 2USP18通過(guò)干擾STAT1的磷酸化作用，抑制干擾素-α抗病毒信號(hào)通路。 3HBV-DNA基線水平、肝臟炎癥分級(jí)及治療前PBMCs中USP18表達(dá)水平與干擾素-α治療療效均存在相關(guān)性，受試者治療前PBMCs中高水平表達(dá)USP18影響干擾素-α治療療效。 4USP18可能是干擾素-α治療慢乙肝過(guò)程中一個(gè)重要的穩(wěn)定的負(fù)性預(yù)測(cè)因子。
[Abstract]:Objective: hepatitis B virus (HBV) infection has become a global public health problem, about 1 million people die of liver diseases associated with HBV infection every year. Active HBV replication is the key factor in liver injury and disease progression. So the maximum long-term inhibition or removal of HBV is the overall goal of chronic hepatitis B treatment. There are two major categories of drugs used for antiviral treatment of chronic hepatitis B: 1, nucleoside (acid) analogues, 2, interferon (IFN). Interferon has the dual effects of antiviral and immunoregulation, the treatment course is short and the drug resistance is rare, so it has gradually become the main choice for the treatment of chronic hepatitis B in the clinic. However, interferon is currently used. The treatment can only reach 30%-40% HBeAg serological conversion, about 60% of the patients still suffer from hepatitis B virus, although they accept interferon antiviral therapy, so it is particularly important to predict the therapeutic effect of interferon in the short term. The latest research focus is interferon stimulating gene in the interferon's antiviral signaling pathway, and ubiquitin specific Sex protease 18 (Ubiquitin-specific protease18, USPl8) is one of the important interferon stimulating genes. This study aims to explore the predictive significance and mechanism of USPl8 in the treatment of chronic hepatitis B by interferon - alpha.
Methods: 70 patients with chronic HBV infection were selected for all patients with immune clearance. All the patients were performed liver biopsy and no contraindication to interferon antiviral therapy. PBMCs was extracted before treatment with the extracorporeal interferon alpha incubation test, and the subjects were treated with interferon alpha antiviral therapy. For 24 weeks, there were 18 cases of nonvirological response (NVR), 32 cases of fully virological response (complete virologicalresponse, CVR), 20 cases of virological response (partial virological response, PVR), and 10 healthy controls.
1 in vitro interferon - alpha incubation PBMCs: 70 cases of fresh peripheral venous blood were left before treatment, PBMCs was separated by density gradient centrifugation, and serum free medium suspension cells were divided into two groups: the first group blank control group (without any factors); the second group added interferon alpha factor to incubate PBMCs, and then collect cells after 4H.
2 all subjects were followed up with interferon - alpha antiviral therapy, and the treatment was 0 weeks, 1 weeks, 12 weeks, and 24 weeks of fresh peripheral blood, and the density gradient centrifugation was used to collect PBMCs..
3Trizol method was used to extract the total RNA of peripheral blood mononuclear cells, and the expression level of USP18 and STAT1mRNA was detected by Real-time PCR.
4RIPA method was used to extract the total protein of the cell. The expression level of USP18, STAT1 and P-STAT1 protein was detected by Western blot.
Result:
1 the expression of USP18 in peripheral blood PBMCs was negative in healthy controls.
Changes of USP18mRNA levels in peripheral blood PBMCs during treatment with interferon alpha 2
70 cases of CHB patients received mRNA level detection during interferon alpha therapy, and the relative expression of GAPDH in PBMCs was set to 1. Compared with GAPDH expression, the expression level of USP18 in NVR group before interferon alpha therapy (0.073 + 0.026) was higher than that in the CVR group (0.043 + 0.023) and PVR group (0.044 + 0.022), and the difference was statistically significant (F=10.665, P). 0.01); at the 1 week of treatment, the expression level of USP18 in each group of PBMCs increased significantly, in group NVR (0.098 + 0.023), in group CVR (0.071 + 0.027), in group PVR (0.074 + 0.027), and in group NVR, the difference was statistically significant (F=6.781, P=0.002), and the expression level of each group was NVR (0.097 0.024) (0.096 + 0.023) at 12 and 24 weeks, CVR group. (0.066 + 0.025), group PVR (0.075 + 0.026) (0.074 + 0.025), and NVR group had the highest level of expression (F1=7.140, p=0.002, F2=8.654, P0.01).
Changes of USP18 protein level in peripheral blood PBMCs during treatment with interferon alpha 3
The relative expression of beta -actin in PBMCs was set to 1. Compared with the expression of beta -actin, the expression level of USP18 in PBMCs in peripheral blood of NVR group before interferon alpha therapy was higher than that in CVR group (0.250 + 0.044) and PVR group (0.248 + 0.048), and the difference was statistically significant (F=11.151, P0.01). After 1 weeks, the expression level of USP18 in each group was clear. In group NVR (0.563 + 0.050), group CVR (0.431 + 0.047), group PVR (0.428 + 0.047), the expression level of group NVR was the highest, the difference was statistically significant (F=51.722, P0.01). At 12 and 24 weeks, the expression level of each group was NVR (0.568 + 0.051) (0.566 + 0.049), CVR group (0.430 + +), NVR group table The level reached the highest level, and the difference was statistically significant (F1=55.958, P0.01; F2=55.841, P0.01).
4 morphological observation of PBMCs stimulated by interferon alpha in vitro
After the PBMCs was adhered to the 2H, some cells were suspended. Most of the cells were B cells and peripheral blood DC. No morphological changes were observed after interferon - alpha was incubated to stimulate 4h, and there was no significant change in the number of cells.
5 changes of USP18 and STAT-1Mrna/ protein levels after stimulation of PBMCs with interferon alpha in vitro
The expression level of USP18 on mRNA/ protein in the interferon - Alpha Group PBMCs was higher than that in the blank control group after interferin - alpha stimulation in the peripheral blood PBMCs. The difference was statistically significant (P0.01), but there was no significant difference in the level of STAT-1mRNA and protein in PBMCs in the group of NVR, CVR and PVR.
6 the correlation between USP18 expression and P-STAT-1 expression after PBMCs stimulation in vitro
The expression level of P-STAT-1 in the blank control group and the interferon - Alpha Group PBMCs were: group NVR (0.25 + 0.02), (0.58 + 0.05), group CVR (0.26 + 0.02), (0.66 + 0.05), PVR group (0.26 + 0.01), (0.65 + 0.05), P-STAT-1 in interferon Alpha Group PBMCs higher than that of blank control group, the difference was statistically significant (P0.01), and the regression analysis showed interference. After PBMCs stimulation, the expression level of USP18 in P-STAT-1 was negatively correlated with the expression level of P-STAT-1, and the correlation coefficient was -0.753 (P0.01).
Regression analysis of 7 factors and efficacy of interferon alpha therapy
The subjects' age, pre treatment HBV-DNA baseline level, liver tissue inflammation classification, pre treatment ALT level, and USP18 expression levels in PBMCs before treatment were analyzed by logisitic regression analysis with interferon - alpha therapy, by stepwise exclusion, viral load, liver tissue inflammation classification and the expression level of USP18 in PBMCs before treatment. Regression equation, y=0.588+20.552x1-39.299x2+19.641x3, regression equation has statistical significance (chi ~2=35.682, P0.01).
Conclusion:
The expression of 1USP18 is strongly induced by interferon.
2USP18 inhibits interferon alpha antiviral signaling pathway by interfering with phosphorylation of STAT1.
3HBV-DNA baseline level, liver inflammation classification and the level of USP18 expression in PBMCs before treatment are correlated with interferon - alpha therapy, and high level expression of USP18 in PBMCs before treatment affects the therapeutic effect of interferon - alpha.
4USP18 may be an important and stable negative predictor of interferon alpha in the treatment of chronic hepatitis B.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.62

【共引文獻(xiàn)】

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