脾切除改善丙型肝炎失代偿期肝硬化抗病毒应答的免疫机制研究

Patients with hepatitis C virus related decompensated cirrhosis are associated with a poor prognosis, and it is a clinical very thorny issues yet to be resolved. Recently, some studies showed that patients can benefit from interferon-based antiviral therapy. However, many patients cannot receive standard treatment due to cytopenia associated with hypersplenism. In our preliminary prospective study, splenectomy resulted in sustained normalization of platelet and leukocyte counts, thereby allowing these patients to undergo antiviral therapy without hematologic disturbance. And the therapeutic effects and safety of antiviral therapy in decompensated cirrhotic patients are favourable after splenectomy, compared with hepatitis C virus related cirrhotic patients without splenectomy.Its mechanism is not yet clear.We assume that the spleen dysfunction may be one of the causes of poor efficacy and splenectomy can reverse splenic dysfunction and improve antiviral response.In this study, we chose hepatitis C virus related decompensated cirrhotic patients with portal hypertension and hypersplenism as our research object.We propose to collect peripheral blood serum samples from these patients before and after splenectomy (2 weeks、one month、prior to antiviral therapy),as well as spleen samples. We will detect the changes of HCV RNA level by RT-PCR. We also detect humoral immunity markers by ELISA and immune turbidimetric method.We will use flow cytometry to detect the separated and purified peripheral blood and spleen mononuclear cells line. Immunohistochemistry and in situ hybridization were used to detect protein and mRNA expression of spleen tissue. Further, We will detect the cytokine differences in expression by cytokines chip, and we plan to screen some cytokine that are highly relevant with the replication of hepatitis C virus, then to observe the effect of these cytokine on HCV replication in vitro. The purpose of this study is to evaluate the immune mechanisms of splenectomy improve antiviral treatment in patients with hepatitis C virus related decompensated cirrhosis,and to focus and clarify splenectomy would be appropriate supportive therapy prior to antiviral therapy in selected patients with portal hypertension and hypersplenism.

丙型肝炎失代偿期肝硬化患者预后差，是临床极待解决的问题。患者可获益于干扰素为基础的抗病毒治疗，但部分因脾亢无法耐受标准方案。课题组前期研究显示脾切除不仅可缓解脾亢，耐受标准治疗，且疗效优于无需脾切除肝硬化患者，其机制尚不清。我们假设脾功能紊乱可能是应答不佳原因之一，脾切除可纠正脾功能紊乱改善抗病毒应答。本课题拟以丙肝肝硬化门脉高压症、脾亢患者为研究对象，收集脾切除前、后（2周、1月、治疗前）外周血及脾脏标本。RT-PCR检测HCV RNA定量，ELISA法检测体液免疫指标，分离、提纯外周血和脾脏单核细胞行流式细胞检测；免疫组化及原位杂交检测脾脏相关蛋白及mRNA表达。基因芯片检测细胞因子差异表达，筛选出与HCV复制高度相关的因子，以不同浓度作用于HCV细胞株，观察其对HCV复制的影响。旨在探讨脾切除改善丙肝肝硬化患者抗病毒应答的免疫机制，为临床上该人群抗病毒治疗前行脾切除手术提供理论依据。

丙型肝炎失代偿期肝硬化患者预后差，是临床极待解决的问题。患者可获益于干扰素为基础的抗病毒治疗，但部分因脾亢无法耐受标准方案，且抗病毒治疗应答率明显低于无肝硬化患者。脾切除对患者免疫细胞功能状态影响如何，以及术后免疫微环境改变对随后接受干扰素联合利巴韦林抗病毒治疗疗效的影响尚不清楚。该项目的研究主要包括：1）脾切除对患者外周血免疫细胞状态的影响；2）评估基于干扰素方案的疗效及其对患者预后的影响；并与脾切除后抗病毒治疗的效果进行比较；3）探讨丙肝肝硬化患者肝脏和脾脏免疫耐受分子的表达，明确脾切除能否改善外周血T淋巴细胞的免疫活性，改善免疫微环境。我们的结果显示：74.2%接受干扰素为基础的抗病毒治疗患者获得治疗结束应答（ETVR），45.5%患者获得SVR，28.8%复发，25.7%无应答。与治疗前比较，获得ETVR患者肝功能明显改善；且随访过程中肝脏失代偿、HCC发生风险降低、存活时间延长；18.2%患者治疗过程中发生严重不良事件，包括2例死亡。但是基因1型和2型患者获得SVR的比例分别为34.1%和68.2%，明显低于脾切除后的44%和100%。丙肝肝硬化患者脾脏中免疫抑制分子PD-1、PD L2在蛋白和mRNA水平表达均明显升高。脾切除术后患者外周血NK、NKT、CD56dim NK细胞活性明显增强，CD4+、CD8+T细胞及其比例趋于正常，CD19+B细胞的比例显著下降。患者脾脏PD-1+CD8+T、PD-1+CD4+T比例明显高于正常脾脏，CD4+CD25+Foxp3+细胞比例较正常脾脏高，但差异无显著性。与健康对照比较，患者外周血CD4+、CD8+T细胞表面PD-1分子表达增加；术后患者外周血CD4+、CD8+T细胞表面PD-1分子表达下降。患者外周血CD4+CD25+Foxp3+细胞比例升高，脾切除术后CD4+CD25+Foxp3+细胞比例降低，但差异无显著性。因此，抗病毒治疗获得应答可改善失代偿期丙肝肝硬化患者的长期预后；脾切除可改善抗病毒治疗的安全性和应答率；门脉高压症脾脏高表达免疫耐受分子，脾切除术后患者外周血免疫细胞功能状态发生明显改变，有利于改善患者免疫微环境，是术后抗病毒应答效果理想的原因之一。该研究可为临床上该人群抗病毒治疗前行脾切除手术提供理论依据。