免疫2号方干预艾滋病患者免疫重建的临床及机制研究

【作者】 汤艳莉；

【导师】 王阶；

【作者基本信息】 北京中医药大学 ， 中西医结合临床， 2012， 博士

【摘要】 艾滋病全称为获得性免疫缺陷综合征(acquired immunodeficiency syndrome,AIDS),由感染人免疫缺陷病毒(human immunodeficiency virus,HIV)而引起,是一种严重危害人类健康的致死性传染病。高效抗逆转录病毒疗法(highly active anti-retroviral therapy,HAART)是目前临床公认的较为有效的治疗方法,可最大限度地抑制艾滋病毒复制,有效降低血浆病毒载量,并在一定程度上促进患者的免疫功能重建。然而,这种免疫重建常常是不完全的,且不同个体间存在很大差异。如何解决HAART后免疫重建不全的问题,发现有效的治疗及干预手段至关重要。中医药从整体调节角度入手,对人体免疫功能具有独特的调节作用,以免疫重建作为中医药干预艾滋病治疗的切入点已成为当前艾滋病领域的研究热点。因此本研究从艾滋病免疫重建的文献和理论梳理入手,确定研究的目标及干预手段,继之以临床流行病学调查结果为支持,深入了解我国HAART治疗的临床特点,最后采用随机、盲法、安慰剂对照的RCT试验对免疫2号方的临床疗效进行了验证,同时在T细胞表面受体基因重排及免疫信号分子通路方面进行了初步的机制探讨,概要如下：1国内HAART治疗对艾滋病免疫重建及死亡率的影响研究目的了解我国近年来接受HAART疗法的人群特征及HAART治疗对艾滋病患者免疫重建和死亡率的影响,为下一步的临床研究寻找目标人群。方法用描述流行病学方法对广西柳州龙潭医院2005年1月至2009年12月5年期间首次接受HAART治疗的2644例艾滋病病例进行流行病学分析,比较不同性别患者年龄、种族以及危险因素的年度分布趋势,描述CD4细胞计数在治疗前后的分布变化,计算标准化死亡率比(SMRs),进行生存分析。结果(1)2005年至2009年,发病患者多数为男性(1740,65.81%),其中30～39岁组最多,与女性患者分布类似,不同之处在于女性20～29岁组在最近3年中有显著增长。性接触成为主要传播方式,2009年占接受治疗人群的85.2%(612/718)。(2)治疗超过1年的患者中,CD4涨幅超过10%的有1064例患者(53%、不包括死亡和失访患者),262例(13%,262/2004)患者治疗后CD4反下降10%以上,免疫重建不全发生率为188例,占17.42%。(3)SMR在20-29岁组最高,为62.42,(95%CI41.44-83.40),而在>=60岁组最低,为1.55(95%CI0.9-2.20)。169例(6.39%,169/2644)患者在随访中死亡,死亡率为3.99/100人年。以不同传播途径分层：静脉吸毒患者死亡率52/475(10.9%),性传播患者死亡率105/2015(5.2%),其他因素感染者死亡率12/154(7.8%)(p=0.000)。结论我国HAART治疗已经极大减少了AIDS所致死亡率,但免疫重建不全患者仍占17.42%,尚需要更多有效的免疫调节手段来弥补HAART治疗的缺陷。2免疫2号方干预艾滋病患者免疫重建的临床及机制研究目的观察免疫2号方对AIDS患者HAART后免疫重建功能的影响,并探讨该疗法对机体免疫调节的作用机制。方法采用随机、双盲、安慰剂对照的临床试验设计原则,264例HAART后免疫重建不全患者随机分为治疗组(131例)和对照组(133例),分别采用免疫2号方联合HAART治疗和安慰剂联合HAART治疗6个月,观察两组治疗前后外周血CD4+、CD4CD45RA+、CD4CD45RO+细胞数量的改变,以及两组治疗前后症状、体征积分及改善率的变化,评价其免疫重建有效率。同时对TCRVβ基因多样性的改变进行检测,并采用Toll样受体(Toll-like receptors, TLR)信号转导基因芯片检测免疫2号方联合HAART对TLR信号传导通路的影响。结果符合纳入标准的病例264例,试验过程中有3例因不良反应脱落,25例自行退出,1例数据不完全,2例因未服用药物剔除。最后完成研究233例,治疗组116例,对照组117例。(1)两组免疫重建有效率及免疫细胞计数比较：干预1个月后,治疗组有效率(18.97%)显著高于对照组(9.40%),(p=0.0209)；疗后3月,治疗组(27.59%)仍然高于对照组(22.22%)(p=0.3076)；疗后6月,治疗组有效率(34.48%)显著优于对照组(21.37%),差异有统计学意义(p=0.0217)。CD4+、CD4CD45RA+、CD4CD45RO+绝对计数治疗组增加幅度显著高于对照组(p<0.05)。(2)两组症状体征改善率及总积分比较：治疗6月以后,两组各项症状、体征均有所改善甚至消失。总积分组内比较差异均有统计学意义(P<0.05),治疗组的总积分改善程度与对照组相比效果更好(P<0.05)。治疗组在改善患者乏力、肌关节痛、皮肤瘙痒、气短等症状时疗效明显优于对照组,差异有统计学意义(P<0.05)。(3)两组病毒载量变化比较：治疗前、治疗3月、治疗6月,两组病毒载量变化组间比较差异无统计学意义,组内前后配对检验结果比较差异无统计学意义。(4)安全性分析：两组均有部分患者发生不良事件,治疗组不良事件发生率为3.08%,对照组6.25%,组间比较差异无统计学意义(P=0.2263)。治疗前后,血常规、尿常规、心电图、B超、胸片检查结果均无明显变化。(5)TCRVP基因多样性改变比较结果显示：①HAART治疗后一年免疫重建不全患者的TCRVβ多个家族的扫描图高斯分布被打破,并发生TCR谱系的偏移,经半年中药联合HAART治疗后,偏移的TCR谱系有所恢复。②以试剂盒提供的定量分析软件计算的距离D(distance)值来量化评价,两组D值改变无明显差异(p>0.05),但联合中药能减少数据的变异性。③CD4+T细胞计数与TCRVβ基因多样性改变两者存在显著的相关关系(r=-0.772,P=0.000)。④相对于正常人,患者一些Vβ内出现了明显的CDR3区的单寡克隆扩增情况,经过治疗后各家族单寡克隆情况有不同程度的改善和恢复。其中,9,11,21,22四个家族的D值两组治疗后均比治疗前下降,治疗组下降幅度较对照组在21,22两个家族更为显著(P<0.05)。治疗组显著降低第18家族D值,而对照组则显著增加(P<0.05)。(6)TLR信号转导通路分析结果显示：筛选出与艾滋病免疫重建不全有关的基因显著上调≥2倍的有5个(如干扰素-γ、IL-6、IL-8等),下调≥2倍的基因有4个(如TLR9、NF-KB等),通路上游以基因下调改变为主(如TLR9、TOLUP、TRIF、IRAK1、TAB1、p105),下游则多为上调改变(如AP-1、IL-6、IL-8)。联合免疫2号方干预后,相关基因显著上调≥2倍的有7个(如IL-2、IL-10、TLR1等),无显著下调基因,对通路上游基因的上调作用最为显著(如TLR1、CD14、TRAF6).结论(1)中药免疫2号方对HAART后艾滋病患者CD4+T细胞免疫重建以及症状体征具有较为明确的疗效,其在改善患者乏力、肌关节痛、皮肤瘙痒、气短等症状方面有较好效果,CD45RA、CD45RO细胞亚群均有一定程度的恢复。(2)免疫2号方具有良好的安全性,无明显不良反应。(3)机制研究表明中药复方对于TCRVβ各家族单寡克隆情况有不同程度的改善和恢复,提示中医药可能促进T细胞部分受体基因重排,丰富受体库,帮助机体免疫细胞有效识别病毒,减少T细胞凋亡；对于toll样受体信号通路的研究表明HAART后免疫重建不全的艾滋病患者细胞内TLR9较正常人表达低下,IRAK1、NF-κB表达下调,提示TLR9介导的信号转导系统异常是艾滋病患者免疫重建不全发生的可能机制之一；免疫2号方调节机体免疫功能的机制可能主要通过上调TLR1和CD14表达,进而引起下游一系列通路信号分子传递而实现。更多还原

【Abstract】 AIDS (acquired immune deficiency syndrome) is a lethal disease infected by the HIV (human immunodeficiency virus) and can caused serious harm to human health. Highly active antiretroviral therapy (HAART) is now recognized as a clinically effective treatment, which can inhibit the HIV replication, effectively reduce the plasma viral load, and to some extent, reconstitute the patients’ immune function. However, this immune reconstitution is often incomplete, and there is big difference between different individuals. How to solve incomplete immune reconstitution after HAART, and find effective treatment and intervention is essential.Chinese medicine has a unique role in the regulation of human immune system. Promoting immune reconstitution by Chinese medicine has become the focus in field of AIDS research. In this study, we started by reviewing the literature and theories of AIDS and immune reconstituiton to identify research objectives and interventions, followed by clinical epidemiological study to know more about the application of HAART in China, and finally using the randomized, blinded, placebo-controlled RCT to confirm the efficacy of Mianyi No.2. At the same time, we examined the T-cell receptor gene rearrangement and TLR signaling pathways to find the molecular mechanism. The research is devided into2parts:PartⅠ Study on effect of immune reconstitution and mortality by HAART in ChinaObjectiveRelatively little is known regarding HIV-positive antiretoviral-naive patients among Chinese people, and how highly active antiretroviral therapy (HAART) influenced immunological outcomes and mortality of HIV positive patients in China We aimed to examine this by analysis of retrospective dataMethodsWe merged data on demographics, risk behavious, CD4cell counts and dates of death based on clinical records of2644HIV-positive antiretroviral-naive patients in Liuzhou district, Guangxi Province, China from2005to2009. We analyzed the trend of HIV prevalence, and calculated standardized mortality ratios (SMRs) standardized by age and sex. CD4counts at start and the end were also compared. Kaplan-Meier methods were used to assess survival over time.ResultsFrom2005to2009, most follow-up was fiom men (1740,65.81%), and the men age30-39years was the most, which was similar as in women, The incidence of young women (20-29years old) was higher than that of young men.1064patients (53%,1064/2004, not including deaths, lost of follow up) had increased magnitudes of CD4more than10percent of origin count, and262patients (13%,262/2004) had10percent decreased CD4count after treatment. The rest678patients (34%,678/2004) had incomplete information. Incomplete immune reconstitution (CD4count increased less than100/ul after one year treatment) were seen in188patients, accounting for17.42%. SMR was highest in patients age20-29at62.42,(95%Q41.44-83.40), and lowest in patients age>=60at1.55(95%CI0.90-2.20).169(6.39%,169/2644) patients died during follow-up, giving a mortality incidence rate of3.99per100person years.156/2241(7.0%) patients with CD4counts of200cells/μl or less died compared with11/314(3.5%) patients with CD4counts above200cells/μl and below350cell/μl, and2/77(2.6%) patients with CD4counts above350cells/μl (p=0.073).2/475(10.9%) patients infected by drug injection died compared with105/2015(5.2%) patients infected by sexual contact and12/154(7.8%) patients by other ways (p0.000).ConclusionHAART has reduced mortality among patients in China with AIDS, but incomplete immune reconstitution was still seen in188patients, accounting for17.42%. We need more effective means to promote the immune reconstitution in patients after HAART.PartⅡ Effect and related mechanism of Mianyi No.2on the Immune Reconstitution in Patients with AIDS after HAARTObjectiveTo observe the Mianyi No.2on the immune reconstitution in patients with AIDS after HAART, and to investigate the possible targets and mechanisms of Mianyi No.2.MethodsA randomized, double-blind, placebo-controlled clinical trial was designed.264patients failure to immune reconstitution after HAART were randomly divided into treatment group (131cases) and control group (133cases), respectively, using Mianyi No.2plus HAART and placebo combined with HAART for6months. CD4+, CD4CD45RA+, CD4CD45RO+cell numbers, as well as the symptoms, signs and integral improvement rates were observed in order to evaluate the immune reconstitution efficiency. At the same time changes in genetic diversity ofTCRVβ and TLR signaling pathways were detected. ResultsDuring the trial,264cases were enrolled and randomized, among which3cases drew out for adverse reactions,2cases for not taking medicine at all, and25cases quit by themselves. There were233cases that accomplished the clinical trial, of which116cases in the experiment group and117in the control group.Results are as follows:1Efficacy of immune function:after the intervention for1months, the effective rate of experiment group (18.97%) was significantly higher than that in control group (9.40%),(P=0.02);3months after treatment, the effective rate of experiment group (27.59%) is still higher than that of the control group (22.22%)(P=0.31);6months after treatment, the effective rate of experiment group (34.48%) was significantly superior to the control group (21.37%)(P=0.02). CD4+, CD4CD45RA+, CD4CD45RO+count of experiment group increased significantly higher than that of control group (P<0.05).2Efficacy of symptoms and signs:The accumulated points of symptoms and signs in treatment group had a better improvement effect compared with the control group (P=0.02), and the improvement of fatigue, muscle and joint pain, pruritus and shortness of breath in treatment group was better than control group (P<0.05).3Efficacy of viral load:There were no significant difference in viral load between or within groups before or after treatment (p>0.05).4Safety analysis:The incidence of adverse events occurred in some patients in both groups, the adverse event rate of experiment group was3.08%, control group6.25%(P=0.2263). There is no significant difference in changes of blood test, urine test, ECG, B-ultrasound, or chest X-ray.5Results ofTCRVP genetic diversity change indicate that:①Gaussian distribution ofTCR V(3families in patients with incomplete immune reconstitution after one year of HAART, had been broken with the occurrence of the offset TCR lineage. After six months of treatment of Traditional Chinese medicine combined HAART, the TCR lineage has been partially restored.②Evaluated by the D (distance) value calculated by a quantitative analysis software which the kit provides, there were no significant difference in D value change (p>0.05) between the two groups, but with traditional Chinese medicine can reduce the data variability.③CD4+T cell counts had a significant correlation (r=-0.772, P=0.000)with TCRV(3genetic diversity.④Compared with the normal group, there appeared some single or oligoclonal amplification of Vβ CDR3region in the patients, which were improved or recovered after treatment Among them, D value of four families (9,11,21,22) decreased after treatment in both groups. The decrease in family21and22was significant (P<0.05) in treatment group compared with the control group. And family18was decreased in treatment group and increased significantly in control group (P<0.05).6Changes on Toll like Receptor Signalling Pathway:There were5genes involved in the complete immune reconstruction significantly raised more than2times (such as interferon-γ, IL-6, IL-8, etc.), and4genes decreased (such as TLR9, NF-κB etc). Genetic changes in upstream were mainly down regulated (such as TLR9, TOLLIP, TRIF, IRAKI, TAB1, p105), while in downstream were mainly up regulated (such as AP-1, IL-6, IL-8). After treatment of immune No2, there were7related genes significantly raised more than2times (such as IL-2, IL-10, TLR1etc), and no genes significant decreased. Genetic changes in upstream were mainly up regulated (such as TLR1, CD14, TRAF6).Conclusion1Mianyi No.2can effectively improve die numbers of CD4+T cell and its subgroups, as well as the main clinical symptoms and signs of patients after HAART, such as fatigue, muscle and joint pain, skin itching, shortness ofbreath, therefore promoting immune reconstitution.2Mianyi No.2is safe to treat AIDS disease, with no significant adverse reactions.3Study of the mechanism showed oligoclonal of TCRVp family can get recovery in some degrees after treated by Mianyi No.2plus HAART, suggesting that the medicine may promote T-cell receptor gene rearrangement, helping immune cells to effectively identify the virus to reduce T-cell apoptosis; TLR9were expressed lowly in AIDS patients with incomplete immune reconstitution that in healthy person, and also IRAK1、NF-κB, suggesting that abnormal signal pathway mediated by TLR9maybe one of the mechanisms of incomplete immune reconstitution of AIDS. The effect of Immune No.2may be realized by up regulating TLR1and CD14expression, resulting changes of signal molecules in downstream.更多还原