从T细胞免疫活化分子及Toll样受体信号通路探究中医药治疗HAART后免疫无应答患者的免疫调节机制

Non-immune response after receiving HAART therapy is a tricky problem puzzled western AIDS treatment, under the premise of ensuring good adherence after receiving HAART therapy, plasma viral load could get undetectable longer control level, but there are still about 5% -30% CD4 + T lymphocytes in patients with no significant growth appears.Traditional Chinese medicine with its multi-target overall regulation has demonstrated preliminary efficacy of improving the immune function and clinical symptoms and signs in patients with no immune response after receiving HAART therapy, but lack of research on deep efficacy mechanism.This project uses flow cytometry and Toll-like receptor signal transduction gene chips methods, through comparison of T cell immune activation molecules and Toll-like receptor signaling pathway between non-immune response patients receiving HAART therapy and HIV-negative healthy subjects, to evaluate abnormal immune response and characteristics of TLR signaling pathways in patients with non-immune response status. This project also compares T cell immune activation molecules and Toll-like receptor signaling pathway between non-immune response patients receiving HAART therapy pre and post traditional Chinese medicine intervention, to evaluate effects of Yiqijianpi Granule on CD cell subtype and TLR signaling pathways, and the correlation between CD cell subtype and TLR signaling pathways and CD4 + T lymphocyte count, HIVVL , clinical symptoms and signs. To reveal the efficacy mechanism of traditional Chinese medicine intervention on non-immune response patients receiving HAART therapy, providing new ideas and efficacy evaluation for Chinese medicine treatment for HIV/AIDS.

HAART（高效抗逆转录病毒疗法）后免疫无应答状态是目前西医艾滋病治疗的难点。接受HAART治疗后，血浆病毒载量较长时间控制在测不出的水平，但仍有约5%-30%的患者CD4+T淋巴细胞数量未出现显著增长，且此类患者并发机会性感染及死亡风险显著增加。中医药多方面多靶点整体调节已在提升HAART后免疫无应答患者免疫功能，改善症状体征等方面展现了初步疗效，但缺乏对疗效机制深入探讨。本项目采用流式细胞术及Toll样受体信号转导基因芯片等方法，通过对HAART后免疫无应答患者中医药治疗前后T细胞免疫活化分子及Toll样受体信号通路的检测，评价益气健脾颗粒对HAART后免疫无应答患者CD细胞亚群和TLR信号通路的可能影响及上述两者与CD4+T淋巴细胞计数、HIVVL、临床症状体征的相关性分析，揭示该药干预HAART后免疫无应答的疗效机制，为中医药治疗HIV/AIDS提供新的思路和疗效评价指标。

艾滋病免疫重建不全机会性感染的发生率显著高于免疫重建良好的人群，对免疫无应答进行干预的尝试成为后HAART时代的研究热点。中医药治疗强调对机体的整体调节，调整阴阳虚实失衡的状态，尽力使免疫病理性应答向生理性应答转化，从而达到改善免疫重建不全状态的目的。本项目共计完成30例中药益气健脾颗粒治疗前及治疗后 6 个月的 HAART 后免疫无应答患者 T 细胞免疫活化分子及 TLR水平的检测，观察上述指标与 CD4+T 淋巴细胞计数的可能相关性。从细胞和基因两个层面探究益气健脾颗粒干预免疫无应答患者的疗效机制。并完成与21例HIV阴性健康对照者的对比研究。初步从T细胞活化分子及TLR通路途径阐明益气健脾法改善免疫功能的作用机制，为提高艾滋病免疫无应答患者的临床疗效奠定基础。结果显示：益气健脾颗粒治疗前后CD4+明显提高，差异有统计学意义（P＜0.05）；治疗前后，艾滋病免疫无应答组CD4CD28、 CD8CD38、 CD4CD25值均较疗前有所增加，但CD4CD28、 CD8CD38前后比较差异没有统计学意义（P＞0.05），CD4CD25治疗前后有统计学差异（P＜0.05）。CD4CD28、 CD8CD38、 CD4CD25三个指标分别与CD4做线性回归相关性分析，CD8CD38与CD4显示出有较强统计学意义的相关性，呈正相关，其余两个指标与CD4之间的相关性没有统计学意义（P>0.05）。益气健脾颗粒治疗前后免疫无应答组TLR1、 3、 4、 7、 8、 9 相关基因mRNA水平检测治疗前后结果分析：艾滋病免疫无应答组经益气健脾颗粒治疗6个月后，TLR4和TLR9均升高，治疗前后比较差异有显著统计学意义, 而TLR1、 3、 7、 8前后比较没有发现明显统计学意义。对来自相同地域（昆明）的22例疗前艾滋病免疫无应答组和21例HIV阴性健康对照组进行外周血TLR4和TLR9检测，结果显示：艾滋病组外周血TLR4和TLR9水平均明显高于健康对照组，差异有显著统计学意义，表明HIV感染后细胞可能通过对HIV进行特异性抗原识别，上调共刺激分子并分泌细胞因子，激活适应性免疫系统。本项目研究可能为评价中医药联合HAART疗法辅助治疗艾滋病免疫无应答患者的疗效机制提供有力证据支持，以TLR为靶点积极开展中药抗感染作用机制研究与新药研发，对于临床治疗疾病和中医药疗效评估具有重要意义。