含转导域重组神经肽PACAP-PTD对小鼠退化胸腺功能的重建作用及其分子细胞学机制的研究

The thymus is a central lymphoid organ for T cell development . Its function is particularly susceptible to various negative influences ranging from age-related involution to atrophy as a consequence of malnutrition, infection or harmful iatrogenic influences such as chemotherapy and radiation. The loss of regular thymus function show na?ve T cell production and output ability was decreased，T-cell repertoire diversity was declined，thymic microenvironment was destroyed. Thymus regeneration plays important roles in anti-aging, anti-infection, anti-tumor and anti-autoimmune disease area. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that was first isolated from an ovine hypothalamic extract, based on its ability to stimulate cAMP formation in anterior pituitary cells. PACAP is reported to play important roles in a large array of physiological and pathophysiological processes related to development, growth, cancer, immune response, circadian rhythms, the control of neuronal and endocrine cells, as well as functions of digestive, respiratory, reproductive and cardiovascular systems.Our Preliminary study demonstrated that PACAP could be a method to enhance and repair atrophy thymus function by producing thymus na?ve T cell and decreasing T cell apoptosis . To expand the application of PACAP and simplify its administration, we constructed a novel recombinant protein PACAP-PTD containing transduction domain PTD at the C-terminus of PACAP and demonstrated PACAP-PTD has increased ability to cross biological barrier. In our future research, after PACAP and PACAP-PTD preparation, we will establish several murine thymus involution animal model and analyze PACAP and PACAP-PTD' function by evaluating production and output of na?ve T cell, expression of functional gene in thymic microenvironment, the secretion level of cytokines, function of nerve repair. Therefore, the thymus regeneration function of PACAP and PACAP-PTD and the molecular and cellular mechanism will be explicitd and compared.

退化的胸腺功能减弱，减少了初始T细胞生成和输出，降低了T细胞受体库多样性，损伤了胸腺微环境。胸腺功能及其可回复性，对抗衰老、抗病毒、抗肿瘤及自免疾病具有重要理论和实践意义。垂体腺苷酸环化酶激活肽PACAP是多功能神经肽。本项目前期研究初步证实PACAP可回复胸腺初始T细胞的生成功能，且和调节特异性受体PAC1表达有关，还明确其抗凋亡作用，也证实含转导域的重组神经肽PACAP-PTD较PACAP有更优活性和穿越生物屏障能力。本项目拟制备纯化PACAP和PACAP-PTD后，设置小鼠胸腺萎缩模型，多手段分析其对瞬时T细胞自稳、胸腺的初始T淋巴细胞的生成和输出功能、胸腺/胸腺微环境中的各种细胞因子和功能基因的表达、细胞因子的分泌、神经修复等方面的作用，明确并比较它们是否具有胸腺功能重建的作用，同时在个体和细胞水平进行分子细胞学机制研究，探讨PACAP-PTD能否成为更有效安全的胸腺功能重建药物。

胸腺是哺乳动物重要的中枢免疫器官，萎缩的胸腺功能减弱。胸腺功能的回复在免疫重建中起着重要作用,对于抗衰老、抗病毒、抗感染、抗肿瘤及自身免疫性疾病都具有重要的理论和实践意义,可为治疗这些疾病提供新的切入点。.本项目在前期研究基础上,优化了重组PACAP-PTD制备体系，证实PACAP-PTD较PACAP具有更有效的保护神经细胞活性、更有效穿越生物屏障的功能，并明确PACAP特异受体PAC1-R在小鼠胸腺内有阳性表达；细胞水平，首先建立药物致淋巴细胞损伤模型，PACAP-PTD和PACAP处理并设置不同组别，明确并比较其对淋巴细胞的作用。其次培养胸腺上皮细胞，构建其和淋巴细胞的细胞共培养体系，模拟胸腺微环境，PACAP-PTD和PACAP处理并设置不同组别，分析并比较细胞因子的分泌、细胞增殖和凋亡的情况，明确PACAP-PTD/PACAP对于胸腺功能细胞、胸腺微环境的作用及其机制；个体水平，建立小鼠D-半乳糖诱导衰老模型和自然衰老模型，腹腔注射PACAP-PTD和PACAP，利用实时定量PCR、流式细胞术、免疫荧光技术等多种检测方法，分析其对于瞬时T细胞自稳、对胸腺的初始T淋巴细胞的生成和输出功能、对胸腺微环境中的各种细胞因子和功能基因的表达、对各类细胞因子的分泌的作用。通过以上研究，本项目已基本明确了PACAP-PTD和PACAP具有胸腺功能的回复作用及其分子和细胞学作用机制。具体来说，PACAP-PTD和PACAP（1）单独作用或通过细胞共培养体系作用于胸腺功能细胞，均促进淋巴细胞的增殖，抑制淋巴细胞的凋亡，且PACAP-PTD较PACAP保护作用更显著（2）胸腺微环境体系中，可提高细胞因子IL-1、IL-4、IL-6、TGF-beta1分泌，且除IL-6之外，PACAP-PTD作用更显著（3）可提高小鼠初始淋巴细胞生成和外周输出的功能，PACAP-PTD作用较明显，但统计学分析差异不显著（4）可改变模型小鼠全血中T细胞亚群，均使CD4/CD8比值显著提升，提示小鼠机体免疫功能的增强。总之，通过本项目的研究，结合PACAP抗炎、免疫调节等作用，“一药多效”，提示PACAP治疗或辅助治疗肿瘤、病毒性疾病、免疫系统疾病的前景。另外，由于含转导肽重组神经肽PACAP-PTD更强活性，可以改善其用药途径。