PI3K/AKT/mTOR信号通路在诱导调节性T细胞扩增、小鼠心脏移植免疫耐受中的作用及柴苓汤的影响

Since traditional Chinese medicine generally exerts anti-rejection effects, most of researches are limited to single drug that has no impact on inducing immune tolerance. The previous study conducted by our team members has shown that traditional Chinese medicine Chailing decoction could induce cardiac allograft immune tolerance, whereas the molecular mechanism was unclear. Adequately based upon the results that regulatory T cells(Tregs) was increased and the expression of mammalian target of rapamycin (mTOR) was down-expressed on mice when they underwent cardiac allograft immune tolerance, we may safely forecast that the mechanism of inducing Tregs increase might via depressing PI3K/AKT/mTOR signal pathway. In the process of study, effective and varied research approaches have been applied,such as MLR, FACS, triple immune stain, real time RT-PCR, Western blot to explore Tregs level, the expression of PI3K/AKT/mTOR signal pathway molecular after mouse cardiac transplantation and the effectiveness of traditional Chinese medicine Chailing decoction. Meanwhile, for the purpose of exploring the mechanism of immune tolerance aroused by traditional Chinese medicine Chailing decoction and the molecular mechanism of inducing Tregs increase in vivo and in vitro, the function of Tregs on cardiac allograft immune tolerance has been further investigated by deleting Tregs via injecting anti-CD25 monoclonal antibody and adoptive transfer. In conclusion, through sufficient and profound academic research, the study aims to provide traditional Chinese medicine for treatment of allograft rejection and promoting transplantation tolerance.

中药具有一定的抗移植排斥反应作用，但仍局限于单药且无诱导免疫耐受之功效。前期研究发现柴苓汤可诱导小鼠心脏移植免疫耐受，但其分子机制仍不清楚。由于心脏移植免疫耐受小鼠调节性T细胞(Tregs)扩增且哺乳动物雷帕霉素靶蛋白(mTOR)的表达下调，据此推测诱导免疫耐受的机制可能是通过抑制PI3K/AKT/mTOR通路导致Tregs间接升高所致。本研究应用混合淋巴细胞反应、流式细胞术、三重免疫染色、实时定量RT-PCR、Western Blot等技术，检测Tregs、PI3K/AKT/mTOR通路分子在小鼠心脏移植后的动态变化以及柴苓汤的影响，应用抗CD25单克隆抗体去除Tregs以及过继转移实验等手段观察Tregs对诱导免疫耐受的影响，从体内、体外实验两方面，在整体、细胞和分子多水平探讨柴苓汤诱导小鼠心脏移植免疫耐受以及Tregs扩增的分子机制，为复方中药在移植免疫耐受领域的应用奠定理论基础。

中药具有一定的抗移植排斥反应作用，但仍局限于单药且无诱导免疫耐受之功效。前期研究发现复方中药柴苓汤可诱导小鼠心脏移植免疫耐受，但其分子机制仍不清楚。由于心脏移植免疫耐受小鼠调节性T细胞(Tregs)扩增且哺乳动物雷帕霉素靶蛋白(mTOR)的表达下调，据此推测柴苓汤诱导小鼠心脏移植免疫耐受的机制可能是通过抑制mTOR通路导致Tregs间接升高所致。体内实验应用流式细胞术、免疫荧光及RT-PCR技术检测发现，柴苓汤具有诱导Tregs扩增的效应。应用RT-PCR、Western-blot技术检测发现，Tregs扩增的受体体内mTOR通路明显受到抑制，其上下游分子（AKT、PI3K、mTOR、p70s6k、4E-bp1）及其磷酸化产物均出现不同程度的下调；通过蛋白芯片法检测发现，相应细胞因子IFN-γ、IL-6、IL-12的表达量下降；IL-10、IL-2、IL-4的表达量升高，符合Tregs扩增条件。体外实验提取受体小鼠脾脏淋巴细胞进行培养，同时使用柴苓汤处理，结果显示应用柴苓汤培养的淋巴细胞其mTOR通路上下游分子（AKT、mTOR、p70s6k、4E-bp1）及其磷酸化产物均出现不同程度的下调。混合淋巴细胞培养检测发现柴苓汤处理后的受体小鼠其淋巴细胞增殖活性明显减弱。综合以上研究表明，柴苓汤可显著抑制PI3K/AKT/mTOR通路活性，诱导受体Tregs扩增从而导致小鼠心脏移植免疫耐受。本研究从体内、体外实验两方面，在整体、细胞和分子水平探讨了柴苓汤诱导小鼠心脏移植免疫耐受的分子机制，为复方中药在移植免疫耐受领域的应用奠定理论基础。目前发表中文核心期刊论文2篇、SCI论文1篇，待发表SCI论文2篇（其中1篇在修回阶段，另外1篇在撰写阶段）。培养硕士硕士生4人，其中3人已取得硕士学位，1人在读。