IFNγ与c-Myc通路交互作用调控门脉高压症脾亢脾MΦ极性转化的分子机制研究

Cirrhotic portal hypertension (PH) and hypersplenism is a common disease in our country. It is harmful for health and hasn’t satisfactory nonoperative treatment method. Splenic macrophages (Mφ) increased phagocytic capacity of hemocytes has been concerdered the most important reason of hypersplenism with PH. The new studis showed that the polarization of Mφ could evaluate the immune function of Mφ. In a former study, we found that LPS-TLR-NF-κB signal pathway was activated in splenic Mφ, it release the possibility of M1 polarization. But many evidences of M2 polarization were found in microarray findings. Until now, it has not got a conclusion about the polarization of splenic Mφ. In this study, we plan to evaluate the polarization of splenic Mφ, to find weather it has some relationship with the staging and typing of hypersplenism. Further more, we will check the polarization of peripheral blood mononuclear cell with PH, to found the relationship between splenic Mφ. It maybe helpful in early diagnosis of the disease. In another side, we will find the target genes and cross talking mechanism between IFNγ and c-myc pathway. To check the relationship of the pathway and the phagocytic capacity of splenic Mφ in vivo. We will feed Mφ specific IFNγ or c-Myc knockout mice and induce PH with CCl4, then admin quantum dots labeled hemocytes. It could be observed in small animals living imaging system to evaluate the function of the two pathways. The study could discover the polarization of splenic Mφ and provide new idea of treating hypersplenism by trans polarization.

门脉高压症及其伴随的脾亢是我国常见病，危害较大缺乏满意的非手术疗法。脾脏Mφ吞噬血细胞过多被认为是脾亢发病的主因，Mφ极性可综合评价Mφ的免疫功能。前期发现脾亢脾Mφ中LPS-TLR-NF-κB途径活化，提示可能存在M1型活化，但多种芯片结果也提示了大量M2型活化的证据，所以脾亢脾Mφ的极化状态及是否存在病程及个体化差异仍未明确。本课题拟从前期发现的脾亢脾Mφ内IFNγ（M1型）和c-Myc（M2型）通路入手，揭示其极化状态，判断是否存在“M1型/期”或“M2型/期”脾亢的分型、分期方法，以及早期诊断的依据。进而检测两条信号通路的靶基因及其交互作用机制，并干预信号通路诱导脾脏Mφ转极化，探索转极化疗法治疗脾亢的可行性。最后用活体成像检测Mφ特异性基因敲除鼠脾脏内量子点标记血细胞的滞留情况，验证两条信号通路对脾亢的作用机制。为进一步揭示脾亢的发病及病理生理特点和从转极化入手治疗脾亢提供思路。

门脉高压症及其伴随的脾亢是我国常见病，脾脏Mφ吞噬血细胞过多被认为是脾亢发病的主因，Mφ极性可评价Mφ的免疫功能。前期发现脾亢脾Mφ中LPS-TLR-NF-κB途径活化，提示存在M1型活化，但多种芯片结果也显示了M2型活化的证据，所以脾亢脾Mφ的极化状态仍未明确。本课主要探讨 IFNγ（M1型）和c-Myc（M2型）信号通路在脾亢患者脾脏Mφ极性转化中的作用并阐明其分子机制。我们发现脾亢脾Mφ中众多M1型炎性因子和少量M2型炎性因子活化明显，转录因子c-Myc表达升高，而IFNγ活化不明显。所以脾亢脾巨噬细胞M1/M2型均有活化，M1由LPS-TLR-NF-κB途径介导，而M2型活化与c-Myc的激活密切相关。通过脾脏单核巨噬细胞高表达基因Pathwey信号通路检测，发现MEK/ERK、PI3K-Alk等信号通路活化。WB证实了脾亢脾巨噬细胞MEK/Erk信号通路激活，参与了M2型巨噬细胞的活化，Jak/Stat信号通路激活，与M1型巨噬细胞的活化有关。体外实验中c-Myc特异性敲除小鼠肝硬化动物实验证实c-Myc敲除小鼠表现出更加严重的肝脏和脾脏炎症,存在MEK信号通路活化。抑制c-Myc的表达，脾脏巨噬细胞呈现M1型活化状态,M2型活化被抑制。外周血单核细胞活化诱导大量趋化因子表达升高，脾亢脾脏对外周血单核细胞有明显的趋化作用。结论：磁珠分选脾亢脾巨噬细胞和c-Myc基因敲除小鼠实验均证实c-Myc 通过MEK信号通路调节脾亢脾M2型巨噬细胞的极化。趋化因子表达是外周血单核细胞向脾脏趋化的主要原因。本课题明确了门脉高压症脾亢脾Mφ的极性，从Mφ极性的角度揭示了脾功能亢进的发生机制和病理生理特点；同时通过临床样本和体外动物实验两方面验证了脾亢脾的发生过程和活化机制，并为后续的生物治疗提供了理论基础。