Tfh细胞转录因子Ascl2/Bcl6在慢性肝炎高球蛋白血症中的作用机制

Chronic hepatitis is prone to form cirrhosis which is always affiliated with hyper-globulinemia. Previous studies have proved that the increase of globulins was due to the function decline of liver detoxication, which would lead to abnormal B cell activation and extraordinary antibody producing. However, the hyperglobulinemia doesn't completely correlated with the extent and severity of cirrhosis. And particularly, the patient with splenectomy still could not acquire a decreased globulins character, which implys that there should be other mechanism contribute to hyperglobulinemia. Currently, it has been demonstrated that the Tfh cells is esentially necessary for supporting B cell differention, expansion, secretion of antibodies, as well as antibody class switch. Currently, Ascl2 and Bcl6 has charactered as important Tfh transcription factors which determine the cell fate of CD4+T cells and lead to differentiation of Tfh cell. Base on these results, our study would focus on the function of Tfh transcraptional factors Ascl2/Bcl6, intend to demonstrate the relation between hyperglobulinemia in Chronic hepatitis and Ascl2/Bcl6 functions in Tfh cells. The project details are designed as follows:1.to investigate Tfh populations, ralative cytokine and receptor levels of the micro-envirement contributed to Tfh differentiation in different types of Chronic hepatitis; 2.to explore the impact of Tfh transcraptional factor Ascl2/Bcl6 signal pathway on B cell differentiation and antibody production with spleen mesenteric lymph nodes and bone marrow specimens from different group of patients; 3.to observe the mechanism and probable relation between Ascl2/Bcl6 level in Tfh cells and hyperglobulinemia in Chronic hepatitis by using gene over expression and silence strategy, accompanied with HBV/HCV antigen stimulation in vitro, or B-cell epitope peptides to stimulate the spleen cells in vitro.

慢性肝炎易合并高丙球蛋白血症，既往认为球蛋白增高与肝功能损害导致B细胞过渡活化有关，但临床所见球蛋白增高与肝脏炎症程度不完全相关，且脾大脾功能亢进者在脾切除术后球蛋白增高不能恢复，提示高球蛋白血症发生原因可能存在其他机制。Tfh细胞可辅助B细胞活化与增殖、抗体产生和Ig类型转换，其分化失衡导致B细胞活化增殖紊乱。新近发现Ascl2是CD4+T细胞向Tfh细胞分化的关键分子,与Bcl6共同发挥作用。因此，我们选择乙肝及丙肝高球蛋白血症患者为研究对象，以Tfh细胞为靶细胞，检测不同临床类型患者外周血、脾脏及肠系膜淋巴结标本中Tfh细胞和相关细胞、细胞受体、细胞因子水平,分析与Ascl2/Bcl6相关性；应用HBV/HCV抗原及抗原肽刺激PBMC、脾和淋巴结细胞，并辅以Ascl2/Bcl6基因沉默及过表达技术，以期阐明Tfh细胞Ascl2/Bcl6相关信号通路在慢性肝病高球蛋白血症中的作用机制。

肝炎病毒进入机体后常引起机体持续性免疫应答，会导致患者的免疫功能紊乱。除肝脏炎症外，部分患者可出现血清球蛋白升高，提示病毒感染可引起免疫失调。血清球蛋白升高与B淋巴细胞的异常分化及增殖有关，而Tfh细胞是参与辅助B淋巴细胞的活化、生发中心的形成的主要细胞群。肝炎病毒感染过程中， Ascl2可能参与启动Tfh细胞的活化，对B细胞分化和球蛋白产生密切相关。课题共选取慢性肝炎患者和健康者血液样本,分析外周血中Tfh细胞各亚群表面分子及细胞中Ascl2 mRNA表达水平，检测细胞因子IL-2、IL-12水平，并应用全自动生物化学分析仪检测肝功。结果显示，CHC患者PBMC Tfh细胞频数与B淋巴细胞频数呈正相关（r=0.5823，P=0.0112）；外周血B淋巴细胞频数与球蛋白呈正相关（r=0.4509，P =0.0316）；Tfh细胞频数与血清球蛋白呈正相关（r=0.5835，P =0.0383）；CHC患者外周血Tfh细胞中Ascl2 mRNA表达显著高于健康对照组(P =0.0051)； CD4+ CCR4+CD294（Crth2）+ T细胞亚群在HCV组显著升高（p=0.0289）。总之，课题明确了外周血Tfh细胞转录因子Ascl2的表达在慢性肝炎感染过程中的作用，为病毒性肝炎的临床治疗提供了实验证据和治疗的靶点。