热休克转录因子2在溃疡性结肠炎粘膜修复中的作用机制研究

Mucosal healing is a treatment target of Ulcerative Colitis (UC). The structure and function restitution of colonic mucosa are the critical process to repair damage colonic mucosa to heal. Unfortunately, the detailed mechanism of colonic mucosal repair is not clear. Thus, series of research had been carried out to solve this problem in our previous work. Our studies found that the heat shock transcription factor 2(HSF2) was an up-expression protein in serum of active phase of ulcerative colitis by gene chip and proteomics. Additionally, the expression level of HSF2 in the serum and intestinal mucosa was positively correlated with the severity of ulcerative colitis. In vivo experiment, HSF2 could reduce expression of TNF-α through inhibiting NF-κB pathway. Additionally, HSF2 could promote the intestinal epithelial cells proliferation, migration and inhibit apoptosis. Those are the molecular and cell biology foundation of colonic mucosal repair, which prompted HSF2 involving in colonic mucosa repair. But the mechanisms of HSF2 in colonic mucosal repair are unclear. So, various research will be carry out in this project : (1)in the clinical level, detection the level of HSF2,calprotectin,TGF-bata, Mayo endoscopy sub-score and before and after mucosal healing of UC patients;(2) in cellular level, knock out and overexpression of HSF2 in intestinal epithelial cells, detection cell factors and signaling pathways associated with cell proliferation, migration and differentiation ;(3) in animal level, breeding HSF2-/- and HSF2+/+ genetically engineered mice, building acute and chronic mucosal damage model, observing the mucosal damage severity and repair time, detecting the cell type, cytokines and signaling pathways involved in each stage of mucosal repair. In order to clarify the mechanisms of HSF2 in colonic mucosal repair of UC, and to provide theoretical basis for HSF2 becoming an therapeutic target in mucosal healing.

粘膜愈合是溃疡性结肠炎（UC）的治疗目标，粘膜修复是达到愈合的关键，但修复的机制不清，我们针对该问题开展系列研究发现：①患者血清基因芯片及蛋白质组学实验确认热休克转录因子2（HSF2）是UC活动期上调表达的蛋白质并与UC严重度呈正相关；②体外实验发现HSF2抑制NF-κB激活而减少TNF-α表达，抑制肠上皮细胞凋亡并促进增殖、迁移、分化。提示HSF2参与肠粘膜修复，但其在修复中的作用机制不清，故本项目通过：①临床水平，检测粘膜愈合前后HSF2和TGF-β水平、MES评分，探索HSF2与粘膜愈合及促粘膜修复因子的相关性；②细胞水平，敲除和过表达肠上皮细胞HSF2基因，检测细胞修复相关的调控因子及通路；③动物水平，建立HSF2基因敲除与过表达小鼠急、慢性肠炎模型，观察粘膜修复进程，检测参与的细胞类型、因子及信号通路。为明确HSF2在UC肠粘膜修复中的作用机制及研发新的治疗靶点提供理论依据。

溃疡性结肠炎（Ulcerative colitis，UC）是一种病因未明确的肠道非特异性慢性炎症性疾病，以反复发作的腹泻、黏液脓血便伴腹痛为主要表现，也是结肠癌的一个重要危险因素。我国UC发病率日益增高。UC粘膜损伤修复及愈合评价是一个复杂的科学问题。课题组前期发现热休克转录因子2（HSF2）是UC活动期差异表达的蛋白质并与UC严重度呈正相关，提示HSF2参与UC肠粘膜修复并且可以量化成为评价粘膜愈合的一个指标。本项目进一步研究HSF2在UC粘膜愈合中的作用和机制。.临床水平，检测HSF2在不同MES（Mayo endoscopic subscore，MES）评分的UC患者粪便中的水平，以肠镜MES≤1为粘膜愈合评价标准，根据约登指数计算取1.97 ng/ml HSF2为截断值，作为预测粘膜愈合的指标，采用诊断试验的评价方法，计算HSF2在预测粘膜愈合中的灵敏度与特异度，阳性预测值与阴性预测值。结果证实粪便HSF2含量能作为预测溃疡性结肠炎粘膜愈合的一个潜在评价指标，具有较高的准确性，其特异度和阴性预测值的临床价值高于敏感度和阳性预测值。.细胞层面，建立HSF2敲除和过表达稳定细胞株，发现HSF2通过抑制ERK、P38及JNK的磷酸化水平，减低LPS诱导结肠上皮细胞炎性因子IL-1β及TNF-α的水平；HSF2通过促进Smad2/3磷酸化水平，提高细胞TGF-β的水平；HSF2促进上皮细胞增殖活性，增加肠粘膜屏障相关蛋白Occludin的表达，促进粘膜损伤修复。证实HSF2能抑制炎症反应并促进粘膜修复因子表达，起到促进粘膜修复的作用。.动物层面，成功构建了hsf2基因敲除小鼠，hsf2基因敲除小鼠表现出更为严重的肠道炎症及DAI评分。.另外，成功重组表达、分离纯化了具有生物活性的人源HSF2蛋白，为制备HSF2多克隆抗体和ELISA试剂盒，大规模检测粪便HSF2水平奠定了良好的基础。.本研究为明确HSF2在溃疡性结肠炎的粘膜损伤修复及评价提供理论依据，具有较高的学术价值和运用价值。