基因重组乳酸乳球菌分泌SDF-1引导骨髓间充质干细胞归巢治疗溃疡性结肠炎及清热燥湿凉血方的协同作用研究

Ulcerative colitis(UC) is a chronic relapsing inflammatory bowel disease characterized by limited therapeutic options. The technology of stem cell and tissue engineering, which can induce stem cells to differentiate toward specific cells and replace dysfunctional cell or tissue, may be the fundamental treatment method of UC . We know that SDF-1 and its receptor CXCR-4 are important factors which can mobilize and guide bone marrow mesenchymal stem cells（BMSCs）.Improving the homing efficiency of BMSCs and the synergy treatment of the prescriptions of clearing heat,removing dampness and cooling blood is a important therapeutic method. We cultivate Lactococcus lactis(LL) which can secrete SDF-1 through genetic recombinant, and improve local microenvironment of SDF-1 / CXCR4 signaling pathways by the prescriptions of clearing heat,removing dampness and cooling blood.In this way, we expect that the signaling pathways of SDF-1 / CXCR4 can guide BMSCs to colon and improve the curative effect of BMSCs in UC. Through the immunohistochemical method, molecular biology, etc, we discusses the treatment of UC by the homing of BMSCs mediated by LL-SDF-1 and the synergy of the prescription of clearing heat,removing dampness and cooling blood. Finally we want to explore and provide new theoretical and research methods to the clinical diagnosis and treatment of the disease，which will have important theory value and promising application perspective.

溃疡性结肠炎（UC）是一种治疗手段比较有限且易复发的炎症性肠病。通过干细胞技术和组织工程技术定向诱导干细胞使其分化为特定的细胞以替代功能障碍的细胞或组织缺损，这可能是治疗UC的根本方法，而基因重组乳酸乳球菌分泌SDF-1 和其受体CXCR-4 是操控骨髓间充质干细胞（BMSCs）动员和牵移的重要因子，通过提高BMSCs归巢效率和清热燥湿凉血方协同作用不失为一条重要的治疗方法，其中本课题通过基因重组培养能分泌SDF-1的乳酸乳球菌，并以清热燥湿凉血方改善SDF-1/CXCR4信号通路的局部微环境，期望能以SDF-1/CXCR4信号通路引导BMSCs定植于结肠病灶部位，从而提高BMSCs治疗UC的疗效。本课题通过免疫组化、分子生物学等方法，研究基因重组乳酸乳球菌分泌SDF-1引导BMSCs归巢治疗UC及清热燥湿凉血方的协同作用，并为开拓该病的临床治疗手段提供新的理论依据和研究方法。

背景: 炎症性肠病(IBD), 包括溃疡性结肠炎(UC)和克罗恩病(CD), 是一种治疗手段有限且易复发的消化系统疾病。通过组织工程技术定向诱导骨髓间充质干细胞(BMSCs)以替代功能障碍的细胞或组织缺损,这可能是治疗肠道疾病的根本办法。外源性的BMSCs输入体内后向受损的肠道归巢的效率非常低下。大量研究表明基质细胞衍生因子1(SDF-1)与其特异性受体趋化因子受体4(CXCR4)能调控BMSCs归巢于受损组织。主要内容：本课题研究SDF-1/CXCR4轴在BMSCs治疗2,4,6-三硝基苯磺酸 (TNBS)诱导的结肠炎中的作用及清热燥湿凉血方的干预作用。观察重组慢病毒技术构建过表达CXCR4的BMSCs向炎症结肠定向迁移情况及其治疗实验性结肠炎的效果及其潜在的黏膜修复、抗炎和免疫调节作用以及清热燥湿凉血方的干预作用。结果: 慢病毒转染后80%的BMSCs能够稳定表达GFP蛋白。相对空白组, 结肠炎大鼠结肠部位SDF-1表达明显上，IFN-γ、TNF-α、IL-6和IL-10的mRNA表达显著上升, STAT-3及磷酸化STAT-3的蛋白表达明显上升(P<0.05)。尾静脉注射治疗后,与模型组比较, Ad-CXCR4-BMSCs能够显著的下降结肠炎疾病活动指数(P<0.01)和病理炎症分数(P<0.01)，结肠部位GFP蛋白表达明显增高(P<0.01), IFN-γ、TNF-α、IL-6的mRNA表达显著下降, IL-10的mRNA表达显著上升(P<0.05); STAT3及磷酸化STAT-3的蛋白表达显著下降(P<0.05)。清热燥湿凉血方可以促进BMSCs归巢于受损结肠并缓解炎症，,机制可能与重新调配外周血和肠黏膜之间的Treg比例有关。意义:SDF-1/CXCR4轴在BMSCs归巢于受损的结肠部位中发挥重要作用, 清热燥湿凉血方可以促进BMSCs归巢于受损结肠并缓解炎症,为炎症性肠病的细胞治疗提供潜在的方法和理论依据。