CCR7/CCL21途径在循环成纤维细胞识别与沉积导致移植肾纤维化的作用及机制研究

Despite current improvements in the longevity of renal allografts, the transplanted kidney typically loses its function before the patient dies. Although a variety of factors contribute to the renal graft loss, chronic allograft nephropathy (CAN) is recognized as the leading cause of renal allograft failure after the first year following transplantation.Histological manifestations of CAN include interstitial fibrosis, tubular atrophy and dilation, accumulation of fibroblasts, glomerulosclerosis, and chronic inflammatory cell infiltration.Fibrosis is the predominant pathological change of chronic allograft kidney.Nevertheless, the precise pathogenesis mechanisms of renal fibrosis remain poorly understood. Since fibroblasts play a critical role in the progression of fibrosis, expression of fibroblast surface protein (FSP), which correlates with the number of fibroblasts, may serve as a biomarker for evaluating fibrosis of kidney allografts. A number of growth factors and cytokines have been implicated in the development of renal fibrosis,Our results indicate that FSP, CCL21, and CCR7 are localized in the interstitial fibroblasts and renal tubular epithelium cells, suggesting the involvement of the CCL21/CCR7 pathway in the progression of renal allograft fibrosis. Hence, these observations provide evidence for the molecular and cellular mechanisms of renal allograft fibrosis, and may help to develop a therapeutic target for allograft failure following kidney transplantation.Chemokines and chemokine receptors are involved in the progression of acute and chronic renal disease . Expression of chemokine receptors, such as CCR7, CCR2 and CXCR4, have been detected in fibrocytes isolated from humans and mice. One of the chemokine receptors, CC-chemokine receptor 7 (CCR7), and its ligand chemokine CC-ligand 21 (CCL21, also known as the secondary lymphoid tissue chemokine) have been shown to regulate trafficking of fibrocytes into the kidney, which leads to renal fibrosis. However, the potential involvement of the CCL21/CCR7 pathway in fibrosis of kidney allografts following transplantation remains undetermined. In the present study, we investigate the distribution and expression of FSP, chemokine CCL21 and chemokine receptor CCR7 in renal allograft biopsies obtained from patients following kidney transplantation. In our study, the model are constructed by using female Fisher344 rat as donator and male Lewis rat as receptor. And the origin of fibroblasts are detected by Y chromatin body detection. Fibroblasts culture，flow cytometry sorting，CCR7siRNA transfection, Fibroblasts intravenous injection and intravenous blocking CCL21/CCR7 pathway are used to study the role of circulating fibrocytes and CCL21/CCR7 signal in interstitial formation of transplant kidney

移植肾间质纤维化是导致慢性移植肾失功、影响移植肾长期存活的最主要原因。虽然已知纤维细胞与纤维化关系密切，但其来源及作用途径尚未阐明。近年文献报道，骨髓来源的循环成纤维细胞可导致器官纤维化，但移植肾间质纤维化过程中，循环成纤维细胞是否参与、如何进行肾脏识别与沉积并不清楚。我们前期工作显示CCL21/CCR7通路对移植肾间质纤维化成纤维细胞的迁移有重要作用。由此，我们假设CCR7/CCL21途径可调控循环成纤维细胞在移植肾中的识别与沉积，并导致间质纤维化。为验证假设，我们拟利用雌性Fisher344大鼠作为供体，雄性Lewis大鼠作为受体构建移植肾间质纤维化的模型，通过Y染色体方法检测成纤维细胞的来源；采用成纤维细胞培养，CCR7siRNA靶向转染等方法，观察CCL21/CCR7通路在循环成纤维细胞肾脏识别与沉积中的作用及机制，为延缓移植肾纤维化的发生、发展及防治慢性移植肾失功提供方法。

慢性肾功能衰竭严重危害着人类的健康，肾移植是治疗肾功能衰竭的最佳选择。移植肾间质纤维化是导致慢性移植肾失功、影响移植肾长期存活的最主要原因。虽然已知纤维细胞与纤维化关系密切，但其来源及作用途径尚未阐明。近年文献报道，循环成纤维细胞可导致器官纤维化，但移植肾间质纤维化过程中，循环成纤维细胞是否参与、如何进行肾脏识别与沉积并不清楚。我们前期工作显示CCL21/CCR7通路对移植肾间质纤维化过程中成纤维细胞的迁移有重要作用。由此，我们假设CCR7/CCL21途径可调控循环成纤维细胞在移植肾中的识别与沉积，并导致间质纤维化。.本项目主要研究内容即为，研究移植肾中成纤维细胞的来源；探讨CCL21/CCR7信号通路在移植肾间质纤维化发生过程中的作用机制；观察抗CCL21治疗是否影响移植肾纤维化进程。.本研究利用一系列的研究方法和实验技术，包括建立肾移植患者随访标准化体系，收集人体移植肾穿刺标本，外周血分离培养循环成纤维细胞，CCR7siRNA靶向转染，构建SD大鼠供给Wistar大鼠的肾移植动物模型，证实了参与移植肾间质纤维化的成纤维细胞可来源于循环成纤维细胞，且循环成纤维细胞可加速移植肾纤维化进程；而循环成纤维细胞可能通过CCL21/CCR7信号途径迁移到移植肾；抗CCL21治疗可延缓移植肾纤维化进程。.本研究通过Y染色体原位杂交检测确定参与移植肾间质纤维化的成纤维细胞可来源于外周血循环成纤维细胞；成功建立移植肾动物模型进一步明确了循环成纤维细胞的作用及途径，并发现抗CCL21治疗可延缓移植肾纤维化进程；人体移植肾穿刺标本及临床资料的综合分析帮助我们在人移植肾中验证了成纤维细胞与肾脏功能改变及纤维化进程具有相关性。.本研究通过观察CCL21/CCR7通路在循环成纤维细胞肾脏识别与沉积中的作用及机制，为延缓移植肾纤维化的发生、发展及防治慢性移植肾失功提供方法，可能会成为延缓移植肾纤维化治疗的新靶点。