Myo1e表达变化影响足细胞抗原递呈功能与蛋白尿发生发展的机制

Proteinuria is not only the common feature of chronic kindey disease, but also the most important risk factor for development of end-stage renal disease and cardio-vascular events. In our previous study, Myo1e, as a cytoskeleton associated protein in podocytes, play an important role in functional integrity of podocytes and glomerular filtration barrier. Impairment of Myo1e in podocytes cause actin cytoskeleton re-organization, alteration of cell shape, and membrane transport, and podocyte drop-out from the glomerular basement membrane, which will eventually lead to an impaired glomerular filtration barrier and proteinuria. meanwhile,overexpression of Myo1e can enhance podocyte migration ability, endocytosis, and attachment to the glomerular basement membrane. Restoration of Myo1e expression in podocytes may therefore strengthen their functional integrity against environmental and mechanical injury. Recently, podocytes are identified as nonhematopoietic professional antigen- presenting cells and participate in the antigen-specific activation of adaptive immune responses within glomeruli. From above all, we hypothesized that Myo1e may play an key role in antigen presenting by podocytes in glomeruli. In present study, we will use conditionally immortalized murine podocytes cell line, isolated primary podocytes from mouse glomerulus, puromycin aminocleoside rat model treated with Myo1e antibody and podocyte-specific knock-out mice, to study the expression of CD80, ICAM-1,TLR, MHC classes and macrophage markers like emr1, sfpi1, MafB, Mpeg1, and Runx1, to study the chemotaxis of podocytes to T lymphocytes by transwell assay, to study if podocytes are able to take up exogenous antigen by endocytosis of ovalbumin and FITC-labeled latex beads, to study the antigen presenting ability of podocytes by mixed lymphocytes reaction, and to determine the function of activated T lymphocytes by measuring interleukin-2, interlukin-12, gama interferon concentration in supernatants of culture. Furthermore, we will study the interaction of podocytes and T lymphocytes in glomerulus from rat and mice model with Myo1e down regulation. Our expected results will reveal the key role of Myo1e in antigen processing and presenting of podocytes, which would be helpful to understand the pathogenesis of common auto-immunological glomerulonephritis in human beings, and be valuable for further development of novel therapy strategy.

蛋白尿不仅是各类慢性肾脏病常见的临床表现，也是终末期肾病及心血管事件的重要危险因素。本小组前期研究表明，肌球蛋白Myo1e对维系足细胞及肾小球滤过膜功能完整性具有重要意义，其表达异常可导致足细胞吞噬、迁移等功能异常及蛋白尿，但其确切机制尚未明确。 各种肾小球肾炎发病常与机体免疫功能紊乱有关，近来有研究表明足细胞可能是肾小球局部重要的抗原递呈细胞。由于Myo1e是重要的足细胞骨架相关蛋白，结合前期基础，我们认为Myo1e可能参与足细胞的抗原递呈功能。本研究以体外培养足细胞及Myo1e特异性敲除小鼠为研究对象，综合分析足细胞Myo1e表达改变前后抗原递呈相关分子表达、对T淋巴细胞趋化功能、抗原处理、递呈能力以及活化T细胞功能的变化，肾小球局部足细胞与T淋巴细胞的空间构象关系演变等，阐明Myo1e参与足细胞抗原递呈能力变化的分子机制，为自身免疫性肾小球肾炎的临床策略提供新的理论依据及治疗靶点。

各类肾小球肾炎发病常与机体免疫功能紊乱有关，近来有研究表明足细胞可能是肾小球局部重要的抗原递呈细胞。由于Myo1e是重要的足细胞骨架相关蛋白，结合前期基础，我们认为Myo1e可能参与了足细胞的抗原递呈功能，对各类免疫性肾小球疾病的发生、发展具有重要临床意义。.本研究采用Myo1e足细胞特异性敲除小鼠作为动物模型，结果发现，抗原递呈相关分子的表达，包括CD80、Toll样受体及主要组织相容性抗原MHCⅡ类分子，与对照组相比较，是存在显著下调的。与此同时，足细胞蛋白尿相关分子的表达变化如Nephrin，骨架相关蛋白Synaptopodin及BMP7等表达也有显著下调。上述研究结果提示，Myo1e在足细胞的特异性敲除，可诱导足细胞在吞噬、处理、递呈抗原等方面的功能异常。.同时，在体外培养的足细胞，包括来自Myo1e足细胞特异性敲除小鼠的原代培养足细胞，或MPC5足细胞株，采用shRNA技术敲低Myo1e表达后，足细胞对OVA的吞噬、对淋巴细胞的趋化作用有所增强。上述研究结果提示，Myo1e在足细胞的特异性敲低，对足细胞的抗原递呈左右在某些方面是有增强作用的。.本研究发现，Myo1e的表达变化，无论是表达下调或上调，无论在实验动物体内实验，还是在培养细胞体外实验，均可能造成抗原提呈功能的紊乱。这些抗原提呈功能的紊乱表现在各方面，如抗原递呈相关分子的表达变化、吞噬、处理、递呈抗原等多方面的功能异常。因此，Myo1e的表达变化造成的免疫功能紊乱是多维度的，复杂的，这些结果跟临床上蛋白尿、大部分肾小球肾炎病人存在的免疫紊乱状态是类似的。本研究结果表明，Myo1e参与了足细胞抗原递呈能力变化的分子机制，Myo1e在肾小球足细胞抗原递呈相关功能的变化可为自身免疫性肾小球肾炎的临床治疗提供新的理论依据及治疗靶点。