肌肉特异性微小RNA联合成肌调节因子MyoD逆转糖尿病大鼠膀胱重构的实验研究

Diabetic cystopathy (DCP), a common and severe complication of diabetes mellitus (DM) in urinary system, diaturbs people's quality of life seriously. Currently there is no effective preventions and threapies for DCP. Bladder remodeling, featured by detrusor muscle fibrosis, is an important step in DCP's pathogenesis. Our preliminary study indicated that ectopic expression of MyoD, an important myogenic regulator, in bladder fibroblast by gene transfection can induce myogenic conversion , although the myogenic efficiency and differentiation level were low. The muscle-specific microRNAs（miR-1,miR-133 and miR-206）, which are expressed in cardiac muscle, skeletal muscle and vascular smooth muscle, have been shown to regulate differentiation and proliferation of these cells, separately?or in collaboration with MyoD. We speculate that upregulating these microRNAs could promote the efficiency of myogenesis and level of differentiation in MyoD-induced myoblast from bladder fibroblast. We propose to develop rat model of DM and culture their primary bladder fibroblast. The levels of expression of these microRNAs will be observed during the process of MyoD-induced myogenic conversion. Based on the results of observation, we will modify the expression levels of these microRNAs separately or collectively using the techologies of qRT-PCR, adenovirus transfection, liposome transfection, immunofluorescence staining and western-blot analysis. The myogenic efficiency and differentiation level will be evaluated. Furthermore, the mechanisms of induced myogenisis will be analyzed and the optimal plan of myogenesis induced by MyoD in combination with different set of miRNAs will be selected. It will provide theoretical and experimental basis for therapy of anti-remodeling of bladder in DM patients.

糖尿病膀胱（DCP）的主要病理改变为膀胱重构（bladder remodeling），其逼尿肌纤维化是重要表现之一，目前尚无有效修复措施。本课题组前期研究显示通过构建成肌调节因子MyoD真核表达载体，转染逼尿肌成纤维细胞转化为成肌细胞，有利于逆转膀胱重构，但转化效率及分化欠佳。有研究证实肌肉特异性微小RNA（miR-1、133、206）在骨骼肌、心肌及血管平滑肌中单独或配合与MyoD协同作用可精细调控肌细胞增殖分化。我们试图通过干预肌肉特异性微小RNA表达并联合MyoD作用于逼尿肌成纤维细胞，验证其可否提高成肌转化效率并促进增殖分化。拟运用qRT-PCR、腺病毒感染、脂质体转染、免疫荧光染色、western-blot等技术研究糖尿病大鼠MyoD诱导逼尿肌成纤维细胞成肌转化过程中三种微小RNA表达变化，定向干预其表达，观察其对成肌转化效率及增殖分化的影响，探讨作用机制并筛选最佳诱导成肌方案。

糖尿病膀胱（DCP）主要病理改变为膀胱重构，逼尿肌纤维化是重要表现，尚无有效修复措施。我们前期研究通过构建MyoD真核表达载体，转染逼尿肌成纤维细胞转化为成肌细胞，有利于逆转膀胱重构，但转化及分化欠佳。有研究证实肌肉特异性微小RNA（miR-1、133、206）在骨骼肌、心肌及血管平滑肌中单独或配合与MyoD协同作用可精细调控肌细胞增殖分化。我们通过干预三种miRNA表达并联合MyoD作用，验证了miRNA表达并联合MyoD可以显著提高成肌转化效率并促进增殖分化。运用qRT-PCR、转染感染、免疫荧光染色、western印迹等技术研究了MyoD诱导成肌转化过程中三种miRNA表达变化，定向干预其表达，观察了其对成肌转化效率及增殖分化的影响，探讨了作用机制并筛选出了最佳诱导成肌方案，为后续临床开展改造自体细胞逆转DCP膀胱重构的潜在疗法提供了实验依据。