去分化脂肪细胞对PBOO所致膀胱损伤中TGF/Smad通路的调控及其机制研究

Bladder outlet obstruction in infants is one of the most commonly reasons of congenital abnormalities of the urinary tract. The natural history of congenital lower urinary tract obstruction in the perinatal period is of high fetal and perinatal morbidity and mortality. fetal obstructive uropathy causes both renal failure and bladder dysfunction after birth. Survivors of the neonatal period may be affected by chronic renal impairment in infancy and adulthood for survivors. Options for intervention antenatally exist but they do not preserve bladder function, and their efficacy is not yet proven by randomised controlled trials (RCTs). In children, bladder outlet obstruction is the primary cause for end-stage bladder, which is characterized by persistent accumulation and deposition of extracellular matrix that lead to widespread tissue fibrosis. Bladder outlet obstruction induces numerous changes in bladder morphology, physiology, and pharmacology. the first step is that the bladder wall muscle is stretched by the dilatation of the bladder as it fills with urine. At the same time, there is increased accumulation of ECM in the submucosal and muscular layer of the bladder wall. obstructed bladder walls at term have thickened, hypertrophied muscle layers interspersed with an increased amount of collagen. TGFβ/Smad signaling is an important pathway leading to EMT, apoptosis and fibrosis in several tissue and organs. BOO may induce the TGFβ/Smad signaling, and the bladder fibrosis may be activated via TGFβ/Smad signaling. Despite significant progress in the understanding of cellular and molecular pathogenesis during recent decades, the treatment of bladder fibrosis remains challenging. Thus, developing a novel therapeutic strategy for more effective treatment of bladder fibrosis and end-stage bladder in patients with PBOO is an important consideration. Because of their multilineage differentiation capabilities, dedifferentiated fat cell ( DFAT ) is considered to have a therapeutic potential in obstructive urethropathy. In our study, DFAT will be cultured with TGF, following the coculture, DFAT will differentiated into smooth muscle cell. DFAT is a potent renotropic factor that plays a critical role in promoting detrusor repair and bladder functional regeneration after injurious stimuli. We will transplant the DFAT with smooth muscle-like phenotype into bladder smooth muscle, then determine the distribution and transition of the transplanted DFAT by in situ hybridization and immunochemistry method. we will use hydrostatic pressure to induce the EMT of BMSCs, RNAi and pc-DNA expression vetor will be explored at different points of TGFβ/Smad signaling by knocking-up and knocking-down Smad. The regulation effort of TGFβ/Smad signaling in injury of BMSCs under hydrostatic pressure will be studied. In vivo study, it will be performed to confirm the in vitro results, and to confirm the effort of anti-fibrosis of the DFAT transplantation. This method may have a synergistic effect in bladder functional regeneration and anti-fibrosis that could contribute to a better outcome.

先天性膀胱出口梗阻是儿童肾功能衰竭的常见原因，其核心致病机制是导致膀胱纤维化最终进展为肾衰竭。除产前干预外，目前尚无理想的治疗方法，且远期预后不佳。膀胱出口梗阻所致膀胱纤维化的关键调控分子和主要致病信号通路尚不清楚。我们前期研究发现：随膀胱出口梗阻时间延长膀胱胶原沉积增加，平滑肌细胞减少且发生表型转化，同时TGF表达增高，将DFAT细胞注射入膀胱，可转化为平滑肌细胞且可减少胶原沉积和TGF表达。故推测膀胱出口梗阻损伤可能与TGFβ/Smad信号通路相关，DFAT对膀胱出口梗阻损伤的修复可能是通过调控TGFβ/Smad信号通路完成的。本研究拟通过细胞培养、制作大鼠膀胱出口梗阻模型，应用DFAT细胞共培养和原位注射治疗，通过构建干扰或表达载体在细胞水平和动物模型体内对TGF/Smad信号通路调控，系统研究DFAT细胞对膀胱出口部分梗阻损伤中的TGFβ/Smad信号通路的调控机制。

背景：膀胱出口梗阻(bladder outlet obstruction，BOO）是儿童泌尿系统最常见的下尿路梗阻疾病，目前该疾病的治疗方法不能从根本上改善患儿膀胱功能及远期终末期肾病的结局，仍会影响患儿生活质量甚至是生命健康。因此，明确膀胱出口梗阻后膀胱病理改变的时序性，寻找治疗该疾病的有效手段，并选择一个合适的节点进行干预，对该疾病所致膀胱损伤的修复有重要意义。随着干细胞研究的深入，去分化脂肪（dedifferentiated fat，DFAT）细胞有可能能够改善先天性膀胱出口梗阻导致的膀胱损伤，为先天性膀胱出口梗阻开辟一个新的治疗方向。主要研究内容：通过两种方法培养DFAT细胞、制作新生大鼠膀胱出口部分梗阻模型，观察BOO后膀胱病理时序性改变，探讨DFAT细胞诱导分化为平滑肌细胞的能力，应用DFAT细胞共培养和原位注射治疗，在细胞水平和动物模型体内对TGF/Smad信号通路进行调控，系统研究DFAT细胞对BOO中的TGFβ/Smad信号通路的调控机制。重要结果：1.新生大鼠 PBOO 后肉眼观膀胱壁增厚，膀胱组织代偿性增生，随着梗阻时间延长，膀胱体积增加，膀胱壁逐渐变薄。HE 染色可见 PBOO 后2天炎细胞浸润，4天肌细胞排列紊乱，黏膜下层和肌层增厚，肌间隙增大。组织免疫荧光检测 PBOO 后膀胱组织中 calponin 1、vimentin、SMMHC 和 collagen Ⅲ 表达量呈现先增加后减少的趋势，且 PBOO 后4天 calponin 1、vimentin 和 SMMHC表达量最高，PBOO 后8天 collagen Ⅲ 表达量最高。2、培养的 DFAT 细胞呈成纤维样、梭形、峰谷样排列。DFAT 细胞流式分析表面抗原标志物表达为 CD29+CD90+CD31-CD45-，阳性细胞百分数分别为 CD29+（96.93±3.61）%，CD90+（98.65±0.84）%，CD31+（0.16±0.13）%，CD45+（0.16±0.10）%。DFAT 细胞经体外成平滑肌细胞诱导后，αSMA 染色阳性，αSMA 阳性细胞率为（73.56±8.44）%。