【作者】 王宁；

【导师】 齐隽； 沈海波；

【作者基本信息】 上海交通大学 ， 外科学（泌尿外科）， 2019， 博士

【摘要】 膀胱出口梗阻是泌尿系统常见的一类疾病,最常见的原因是前列腺增生,患者往往伴有下尿路症状,严重影响生活质量。现已发现BOO后的膀胱在结构和功能上均发生变化,并从炎症改变进展到膀胱的纤维化改变。MicroRNA-101在肝脏等器官纤维化中发挥重要作用,但是其在膀胱纤维化中的作用仍不清楚;ALK4是TGFβ超家族中一员,其在心脏纤维化中发挥重要作用,但是其是否参与PBOO引起的膀胱纤维化中仍不是很清楚;本课题从以上两个方面研究PBOO引起膀胱重塑的具体机制:第一部MicroRNA-101在低氧诱导的膀胱纤维化中的作用及机制研究目的现已发现BOO引起的膀胱重塑是一种从炎症到纤维化的转变过程,本研究是探讨microRNA-101在低氧引起的膀胱纤维化中的作用机制。材料及方法1.构建大鼠BOO动物模型,通过免疫组化、western blot及realtime PCR检测相关分子及microRNA-101表达水平;2.原代培养大鼠膀胱平滑肌细胞,给予低氧刺激,通过Western blot及realtime PCR检测纤维化相关表型变化;3.转染microRNA-101 mimic及microRNA-101inhibitor,并在低氧下进行培养,通过western blot检测smad2/3信号通路是否激活;4.通过双荧光素酶实验验证TGFβR1是microRNA-101的靶向分子。结果1.大鼠BOO4W及6W后,膀胱湿重由sham组的0.158±0.006g增长至BOO4W的0.63±0.02g及BOO6W的0.84±0.046g,膀胱与体重的比例也由sham组的0.03±0.003%增长至BOO 4W的0.15±0.007%;collagen/smooth muscle比例sham组为0.513±0.007%,BOO4W组collagen/smooth muscle比例为0.3657±0.02%,BOO 6W组0.975±0.1%,膀胱纤维化程度明显升高;2.与sham组相比,BOO 4W及6W后膀胱中HIF1α及TGFBR1表达水平分别升高,并且microRNA-101表达水平升高。3.低氧刺激膀胱平滑肌细胞后,细胞外基质水平表达升高,TGFBR1表达水平也升高,P-smad2/3表达水平升高。4.过表达microRNA-101后,膀胱平滑肌细胞低氧诱导的纤维化改变被逆转,P-smad2/3表达水平下降,smad2/3信号通路受到抑制。5.MicroRNA-101逆转低氧诱导的TGFBR1升高,双荧光素酶实验显示TGFBR1是microRNA-101的靶向分子。结论MicroRNA-101通过TGFBR1抑制低氧诱导的膀胱纤维化第二部分ALK4在BOO引起的膀胱纤维化中的的作用及机制研究目的BOO可以引起膀胱的结构性重塑,以膀胱平滑肌的增生和ECM的堆积为主要特点。ALK4是TGF超家族中的一种受体蛋白,有研究发现其在心肌病理性增生及纤维化中表达升高,并激活smad2/3信号通路,具有关键性作用。但ALK4-smad2/3信号通路在BOO引起的膀胱纤维化中的作用机制还不清楚。本研究主要目的是研究ALK4在BOO引起的膀胱纤维化的作用机制。材料及方法1.构建BOO小鼠动物模型,验证ALK4表达水平在BOO后4W及6W后的膀胱中的变化。2.构建ALK4+/-的转基因小鼠,构建BOO模型,分为BOO 4W、BOO 6W及sham三组,并通过组化染色,realtime PCR及western blot检测相关分子的表达水平,所有动物实验均被动物伦理委员会批准。3.原代培养BSMCs,过表达ALK4,通过western blot及realtime PCR检测纤维化相关分子表达水平及samd2/3信号通路变化,并检测其对膀胱平滑肌细胞细胞周期及增殖的影响。结果1.在WT小鼠中,ALK4在BOO4W及6W后表达水平明显升高。2.western blot发现ALK4半敲除小鼠中ALK4表达水平明显降低。3.realtime PCR及免疫组化发现,BOO 6W后的ALK4+/-小鼠胶原纤维表达水平明显降低,膀胱纤维化受到明显抑制。4.通过western blot检测发现,BOO6W后的WT小鼠MAPK及P-smad2/3表达水平升高,相关信号通路被激活。BOO 6W后的ALK4+/-小鼠中P-smad2/3表达水平降低,具有统计学差异(P<0.05),ALK4对BSMCs的增殖和周期没有影响,而过表达ALK4后,纤维化相关分子表达升高,P-smad2/3表达水平升高,samd2/3信号通路被激活。结论ALK4通过smad2/3信号通路调节BOO引起的膀胱纤维化,ALK4可能作为BOO引起的膀胱重塑的一个治疗靶点。更多还原

【Abstract】 Bladder outlet obstruction is common in the urinary system.The most common cause is BPH.Patients often suffer from lower urinary tract symptoms,which seriously affect the quality of life.It has been found that the structure and function of the bladder makes a difference after BOO,while fibrotic changes were also found.MicroRNA-101 is a small class of RNA that plays an important role in fibrosis of liver and so on.The role of MicroRNA-101 in bladder fibrosis caused by BOO is unclear.ALK4 is a receptor in the TGFβsuperfamily and has been found to promote the heart fibrosis.However,whether it participate in bladder fibrosis caused by BOO is not clear.Then from the above two aspects,we do some research about BOO-induced bladder remodeling.Part1 The effect and mechanism of microRNA-101 in the bladder fibrosis induced by hypoxiaObjectivesIt has been found bladder remodeling induced by BOO is a transition from inflammation to fibrosis and a multifactorial disorder.This study is to explore the mechanism of microRNA-101 in hypoxia-induced bladder fibrosis.Materials and methods1.Rat model of BOO was constructed,and the expression of related molecules and microRNA-101 was detected by immunohistochemistry,western blot and realtime-PCR;2.Bladder smooth muscle cells were primary cultured,and were cultured under hypoxia.Fibrosis-related phenotypic change was detected by western blot and realtime PCR;3.MicroRNA-101 mimic and microRNA-101 inhibitor were Transfected into BSMCs.Activation of smad2/3 signaling pathway was detected by western blot and realtime PCR in BSMCs cultured under hypoxia;4.Detect whether TGFβR1 is a targeting molecule of microRNA-101 by dual luciferase assay.Results1.The wet weight of bladder increased from 0.158±0.006g in sham group to 0.63±0.02g in BOO4W and 0.84±0.046g in BOO6W.The ratio of bladder to body weight also increased from 0.03±0.003%in sham group to BOO 4W 0.15±0.007%.Collagen/smooth muscle ratio of sham group was 0.513±0.007%,collagen/smooth muscle ratio of BOO4W was0.3657±0.02%,and collagen/smooth muscle ratio of BOO 6W was0.975±0.1%.2.The expression of HIF1αand TGFBR1 in the bladder after BOO 4W and BOO 6W increased respectively.And the expression of microRNA-101a was up,too.3.After bladder smooth muscle cells were cultured under hypoxia,the expression of extracellular matrix,TGFBR1and P-Smad2/3 increased,and the signaling pathway was activated.4.After microRNA-101b was transferred into BSMCs,the expression of ECM induced by hypoxia in BSMCs decreased,and BSMCs fibration was significantly inhibited.The expression of P-Smad2/3 decreased,and Smad2/3 signaling pathway was inhibited by microRNA-101b.5.TGFBR1 was induced by hypoxia in BSMCs and the progress was inhibited by microRNA-101b.Dual luciferase assay showed that TGFR1 is a targeting molecule of microRNA-101b.ConclusionMicroRNA-101 inhibits hypoxia-induced bladder fibrosis via TGFBR1Part 2 The effect and mechanism of activin receptor-like kinase 4 in bladder fibrosis caused by bladder outlet obstructionObjectivesThe urinary bladder underwent profound structural alterations after bladder outlet obstruction(BOO),characterized by hypertrophy of bladder wall and accumulation of extracellular matrix(ECM).Activin receptor-like kinase 4(ALK4)is a downstream receptor of transforming growth factor-βsuperfamily.ALK4 and smad2/3 signaling have been reported to play a pivotal role in pathogenesis of cardiac hypertrophy and cardiac fibrosis.However,the role of ALK4–Smad2/3 pathway in the pathogenesis of bladder hypertrophy and bladder fibrosis caused by BOO remains unknown.This study is focused to figure out the role of ALK4 in the progress of bladder fibrosis caused by BOO.Materials and Methods1.WT mouse model of BOO was constructed,and the expression of ALK4 in bladder after BOO 4W and 6W was detected.2.Mice of ALK4+/-were constructed,and three groups were divided:BOO 4 weeks,BOO 6 weeks and sham.Histochemical staining,realtime PCR and western blot were used during experiment.All animal experiments were approved by the Animal Ethics Committee.3.BSMCs were primary cultured.The expression of fibrosis-related molecules and samd2/3signaling pathway was measured in BSMCs after overexpression of ALK4.Then we also detect the effect on cell proliferation and cell cycle in BSMCs after overexpression of ALK4.Results1.In WT mice,the expression of ALK4 was significantly increased after BOO4W and BOO6W compared with sham group.2.The expression of ALK4 in ALK4+/-mice was significantly decreased by western blot.3.It was showed that the expression of ECM in ALK4+/-mice after BOO6W was significantly decreased,and fibrosis of bladder was significantly inhibited.4.Compared with sham group,the signaling pathways of MAPK and P-smad2/3 in bladder of WT mice after BOO 6W was activated.Compared with WT group after BOO 6W,the signaling pathways of P-smad2/3 in ALK4+/-mice after BOO 6W depressed,which had statistical difference.5.The expression of fibrosis-related molecules and P-smad2/3 in BSMCs increased,after the overexpression of ALK4.The overexpression of ALK4 did not affect cell proliferation and cell cycle of BSMCs.ConclusionsOur results demonstrate that ALK4 sufficiency alleviates bladder fibrosis in a mouse model of BOO through the suppression of Smad2/3activity,and suggest that ALK4 may be a novel target in treating bladder remodeling induced by BOO.更多还原