应用PB转座子插入的突变小鼠研究Robo2与先天性肾脏及尿路畸形的关系

Congenital anomalies of the kidney and urinary tract anatomy (CAKUT) are the most common cause of renal failure in childhood and represent approximately 30% of all prenatally diagnosed malformations. ROBO2 belongs to the ROBO family，it is a transmembrane receptor involved in the urinary and many other systems development process and is one of the candidate genes of CAKUT. Previous Robo2 homozygous knockout mice the gene expression were completely blocked and they died after birth due to severe abnormalities of the urinary tract, but making the long-term surviving mosaic ones takes a long time and is complicated. Therefore,our group choose Robo2 PB/PB and Robo2 PB/+ mice made by PB transposon. Efficient PB transposition in germline allows rapid generation of mutant animals by simple crossing. We plan to observe the relationship between the different degrees of the Robo2 expression and the variety of CAKUT clinical phenotype; identify the position of Robo2 expression in the mouse urinary system; the relationship between the changed Robo2 expression and the clinical phenotype of CAKUT; analyze the decreased degree of Robo2 expression influence the morphology and function of smooth muscle through the in vitro functional experiments;study on the role of ROBO2 in the signaling pathways of ureteral smooth muscle cytoskeleton. So as to clarify Robo2 involved in the pathophysiological mechanisms of the incidence of CAKUT and provide a theoretical basis for the early clinical molecular diagnosis of related diseases.

先天性肾脏及尿路畸形（CAKUT）是造成儿童终末期肾功能衰竭(ESRD)的主要原因，在产前检查提示畸形的胎儿中约占30% 。ROBO2编码发育相关的跨膜蛋白受体,参与泌尿系统等多个系统发育，是CAKUT的候选致病基因之一。纯合动物模型完全阻断基因表达，多由于严重泌尿系统畸形生后即死亡；而可存活的同源性嵌合体模型构建复杂，周期长。因此本课题拟用可高效、大量稳定传代的PB转座子插入技术而随机获得的Robo2第一内含子插入小鼠模型为研究对象，观察Robo2不同缺失程度造成的CAKUT的临床表型差异；明确Robo2在小鼠泌尿系统中的定位，观察其表达改变与临床表型间的关系；通过体外实验研究Robo2缺失对输尿管平滑肌形态、结构和功能的影响,及Robo2缺失导致输尿管平滑肌细胞骨架改变的可能信号通路。进而阐明Robo2参与CAKUT发病的病理生理机制，为临床开展相关疾病的早期分子诊断提供依据。

背景：在膀胱输尿管反流（VUR）患者中已有许多Robo2基因杂合点突变的报道。纯合Robo2动物模型因严重的泌尿系统畸形多于生后48小时内死亡；而可存活的杂合小鼠研究较少，仅提示15%出现单侧VUR；嵌合的小鼠可能因基因表达量不稳定出现程度不一的CAKUT。PB是一种DNA转座子，其插入通常会减少但不消除基因表达，便于特定基因功能的识别。Robo2插入突变小鼠能稳定存活且易传代，可用于研究VUR/CAKUT的发生情况，探讨Robo2表达量与发生和严重程度之间的关系。.研究内容： 运用Vevo 770超声显象技术评估新生小鼠泌尿系统的解剖表现。异常的小鼠随访至成年，记录泌尿系统解剖异常的类型及程度，行膀胱输尿管反流（VUR）检测；肾盂积水的小鼠进行泌尿道梗阻的检测。泌尿系统解剖正常的小鼠也均进行VUR检测。光镜和透射电镜分别用于观察肾脏及输尿管的组织和超微结构变化。构建Robo2 PB/PB；Hoxb7/myr-Venus小鼠观察输尿管膀胱连接处形态。.结果: PB转座子插入了Robo2的第一内含子，导致了PB纯和小鼠Robo2表达降低约50%。共34.06%（78/229）的PB纯和小鼠存在CAKUT，其中主要表型为单独VUR（27.07%），发生率均明显高于野生型小鼠6.84%和PB杂合小鼠7.87%。还存在肾发育不良/不全，重复畸形或多余输尿管，肾盂输尿管连接处狭窄（UPJO）和输尿管膀胱连接处狭窄（UVJO）等。超声随访至成年均无自愈倾向。PB杂合小鼠未发现严重的肾脏和尿路异常。成年单独反流PB纯和小鼠肾脏病理提示肾小球肥大，没有疤痕出现的迹象；UVJ插入位置与野生型相似，膀胱水平输尿管的大体结构层次正常，但平滑肌纤维结构紊乱。透射电镜提示输尿管平滑肌细胞数量下降，肌层可见大的细胞间水肿区域和细胞内空泡变。VUR合并肾发育不良等其他尿路畸形的PB纯和小鼠膀胱水平的输尿管病理改变更为明显。.结论：PB纯合小鼠VUR为主的多种泌尿系统异常发生率明显升高，这提示我们在此类患者中行Robo2基因检测可能会发现更多的有害变异。推测临床由于基因突变导致的VUR/CAKUT有可能很难自愈；单纯VUR的PB纯和小鼠随访至成年预后尚可，为临床制定合理策略提供理论依据。Robo2突变小鼠VUR/CAKUT发生和严重程度可能和Robo2表达量相关。