【作者】 季佳瑶；

【导师】 陈香美；

【作者基本信息】 南开大学 ， 内科学， 2012， 博士

【摘要】 背景：肾脏的发育是一个复杂过程，需要输尿管芽（UB）和后肾间充质（MM）两种细胞群精确的相互作用来完成的。某一环节出现错误会导致肾脏发育不良、膀胱输尿管反流等严重的肾脏和尿道发育异常。Robo2通过其配体Slit2在肾脏发育过程中发挥重要作用。Robo2/Slit2基因缺失可以导致调节肾脏发育的主要因素Gdnf的表达异常，从而使肾脏和输尿管发育异常，引起多个肾脏、多个输尿管和输尿管扩张改变。但是目前对于Robo2调控肾脏和输尿管发育的研究仍比较缺少，主要是对肾脏发育初期Robo2/Slit2基因敲除导致输尿管芽萌出异常的研究，缺乏Robo2对肾脏发育后期的影响以及Robo2基因缺失导致肾脏和输尿管发育异常的机制研究。目的：通过观察和分析Robo2在肾脏发育过程中的表达变化、体外过表达Robo2对肾脏发育的影响、Robo2基因敲除对小鼠肾脏和输尿管发育的影响等现象，揭示Robo2在肾脏和输尿管发育过程中的作用及其机制。方法：(1)显微分离胚龄E12.5d、E13.5d、E14.5d、E15.5d、E16.5d、E17.5d、E18.5d胎鼠肾脏和生后日龄P1d、P1w、P4w小鼠肾脏，石蜡切片进行PAS染色系统观察小鼠肾脏发育各阶段的形态学变化特点；应用实时定量RT-PCR技术对肾脏组织中Robo2mRNA水平表达进行半相对定量分析；应用免疫荧光技术检测小鼠肾脏发育过程中Robo2蛋白水平的表达部位。(2)显微分离胚龄E12.5d小鼠胚胎肾组织，应用显微注射及电转染方法，进行表达载体显微注射、转染。实验分为三组，正常对照组、空载体组、转染GFP-Robo2组。将体外转染的胚肾置于Transwell膜上，以气液交界面的方式培养3天后，进行肾小球和输尿管芽标志物（WT1、E-cadherin）和细胞增殖和凋亡（PH3、TUNEL）免疫荧光染色，观察输尿管芽分支、肾小球数目及后肾间充质细胞增殖、凋亡变化情况。(3)取野生型胚龄E13.5d和E14.5d小鼠胚胎输尿管膀胱组织，应用免疫荧光染色和实时定量RT-PCR的方法检测Robo2在输尿管、膀胱和中肾管（WD）的表达情况；应用组织PAS染色和免疫荧光染色的方法，观察Robo2基因敲除小鼠胚胎肾脏和输尿管发育异常的情况。(4)取野生型胚龄E12.5d小鼠胚胎肾脏、输尿管和膀胱组织，分别进行分离培养，分为正常培养组、去肾组、去膀胱组和去肾去膀胱组，共4组，每组10个，于Transwell气液交界面上培养6天后，进行E-cadherin和α-SMA免疫荧光染色，观察各组输尿管长度的变化情况；并且应用显微注射和电转染的方法，对野生型E12.5d胚胎的膀胱三角区进行GFP-RNAiRobo2质粒转染，体外培养6天后进行E-cadherin和α-SMA免疫荧光染色，观察输尿管长度的变化情况。(5)取同窝E13.5d Robo2基因敲除野生型和纯合子胚胎，冰冻包埋连续切片，TUNEL染色观察CND（common nephric duct)凋亡情况。结果：(1)肾脏发育过程中不同时间点的形态特征：①小鼠肾脏发育各阶段PAS染色结果显示，UB于E11.5d开始分支，E15.5d可见到初步形成的肾盂、肾盏；E16.5d有发育成熟的集合管、肾盂、肾盏；②M M在整个后肾发育阶段围绕UB呈“帽”状分布；E13.5d UB周围的MM聚集成团，可见逗号形体、S形体；E14.5d出现肾小囊腔；E15.5d出现发育成熟的肾小球；E16.5d可见发育成熟的肾单位（肾小球、近端小管、远端小管等）；出生后1.5d至1w肾小球数目继续增多；出生后2w肾脏发育成熟。(2)肾脏发育过程中Robo2的表达变化：①实时定量RT-PCR结果显示，Robo2在发育的E12.5d～E14.5d表达水平最高，随后水平明显下降，在出生后维持在低水平表达。②免疫荧光染色结果显示,Robo2最初表达在胚肾的后肾间充质，而不在输尿管芽表达。随着胚胎肾脏发育，Robo2表达于后肾间充质细胞膜、围绕输尿管芽的浓缩帽状间充质、逗号形体和“S”形体以及毛细血管袢，最终表达于肾小球足细胞。另外，部分早期肾小管上皮细胞也有微弱Robo2表达。(3)过表达Robo2对肾脏发育的影响：①输尿管芽标志物E-cadherin和肾小球标志物WT1免疫荧光染色显示，正常对照组、转染空载体组肾脏输尿管芽分枝和肾小球数目无明显异常；而转染GFP-Robo2组的输尿管芽分枝减少，肾小球数目减少（p <0.05）。②与对照组相比，过表达Robo2组出现后肾间充质减少，输尿管芽顶端缺少聚集的帽状间充质。③与正常对照组和转染空载体组比较，过表达Robo2组肾脏后肾间充质细胞凋亡情况未见明显异常（p＞0.05），并且E-cadherin染色显示输尿管芽上皮细胞形态及排列无异常。(4) Robo2基因敲除对小鼠肾脏和输尿管发育的影响：①免疫荧光染色显示Robo2表达在膀胱三角区的疏松结缔组织、CND和输尿管；实时定量RT-PCR显示，Robo2在输尿管、膀胱、脑、肺组织中均有不同程度的表达，在膀胱和输尿管的含量多于肺组织但少于脑组织的含量。②与同窝野生型小鼠比较，Robo2基因敲除小鼠表现为肾脏明显变小，有多个肾脏、多个输尿管，输尿管缩短，输尿管积水和输尿管与膀胱连接异常。(5)输尿管发育的影响因素及Robo2影响输尿管发育的可能机制：①野生型E12.5d小鼠胚胎肾脏、输尿管和膀胱组织体外培养发现，与正常培养组相比，去肾组输尿管长度延长无明显差异（p＞0.05），去膀胱组和去肾去膀胱组输尿管长度延长明显变短（p<0.05）。②在膀胱三角区转染GFP-RNAiRobo2质粒后，与正常培养组相比，输尿管长度延长无明显异常（p＞0.05）。③R obo2基因敲除纯合子小鼠膀胱三角区的CND末端可见到凋亡，但异常部位的CND无凋亡。结论：Robo2在肾脏和输尿管中发育过程中均有表达，对肾脏和输尿管的发育有重要作用；Robo2体外过表达可以导致肾脏输尿管芽减少，后肾间充质聚集减少等异常；体内Robo2基因敲除可以引起输尿管的发育异常，主要是由于异常部位的输尿管未能与膀胱正确连接所致。更多还原

【Abstract】 Background：The development of kidney is a complex process,which requiresthe precise interaction of the ureteric bud (UB) and metanephricmesenchymal (MM). A minim error in this process will lead to renaldysplasia, vesicoureteral reflux, severe kidney and urinary tractdevelopment abnormalities. Roundabout2(Robo2) and its ligand Slitsplay an vital role in the process of kidney development. Slit2or Robo2mouse mutants exhibit multilobular kidneys, multiple ureters, anddilatation of the ureter, renal pelvis, and collecting duct system, whichlead to vesico ureteral reflux. However, there were very little studyabout Robo2regulate kidney and ureter development,mainly about the UB outgrown from WD,and was lack of the mechanism of the Robo2gene deletion lead to kidney and ureter development defect.Objective:To observe expression characteristics of Robo2and further exploreits role during murine kidney development.To understand the effect ofRobo2overexpression on kidney development in vitro.Methods:(1) PAS staining for embryonic mouse E12.5d～18.5d and afterbirth P1d、1w、5w, to observed the characteristics of the morphologicalchanges of the kidneys of mice in different developmental stages.qRealTime RT-PCR technique was used to measure the expression level ofRobo2mRNA in the developing murine kidney from12.5-18.5d postcoitum (d.p.c.) and postnatal day1d、1w、5w; Immunofluorescencestaining were used to examine the expression locations of Robo2proteinat different stages of embryonic and adult mice kidney. (2) Embryonic kidney was extracted from timed-pregnant femalemice at embryonic day12.5(E12.5d).Using microinjection and electrictransfection method to transfer the expression vector into E12.5dmouse embryonic kidney. The experiment was divided into three groups:normal control group, empty vector group and GFP-Robo2group. Afterthe electroporation, kidneys were immediately placed in Transwells andcultured at the air-liquid interface for3days, immunofluorescencestaining with WT1、E-cadherin、PH3and TUNEL. To observe thebranching of the ureteric bud, glomerular number and and theproliferation and apoptosis of mesenchymal cell.(3) Embryonic ureter and bladder was extracted fromtimed-pregnant female mice at E13.5d and E14.5d, usingimmunofluoresence and quantitative real-time PCR method to detect theexpression of Robo2in the ureter, bladder, and WD. Using PAS stainingand immunofluorescence staining method to observe the kidney and ureter development of the Robo2gene knockout mouse.(4) Embryonic mouse embryonic kidney, ureter and bladder wasextracted from timed-pregnant female mice at E12.5d, then dividedthem into four groups,normal culture group, removing bladdergroup,removing kidney group and removing kidney and bladdergroup(n=10). After cultured for6days in vitro, immunofluorescencestaining with E-cadherin andα-SMA, observe the ureter growth in eachgroup. Using microinjection and electric transfection method totransfer the GFP-RNAiRobo2plasmid into E12.5d mouse embryonicBladder trigone, After cultured for6days in vitro, immunofluorescencestaining with E-cadherin andα-SMA, observe the ureter growth.Microdissect E12.5d Robo2gene knockout mouse embryonic kidney,ureter and bladder, cultured for1day in vitro, immunofluorescencestaining with E-cadherin, observe the development of the ureter andCND. (5) Immunofluorescence staining for the frozen tissue sections ofE13.5d and E14.5d Robo2gene knockout mouse embryonic, observe theapoptosis of the CND.Results:(1) Morphological characteristics of the different time points in theprocess of developing kidney:①UB began to branchin E11.5d.The renalpelvis, calyceal initial formation in E15.5d. In the whole of the kidneydevelopment, MM surrounded the UB form the cap mesenchyme. MMsurrounding the UB gathered in E13.5d，The comma, s-shaped body andthe renal capsule was seen in E15.5d. The mature nephron was seen inE16.5d. The mouse kidney mature after birth2w.(2) Expression of Robo2during the kidney development:①Compared to5w group, Robo2was highly expressed at E12.5d,E13.5d and E14.5d, but which were quickly reduced from E15.5andkept at very low level after Newborn. The expression of Robo2mRNA was reduced with ages.③Immunofluorescence staining showed thatRobo2protein expression could be observed in metanephricmesenchyme,cap mesenchyme,comma, s-shaped body and ultimatelyexpressed in the podocytes, which was also weakly expressed in part ofthe proximal tubular epithelial cells. Robo2deficiency casued nephrondevelopment defect, some tubules and collect ducts were dilated.(3) The impact of Robo2overexpression in the kidneydevelopment:①Results show reduced UB branching and decreasedglomerular number after in vitro Robo2overexpression in the embryonickidneys（p<0.05）.②We found fewer metanephric mesenchymal (MM)cells surrounding the UB but no abnormal morphology in the branchingepithelial UB.③Meanwhile, no significant change in MM proliferationor apoptosis was observed（p＞0.05）.(4) The impact of Robo2knockout in the kidney and ureterdevelopment:①Immunofluorescence staining shown that Robo2 expressed in the trigone of the bladder,CND. The result of quantitativereal-time PCR shown the different levels of Robo2expressed in theureter, bladder, brain and lung tissue, the most expression is brain, thelest expression is lung,followed by bladder and ureter.②Comparedwith the widetype, Robo2gene knockouts mouse developedsupernumerary ureteric buds,duplex kidneys, hydroureter and renaldysplasia.(5) Impact factors and possible mechanism of Robo2in the ureterdevelopment:①There was no significant difference between the normalculture group and the removing kidney group（p＞0.05）. Comparedwith the normal culture group, the extended length of ureter wassignificantly shorter in removing bladder group and removing kidneyand bladder group（p<0.05）.②After transfection the GFP-RNAiRobo2plasmid, compared with the normal culture group, there was nosignificant difference between the normal culture group and the GFP-RNAiRobo2group（p＞0.05）.⑤There were apoptosis of CND inWT and Robo2gene knockout mouse bladder trigone.Conclusions:Robo2expresses in the kidney,trigone,CND and ureter during themouse embryonic development,and played an important role duringkidney and ureter development. Overexpression of Robo2causes defectsin the recruitment of metanephric mesenchymal cells and ureteric budbranching morphogenesis.Robo2gene deletion in mice resultsupernumerary ureters,duplex kidneys, hydroureter ureteral lengthshortened, and the renal collecting system and hydroureterexpansion,that’s because the ureter failed linking to the bladder.更多还原