膀胱输尿管反流基因Robo2在小鼠肾脏发育过程中的表达

Expression of Robo2 gene during the renal development in mice

目的观察Robo2在小鼠肾脏发育过程中的表达情况,探讨其在肾脏发育中的作用。方法应用实时定量RT-PCR技术对胚龄12.5、13.5、14.5、15.5、16.5、17.5、18.5d和生后日龄1d、1周、5周共10组小鼠肾脏组织中的Robo2 mRNA表达水平进行半定量分析。应用免疫荧光技术检测胚龄12.5、13.5、14.5、15.5、16.5、17.5d和成年小鼠肾脏Robo2蛋白表达部位。结果实时定量RT-PCR结果显示,Robo2蛋白表达水平在胚龄12.5、13.5、14.5d时最高,随后明显下降,在出生后维持低水平表达。免疫荧光染色结果显示,Robo2最初在胚肾的后肾间充质表达,而不在输尿管芽表达,随着胚龄增加及肾脏发育,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形体以及肾小囊体,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。在Robo2基因缺失后,肾单位发育异常,部分肾小管、集合管扩张。结论 Robo2通过调控后肾间充质与输尿管芽相互作用在小鼠肾脏发育过程中对肾单位发育起重要作用。

Objective To observe the expression of Robo2 gene,and explore its role during the renal development of mice.Methods Real-time quantitative RT-PCR was used to semi-quantitatively measure the expression level of Robo2 mRNA in the developing murine kidney at fetal age of 12.5,13.5,14.5,15.5,16.5 and 17.5 days,and also 1 day,1 week,5 weeks after birth.Immunofluorescence staining was used to examine the expression location of Robo2 protein at different stages of embryonic and postnatal kidney.Results Real-time quantitative RT-PCR analysis revealed that Robo2 was highly expressed in embryonic kidney at fetal age of 12.5,13.5 and 14.5 days,while the expression level declined quickly thereafter and maintained at very low level after birth.Immunofluorescence staining showed that the expression of Robo2 protein could be primarily detected in metanephric mesenchyme of the developing kidney,but not in the ureteric bud.With the development of embryonic kidney,Robo2 protein was expressed in cell membrane of metanephric mesenchyme,condensed cap mesenchyme surrounding the tip of the ureteric bud,comma-shaped body,S-shaped body and renal capsule,finally expressed in the podocytes.Besides,Robo2 protein was also weakly expressed in part of the proximal tubular epithelial cells.Absence of Robo2 gene resulted in abnormal development of nephron,and broadening of some renal tubules and collecting ducts.Conclusion Robo2 plays an important role in the nephron development in mice by regulating the interaction of metanephric mesenchyme and ureteric bud.