【作者】 侯双兴；

【导师】 邓艳春；

【作者基本信息】 第四军医大学 ， 神经病学， 2013， 博士

【摘要】 NDRG（N-Myc downstream-regulated gene）是近年来发现的一个新的基因家族，该基因家族由NDRG1、NDRG2、NDRG3和NDRG4组成；此基因家族成员间有57-65%氨基酸同源性，经生物信息学分析表明，NDRG2编码的蛋白分子内含有一个可以携带酰基的酰基携带蛋白（ACP）结构域和多种核转录因子的结合位点，这是NDRG家族共同特有的结构域。此特殊结构或许决定了它们有着共同的表达及部分一致的功能，该基因家族参与了细胞的生长，但具体机制仍不清楚。既往文献报道NDRG家族在成年大鼠、小鼠及成人脑中都有不同程度表达；NDRG2在抑郁症、阿尔海默氏病、应激及缺氧时，表达异常，说明NDRG2与脑部疾病具有高度相关性；目前NDRG1、NDRG2都被称为脑分化相关基因；NDRG4在神经前体细胞也有较高表达，受视黄酸（RA）等因子调控；NDRG3在脑发育中表达无明确报道。因此，推测其家族在脑组织中的广泛表达可能与神经系统发育有关。目前研究已经证实，此基因家族与肿瘤细胞的异常增殖、分化密切相关，NDRG2已被明确列为抑癌候选基因。神经前体细胞（NPC）的正常增殖、分化、迁移对于脑的发育至关重要，但其机制还不清楚。我们在前期验证中发现，NDRG2在小鼠神经前体细胞发生区高表达，提示其可能是调控NPC增殖、分化和迁移的重要基因。但NDRG2在人脑发育中是否有相同的表达特点，还尚不清楚；此外，NDRG家族的4成员具有高度氨基酸同源性且在进化中高度保守性，为了探索它们在脑发育中的作用，需首先明确其在脑内的分布及细胞定位。目的通过研究NDRG2在在人胎脑及NDRG家族4个成员在小鼠生后各个脑区的表达特点及细胞定位，为探索NDRG家族在脑发育中的作用奠定基础。方法应用免疫组化、免疫荧光、RT-PCR、Western Blot、细胞培养及电镜等实验技术，研究NDRG基因家族在不同时间点人胎脑和小鼠生后各脑区的表达变化及细胞定位。（1）RT-PCR法检测NDRG2mRNA在16-28周人胎脑不同脑区中的表达；（2）Western blot技术检测NDRG2在28周各脑区的表达；（3）免疫组织化学法检测NDRG2在人胎脑各脑区中的表达；（4）NDRG2与GFAP或NeuN的荧光双标法研究其在胎脑中的细胞定位；（5）Western Blot、免疫荧光技术检测NDRG家族4成员在出生后小鼠各脑区的表达及细胞定位；并在培养神经前体细胞及其分化细胞中检测NDRG2表达情况；（6）电镜技术检测NDRG2的亚细胞定位。结果（1）通过RT-PCR、Western Blot分别在RNA和蛋白水平检测NDRG2在不同时间点的人胎脑脑区表达水平的变化。随着胎龄成熟，NDRG2表达升高，尤其在脑室下带、脑室管膜区域表达最为突出；（2）免疫组化从细胞水平进一步验证了NDRG2主要在人胎脑脑室下带、脑室管膜表达；免疫荧光双标还明确了NDRG2可表达于星形胶质细胞和相对成熟的神经元中，主要定位于细胞质中，极少数星型胶质细胞细胞核也有表达；（3）用Western Blot明确了NDRG1-4成员在出生后小鼠不同时间点、不同脑区表达特征：21天鼠脑海马区、脑室下带等神经发生区NDRG2有特异表达；在120天小鼠，表达明显减弱；NDRG1、3、4在脑室下带、海马亦有表达，但无明确规律；（4）免疫荧光显示NDRG1、3、4在出生后神经前体细胞发生区无表达，NDRG2在小鼠脑海马区以星形胶质细胞胞浆特异性表达为主。NDRG2在培养分化的星形胶质细胞胞浆表达；（5）在NDRG2在亚细胞定位研究中，发现NDRG2表达在神经前体细胞及分化的星形胶质细胞线粒体嵴和包膜上。结论（1）NDRG2在人胎脑脑功能区表达广泛；随着胎龄成熟，表达增强；在神经前体细胞发生区最强，说明NDRG2与细胞增殖、分化及成熟密切相关。（2）NDRG家族在出生后鼠脑区表达有明确区别，NDRG2主要表达于神经发生区，NDRG1、3、4在神经前体细胞发生区参与了分化，但与神经前体细胞发生无特异性关系。（3）NDRG2可能通过影响神经前体细胞的线粒体功能调控其增殖、分化过程。更多还原

【Abstract】 NDRG（N-Myc downstream-regulated gene） family is a set of new genes foundrecent years which consists of4family members: NDRG1~4. These members share57-65%amino acid homology. Through bioinformatics analysis, the NDRG2codingprotein has an ACP domain that can carry the acyl and a variety of nuclear transcriptionfactor binding sites. This is a common character of the NDRG family structure domain.This special structure may result in a common expression and some consistent functionamong the family members. The gene family is involved in the growth of cells.However,the exact mechanism is not very clear yet.Previous ariticals reported NDRG family has a different degree of expression inadult rat, mice and human brains. The expression of NDRG2in depression, stress andhypoxia are abnormal, indicating that NDRG2is highly correlated with brain diseases.Currently NDRG1,2are considered as brain differentiation related genes. NDRG4alsohas a higher expression in neural precursor cells, and is regulated by retinoic acid(RA) andother factors. There is no clear evidence of the expression of NDRG3during brain development. So, presumably, the wide expression of the family in the brain may beassociated with nervous system development. The present study has confirmed that thisgene family is closely related to the abnormal proliferation and differentiation of tumorcells. NDRG2is confirmed as a candidate tumor suppressor gene.The normal proliferation,differentiation and migration of neural progenitor cells iscritical for the development of embryonic brain. However, the mechanism is still not clear.Our previous study have found that NDRG2, a candidate tumor suppressor which is veryimportant for the proliferation and differentiation of cancer cells, is highly expressed inNPCs in mouse’s fetal brain tissue, but it’s not clear whether NDRG2has similarexpression pattern during human brain development. Besides, the four family members ofNDRG has highly amino acid homology and highly conservative. To explore theirfunction during brain development, it is necessary to co nfirm the distribution and cellularlocalization of them.ObjectsTo study the expression characteristics and the cell localization of NDRG2in humanfetal brain and the four NDRGs in mice after birth, then to lay the foundation for exploringthe NDRG family role in brain development.MethodsWe used experimental techniques such as immunohistochemical， immunefluorescence，RT-PCR，Western Blot，cell culture and electron microscopy to study theexpression and the cell localization of NDRG family in human fetal brain and mice invarious regions.(1)Using RT-PCR method to detect NDRG2mRNA expression in16to28weeks in different brain regions of fetal brain.(2) Using Western blot to detectNDRG2expression in28weeks in various regions.(3) Using Immune histochemicalmethod to detect NDRG2protain expression in human fetal brain.(4) Using NDRG2withGFAP or NeuN fluorescent double labeling to study its positioning in fetal brain cells.(5)Using Western Blot and immunofluorescence technology to detect the expression of fourNDRGs in the postnatal mouse brain and cellular localization,and to detect NDRG2 expression in neural precursor cells and their differentiated cells in vitro.(6) Using electronmicroscopy technology to detect the subcellular localization of NDRG2.Results(1)We detected the expression levels of NDRG2in different time of human fetalbrain by RT–PCR and Western Blot. We found that the NDRG2expression increased asgestational age matured,especially in the ventricular zone and subventricular zone(2)Using immunohistochemical method, we further confirmed that NDRG2mainlyexpressed in the ventricular zone and subventricular zone.Using double-labellingimmunofluorescence technology,We proved that NDRG2was expressed in astrocytes andrelatively mature neurons, mainly located in cytoplasm, some in the nucleus ofastrocytes.(3)Using Western Blot, we found the expression pattern of NDRG1-4membersat different time points in different brain regions of postnatal mice: NDRG2was expressedin hippocampus and subventricular zone in21day mice，the expression in120day micewas decreased significantly;NDRG1,3,4were also expressed in the subventicular zoneand hippocampus with no regular pattern.(4)Using immunofluorescence, we showed noexpression of NDRG1,3,4in neural precursor cells. NDRG2was mainly expressed in theastrocytes cytoplasm in mouse hippocampus.(5) In subcellular level, we found thatNDRG2was expressed in mitochondrial crest of astrocytes of neural precursor cells.Conclusion(1)NDRG2was widely expressed in human fetal brain functional area. Along withthe gestational age mature, the expression of NDRG2enhanced.The strongest expressionarea was in neural precursor cells,indicating that NDRG2was closely related to cellproliferation, differentiation and mature.(2) The expression patterns of NDRG familymembers were distinguished. NDRG2was mainly expressed in the neurogenesis areawhile NDRG1,3,4were involved in the differentiation of neural precursor cells.(3)NDRG2might regulate the proliferation and differentiation of precursor cells by affectingthe mitochondrial function.更多还原