NDRG2在大鼠睾丸发育中的表达及其与生精细胞凋亡的关系

发布时间：2018-01-03 13:09

本文关键词：NDRG2在大鼠睾丸发育中的表达及其与生精细胞凋亡的关系　出处：《第四军医大学》2009年博士论文　论文类型：学位论文

【摘要】： 目的 NDRG2与NDRG1,NDRG3和NDRG4共同组成NDRG家族,该家族成员可能与细胞的增殖和分化相关,但其具体的生物学功能有待于进一步研究。在前期的研究中我们通过免疫组织化学染色观察到NDRG2在成年人和小鼠睾丸内均表达定位于睾丸间质细胞,而在生精细胞内未见明显的阳性着色,推测其可能参与睾丸间质细胞睾酮的分泌与调节,进而调控精子的发生,因此本实验的目的在于探讨NDRG2在睾丸发育与精子发生过程中的作用及其机制。0 方法 1.利用RT-PCR、蛋白质免疫印迹杂交及免疫组织化学等方法对比研究出生后大鼠睾丸发育不同阶段(1、5、15、21、35和75 d)中NDRG2的表达与定位;并分离21、35和75 d大鼠睾丸间质细胞和生精细胞,利用RT-PCR和蛋白质免疫印迹杂交分析和验证NDRG2在大鼠睾丸不同发育阶段中表达的差异性。 2.建立成年大鼠单剂量MAA(650 mg/kg)腹腔注射诱导精母细胞凋亡模型,采用TUNEL、RT-PCR、蛋白质免疫印迹杂交、免疫组织化学、间接免疫荧光和激光显微切割技术分析NDRG2在生精细胞凋亡过程中表达的变化,探讨NDRG2与生精细胞凋亡之间的关系。 3.青春期大鼠睾丸内生精细胞自发性凋亡达到顶峰,建立青春期(21 d)大鼠隐睾模型,通过TUNEL染色验证该模型、运用免疫组织化学和间接免疫荧光等方法分析NDRG2在隐睾和对照侧睾丸表达的差异,并且分离术后不同时间点的间质细胞和生精细胞,运用免疫印迹杂交检测NDRG2和p53的表达变化。结果 1.RT-PCR和Western blot结果均显示NDRG2在出生后1 d大鼠睾丸内即有表达,随后逐渐升高,至青春期达到顶峰(21和35 d),在成年期其表达反而下降。免疫组织化学结果显示NDRG2持续表达于睾丸间质细胞,但在睾丸发育不同阶段,其在生精小管内的表达却存在明显的差异。在新生期(1和5 d)主要表达于原始生殖细胞,而不表达于支持细胞;在15和21 d大鼠睾丸,NDRG2在生精小管内主要表达于精母细胞,而精原细胞和支持细胞未见阳性着色;在35 d大鼠睾丸,NDRG2在生精小管内主要表达于精母细胞和圆形精子细胞;在成年(75 d)大鼠,生精小管内几乎未见NDRG2阳性着色。分离纯化21,35和75 d大鼠睾丸生精细胞,采用RT-PCR和Western blot进一步验证了NDRG2在青春期大鼠生精细胞内高表达,而在成年大鼠生精细胞内几乎不表达。分离纯化21,35和75 d大鼠睾丸间质细胞,结果显示NDRG2在这三个时间点间质细胞内均有较强的表达,但相互间无明显差异。 2.MAA腹腔注射3和6 h与对照组相比未见显著性差异,但MAA诱导12 h后,成年大鼠睾丸内出现大量初级精母细胞的凋亡,主要集中在生精周期的第X-XIII期,通过分离生精细胞和激光显微切割技术均检测到NDRG2在MAA诱导12 h后在生精细胞的表达上调,免疫组织化学和间接免疫荧光结果均显示NDRG2在自发性和MAA诱导凋亡的生精细胞中均高表达。 3.建立21 d大鼠单侧隐睾模型,术后7 d和14 d,隐睾侧睾丸重量明显减轻,凋亡生精细胞数明显升高,免疫组织化学与间接免疫荧光结果显示NDRG2在自发性和热刺激诱导凋亡的生精细胞内表达均明显上调,但在一些正常的精母细胞和圆形精子内仍存在一定量的表达,并且NDRG2在隐睾侧间质细胞的表达强度明显降低。分离纯化术后不同时间点隐睾侧和对照侧睾丸生精细胞,发现NDRG2和p53在术后7 d隐睾侧生精细胞的表达量明显上调,而在隐睾后14 d未见明显差异,可能与生精细胞的凋亡与脱落有关;分离纯化术后不同时间点隐睾侧和对照侧睾丸间质细胞,进一步证实了NDRG2在术后7和14 d隐睾侧间质细胞的表达量减少。结论 1. NDRG2在大鼠睾丸发育不同阶段持续表达于间质细胞,可能参与睾丸早期发育和睾酮的分泌与调节。 2. NDRG2在大鼠睾丸发育过程中在生精细胞的表达存在较大差异,在青春期前在生精细胞有较高水平的表达,但在成年期表达量很低,提示NDRG2可能参与睾丸发育早期生精细胞增殖与分化的调节。 3. NDRG2在MAA和热刺激诱导的生精细胞凋亡中表达均明显上调,并且在青春期和成年期大鼠睾丸自发性生精细胞凋亡中均存在较强的表达,提示NDRG2可能参与了生精细胞凋亡的调控。
[Abstract]:objective
NDRG2 and NDRG1, NDRG3 and NDRG4 composed of the NDRG family, the family members may be related to cell proliferation and differentiation, but its biological function remains to be further studied. In previous study we observed by immunohistochemical staining to NDRG2 in testis of adults and are expressed in mouse Leydig cells however, in spermatogenic cells no obvious positive staining, that it may be involved in the secretion and regulation of Leydig cell testosterone, thereby regulating spermatogenesis, therefore the purpose of this experiment is to explore the role of NDRG2 in testicular development and Spermatogenesis Function and its mechanism in the process of.0
Method
1. by RT-PCR, Western blot and immunohistochemistry method in comparative study of postnatal rat testis of different developmental stages (1,5,15,21,35 and D 75) expression and localization of NDRG2; 21,35 and D 75 in rat Leydig cells and spermatogenic cells were isolated and differentially expressed in the testes of rats at different developmental stages using RT-PCR and Western blot analysis and verification of NDRG2.
2. of adult rats were single dose of MAA (650 mg/kg) induced by intraperitoneal injection of spermatocyte apoptosis model, using TUNEL, RT-PCR, Western blot, immunohistochemistry, indirect immunofluorescence and laser microdissection analysis of NDRG2 changes in spermatogenic cell apoptosis, to investigate the relationship between NDRG2 and spermatogenesis the apoptosis of the cells.
Reached a peak of 3. in the testis of adolescent rats spontaneous apoptosis of spermatogenic cells, the establishment of puberty (21 d) rat model of cryptorchidism, the model is validated through TUNEL staining, immunohistochemistry and indirect immunofluorescence analysis NDRG2 in cryptorchidism and control side testis expressed the difference at different time points after surgery in stromal cells spermatogenic cells and the expression of separation and, by Western blot detection of NDRG2 and p53.
Result
1.RT-PCR and Western blot showed that NDRG2 in 1 d after birth in the rat testis was expressed, then increased gradually until puberty reached its peak (21 and 35 d), the expression decreased in adulthood. Immunohistochemistry showed that NDRG2 was constitutively expressed in Leydig cells in the testis, but in different developmental stages however, there are obvious differences in its expression in the seminiferous tubule. In the neonatal period (1 and 5 d) is mainly expressed in primordial germ cells, but not expressed in Sertoli cells in the testis; 15 and 21 d rats, NDRG2 in seminiferous tubules mainly expressed in spermatocytes and spermatogonia. No positive staining and Sertoli cells in the testis; 35 d rats, NDRG2 in seminiferous tubules mainly expressed in spermatocytes and round spermatids; (75 d) in the adult rat, the seminiferous tubule almost no positive staining of NDRG2 21,35 and 75 D. The separation and purification of rat spermatogenic cells,. Using RT-PCR and Western blot further verified the high expression of NDRG2 in adolescent rats spermatogenic cells in spermatogenic cells, almost no expression in the adult rat. The separation and purification of 21,35 and 75 D of rat Leydig cells, results showed that the expression of NDRG2 in the three time points in stromal cells have a strong however, no significant difference between.
Intraperitoneal injection of 2.MAA 3 and 6 h compared with the control group had no significant difference, but the MAA 12 h after induction of adult rat testis in massive apoptosis of primary spermatocytes, mainly concentrated in the spermatogenic cycle phase X-XIII, the separation of spermatogenic cells and laser microdissection were detected in NDRG2 MAA 12 h after induction in upregulation of spermatogenic cells expression, immunohistochemistry and immunofluorescence results showed that NDRG2 in spontaneous and MAA induced apoptosis in spermatogenic cells were highly expressed.
3. 21 d rats with unilateral cryptorchidism model, after 7 d and 14 d, cryptorchid testicular weight significantly reduced, apoptosis of spermatogenic cells increased obviously, immunohistochemistry and indirect immunofluorescence showed that NDRG2 in the spontaneous and thermal stimulation induced apoptosis of spermatogenic cells expression were up-regulated expression, but there are still a certain amount in some normal spermatocytes and round spermatids within, and decreased NDRG2 in cryptorchid stromal cells. The expression intensity of the separation and purification of different time points after surgery of cryptorchidism and the control side of spermatogenic cells, found that the expression of NDRG2 and p53 in 7 d after operation in experimental cryptorchidism spermatogenic cells significantly the rise, in cryptorchidism after 14 d showed no significant difference, and apoptosis of spermatogenic cells and shedding; separation and purification of different time points after surgery of cryptorchidism and the control side of Leydig cells, further confirmed that NDRG2 after 7 and 14 d of cryptorchidism The expression of lateral stromal cells was reduced.
conclusion
1. NDRG2 is expressed in stromal cells at different stages of rat testicular development, which may be involved in the early development of testis and the secretion and regulation of testosterone.
The expression of 2. NDRG2 in spermatogenic cells was quite different in the process of testicular development in rats. It had a high level of expression in spermatogenic cells before puberty, but the expression level was low in adulthood, suggesting that NDRG2 may participate in the regulation of proliferation and differentiation of spermatogenic cells at early stage of testicular development.
3. NDRG2 expression was significantly up-regulated in MAA and heat induced apoptosis of spermatogenic cells, and there was a strong expression in the apoptosis of testicular spontaneous spermatogenic cells in pubertal and adult rats, suggesting that NDRG2 may be involved in the regulation of spermatogenic cell apoptosis.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【共引文献】