本文關(guān)鍵詞：NDRG2在大鼠睪丸發(fā)育中的表達(dá)及其與生精細(xì)胞凋亡的關(guān)系　出處：《第四軍醫(yī)大學(xué)》2009年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： NDRG2 間質(zhì)細(xì)胞 精子發(fā)生 凋亡 MAA 隱睪 睪丸 大鼠

【摘要】： 目的 NDRG2與NDRG1,NDRG3和NDRG4共同組成NDRG家族,該家族成員可能與細(xì)胞的增殖和分化相關(guān),但其具體的生物學(xué)功能有待于進(jìn)一步研究。在前期的研究中我們通過免疫組織化學(xué)染色觀察到NDRG2在成年人和小鼠睪丸內(nèi)均表達(dá)定位于睪丸間質(zhì)細(xì)胞,而在生精細(xì)胞內(nèi)未見明顯的陽性著色,推測其可能參與睪丸間質(zhì)細(xì)胞睪酮的分泌與調(diào)節(jié),進(jìn)而調(diào)控精子的發(fā)生,因此本實(shí)驗(yàn)的目的在于探討NDRG2在睪丸發(fā)育與精子發(fā)生過程中的作用及其機(jī)制。0 方法 1.利用RT-PCR、蛋白質(zhì)免疫印跡雜交及免疫組織化學(xué)等方法對比研究出生后大鼠睪丸發(fā)育不同階段(1、5、15、21、35和75 d)中NDRG2的表達(dá)與定位;并分離21、35和75 d大鼠睪丸間質(zhì)細(xì)胞和生精細(xì)胞,利用RT-PCR和蛋白質(zhì)免疫印跡雜交分析和驗(yàn)證NDRG2在大鼠睪丸不同發(fā)育階段中表達(dá)的差異性。 2.建立成年大鼠單劑量MAA(650 mg/kg)腹腔注射誘導(dǎo)精母細(xì)胞凋亡模型,采用TUNEL、RT-PCR、蛋白質(zhì)免疫印跡雜交、免疫組織化學(xué)、間接免疫熒光和激光顯微切割技術(shù)分析NDRG2在生精細(xì)胞凋亡過程中表達(dá)的變化,探討NDRG2與生精細(xì)胞凋亡之間的關(guān)系。 3.青春期大鼠睪丸內(nèi)生精細(xì)胞自發(fā)性凋亡達(dá)到頂峰,建立青春期(21 d)大鼠隱睪模型,通過TUNEL染色驗(yàn)證該模型、運(yùn)用免疫組織化學(xué)和間接免疫熒光等方法分析NDRG2在隱睪和對照側(cè)睪丸表達(dá)的差異,并且分離術(shù)后不同時間點(diǎn)的間質(zhì)細(xì)胞和生精細(xì)胞,運(yùn)用免疫印跡雜交檢測NDRG2和p53的表達(dá)變化。 結(jié)果 1.RT-PCR和Western blot結(jié)果均顯示NDRG2在出生后1 d大鼠睪丸內(nèi)即有表達(dá),隨后逐漸升高,至青春期達(dá)到頂峰(21和35 d),在成年期其表達(dá)反而下降。免疫組織化學(xué)結(jié)果顯示NDRG2持續(xù)表達(dá)于睪丸間質(zhì)細(xì)胞,但在睪丸發(fā)育不同階段,其在生精小管內(nèi)的表達(dá)卻存在明顯的差異。在新生期(1和5 d)主要表達(dá)于原始生殖細(xì)胞,而不表達(dá)于支持細(xì)胞;在15和21 d大鼠睪丸,NDRG2在生精小管內(nèi)主要表達(dá)于精母細(xì)胞,而精原細(xì)胞和支持細(xì)胞未見陽性著色;在35 d大鼠睪丸,NDRG2在生精小管內(nèi)主要表達(dá)于精母細(xì)胞和圓形精子細(xì)胞;在成年(75 d)大鼠,生精小管內(nèi)幾乎未見NDRG2陽性著色。分離純化21,35和75 d大鼠睪丸生精細(xì)胞,采用RT-PCR和Western blot進(jìn)一步驗(yàn)證了NDRG2在青春期大鼠生精細(xì)胞內(nèi)高表達(dá),而在成年大鼠生精細(xì)胞內(nèi)幾乎不表達(dá)。分離純化21,35和75 d大鼠睪丸間質(zhì)細(xì)胞,結(jié)果顯示NDRG2在這三個時間點(diǎn)間質(zhì)細(xì)胞內(nèi)均有較強(qiáng)的表達(dá),但相互間無明顯差異。 2.MAA腹腔注射3和6 h與對照組相比未見顯著性差異,但MAA誘導(dǎo)12 h后,成年大鼠睪丸內(nèi)出現(xiàn)大量初級精母細(xì)胞的凋亡,主要集中在生精周期的第X-XIII期,通過分離生精細(xì)胞和激光顯微切割技術(shù)均檢測到NDRG2在MAA誘導(dǎo)12 h后在生精細(xì)胞的表達(dá)上調(diào),免疫組織化學(xué)和間接免疫熒光結(jié)果均顯示NDRG2在自發(fā)性和MAA誘導(dǎo)凋亡的生精細(xì)胞中均高表達(dá)。 3.建立21 d大鼠單側(cè)隱睪模型,術(shù)后7 d和14 d,隱睪側(cè)睪丸重量明顯減輕,凋亡生精細(xì)胞數(shù)明顯升高,免疫組織化學(xué)與間接免疫熒光結(jié)果顯示NDRG2在自發(fā)性和熱刺激誘導(dǎo)凋亡的生精細(xì)胞內(nèi)表達(dá)均明顯上調(diào),但在一些正常的精母細(xì)胞和圓形精子內(nèi)仍存在一定量的表達(dá),并且NDRG2在隱睪側(cè)間質(zhì)細(xì)胞的表達(dá)強(qiáng)度明顯降低。分離純化術(shù)后不同時間點(diǎn)隱睪側(cè)和對照側(cè)睪丸生精細(xì)胞,發(fā)現(xiàn)NDRG2和p53在術(shù)后7 d隱睪側(cè)生精細(xì)胞的表達(dá)量明顯上調(diào),而在隱睪后14 d未見明顯差異,可能與生精細(xì)胞的凋亡與脫落有關(guān);分離純化術(shù)后不同時間點(diǎn)隱睪側(cè)和對照側(cè)睪丸間質(zhì)細(xì)胞,進(jìn)一步證實(shí)了NDRG2在術(shù)后7和14 d隱睪側(cè)間質(zhì)細(xì)胞的表達(dá)量減少。 結(jié)論 1. NDRG2在大鼠睪丸發(fā)育不同階段持續(xù)表達(dá)于間質(zhì)細(xì)胞,可能參與睪丸早期發(fā)育和睪酮的分泌與調(diào)節(jié)。 2. NDRG2在大鼠睪丸發(fā)育過程中在生精細(xì)胞的表達(dá)存在較大差異,在青春期前在生精細(xì)胞有較高水平的表達(dá),但在成年期表達(dá)量很低,提示NDRG2可能參與睪丸發(fā)育早期生精細(xì)胞增殖與分化的調(diào)節(jié)。 3. NDRG2在MAA和熱刺激誘導(dǎo)的生精細(xì)胞凋亡中表達(dá)均明顯上調(diào),并且在青春期和成年期大鼠睪丸自發(fā)性生精細(xì)胞凋亡中均存在較強(qiáng)的表達(dá),提示NDRG2可能參與了生精細(xì)胞凋亡的調(diào)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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R329

【共引文獻(xiàn)】

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