本文關(guān)鍵詞：睪丸去神經(jīng)支配對(duì)雄性生殖細(xì)胞的影響及其機(jī)制　出處：《南京醫(yī)科大學(xué)》2008年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 睪丸 神經(jīng) 配對(duì) 雄性 生殖 細(xì)胞 影響 及其 機(jī)制

【摘要】： 目的 研究睪丸支配神經(jīng)(精索神經(jīng))在睪丸組織內(nèi)的分布特征及去神經(jīng)支配后對(duì)雄性生殖細(xì)胞凋亡與分化的影響,以及對(duì)性激素分泌的影響。 方法 1)免疫組織化學(xué)ABC法,以特異性的抗體標(biāo)記人睪丸組織內(nèi)蛋白基因產(chǎn)物9.5(PGP9.5)、C末端神經(jīng)多肽Y(CPON)、神經(jīng)肽Y(NPY)和血管活性腸肽(VIP)等肽能神經(jīng),了解其在人睪丸組織內(nèi)的分布特點(diǎn) 2)通過(guò)顯微外科技術(shù),同時(shí)切除SD大鼠(300-350g)精索上神經(jīng)和精索下神經(jīng),分別于1、4、8天采取睪九組織,采用脫氧核搪核酸末端轉(zhuǎn)移酶介導(dǎo)的dUTP的缺口末端標(biāo)記技術(shù)(TUNEL)觀察精原細(xì)胞和Leydig細(xì)胞的凋亡情況。 3)切除精索神經(jīng)對(duì)性激素的影響,解剖顯微鏡下建立睪丸去神經(jīng)支配大鼠模型,分別于術(shù)前7天、術(shù)后1、7、14、21天和術(shù)后28天進(jìn)行麻醉下大鼠眼眶后靜脈叢處取血,放射性免疫的方法檢測(cè)血清中睪酮(T)、卵泡刺激素(FSH)、黃體生成素(LH)、泌乳素(PRL)和雌二醇(E2)的濃度。了解性激素分泌的變化。 4)以EDS建立Leydig細(xì)胞特異性敲除模型,不同劑量EDS腹腔內(nèi)注射給藥,注射后3天、7、14天處死大鼠,取出睪丸組織,行HE染色光鏡下組織學(xué)觀察和P450scc免疫組化觀察及灰度分析。觀察Leydig細(xì)胞特異性敲除情況。 結(jié)果 1)PGP9.5肽能神經(jīng)和CPON肽能神經(jīng)主要分布在睪丸間質(zhì)內(nèi),與曲細(xì)小管相鄰,CPON陽(yáng)性纖維尚見(jiàn)于血管周?chē)?其分布密度低于PGP9.5;NPY肽能神經(jīng)在人睪丸實(shí)質(zhì)內(nèi)主要見(jiàn)于睪丸血管周?chē)?VIP肽能神經(jīng)在睪丸實(shí)質(zhì)內(nèi)未見(jiàn)分布。 2)術(shù)后1、4、8天,精原細(xì)胞和間質(zhì)細(xì)胞均發(fā)生顯著凋亡。其中精原細(xì)胞的凋亡峰值出現(xiàn)在術(shù)后第一天,間質(zhì)細(xì)胞的凋亡在這3個(gè)時(shí)點(diǎn)沒(méi)有明顯差異 3)血清中睪酮測(cè)定:去精索神經(jīng)組術(shù)后睪酮持續(xù)下降,與手術(shù)前相比差異有顯著性(P＜0.05),去精索上下神經(jīng)組較去精索上神經(jīng)組和去精索下神經(jīng)組睪酮下降更為明顯,于28天時(shí)有顯著差異性(P＜0.05);血清中的黃體生成素測(cè)定:去精索神經(jīng)組術(shù)后黃體生成素持續(xù)上升,與手術(shù)前相比差異有顯著性(P＜0.05),去精索上下神經(jīng)組較去精索上神經(jīng)組和去精索下神經(jīng)組上升更為明顯,于28天時(shí)有顯著差異性(P＜0.05);血清中的卵泡刺激素測(cè)定:均于手術(shù)后1天迅速升高(P＜0.05),此后逐漸降低至手術(shù)前的水平;血清中泌乳素測(cè)定:去精索神經(jīng)各組血清中泌乳素于手術(shù)后逐漸升高,術(shù)后28天達(dá)最高,其中去精索下神經(jīng)組術(shù)后21天和術(shù)后28天與術(shù)前比差異有顯著性(P＜0.05),去精索上神經(jīng)組術(shù)后14天、術(shù)后21天和術(shù)后28天與術(shù)前比差異有顯著性(P＜0.05),去精索上下神經(jīng)組術(shù)后1天、術(shù)后7天、術(shù)后14天、術(shù)后21天和術(shù)后28天與術(shù)前比差異有顯著性(P＜0.05);血清中雌二醇測(cè)定:去精索神經(jīng)各組血清中雌二醇均于術(shù)后1天急劇下降并于術(shù)后14天降至最低(P＜0.05),而后稍有回升并保持穩(wěn)定,但是仍低于手術(shù)前(P＜0.05)。 4)EDS處理3天后,均觀察到Leydig細(xì)胞的減少,其中40mg/kg劑量組的減少程度不明顯;EDS處理后7天,曲細(xì)精管內(nèi)生精細(xì)胞出現(xiàn)排列紊亂的現(xiàn)象,以90mg/kg劑量組更為明顯;EDS處理后14天,60mg/kg劑量組出現(xiàn)一種核圓形、核染色淡、體積較大的新形成Leydig細(xì)胞,而在75mg/kg劑量組沒(méi)有發(fā)現(xiàn)此細(xì)胞。P450scc免疫組化與光鏡結(jié)果一致。灰度分析結(jié)果顯示,EDS處理后3天及7天,各劑量組與正常對(duì)照相比差異均有顯著性(P＜0.05);60mg/kg劑量組14天與3天、7天相比差異有顯著性(P＜0.05),且與正常對(duì)照差異無(wú)顯著性(P＞0.05)。 結(jié)論 1)人睪丸間質(zhì)內(nèi)分布著PGP9.5、CPON和NPY肽能神經(jīng)纖維,為精索神經(jīng)參與精子發(fā)生過(guò)程提供形態(tài)學(xué)基礎(chǔ)。 2)在睪丸去神經(jīng)早期階段,精原細(xì)胞和間質(zhì)細(xì)胞即有大量凋亡,這可能是去神經(jīng)支配后期睪丸組織發(fā)生病理改變的重要原因。 3)睪丸去精索神經(jīng)支配對(duì)大鼠性激素的產(chǎn)生有明顯的影響,精索神經(jīng)對(duì)精子的發(fā)生具有重要的調(diào)控作用。 4)EDS確實(shí)能特異性地敲除成年大鼠Leydig細(xì)胞,60mg/kg的劑量可以更好的模擬青春期Leydig細(xì)胞增殖分化過(guò)程。
[Abstract]:objective
The distribution characteristics of testicular innervation (spermatic cord nerve) in testicular tissue and the effect of denervation on the apoptosis and differentiation of male germ cells and the effects of sex hormones secretion were investigated.
Method
1) immunohistochemical ABC method was used to label the peptidergic nerve of human testicular tissue, protein gene product 9.5 (PGP9.5), C terminal neuropeptide Y (CPON), neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) with specific antibodies, and to understand its distribution in human testicular tissue.
2) by microsurgical technique, simultaneous resection of SD rats (300-350g) spermatic nerve and spermatic nerve, take nine testis tissue on 1,4,8 days respectively, TUNEL using DNA RNA TDT mediated dUTP (TUNEL) to observe the apoptosis of spermatogenic cells and Leydig cells.
3) resection of spermatic nerve effect on sex hormone, under a dissecting microscope to establish the rat model of testicular denervation, respectively in the 7 days before surgery, 28 1,7,14,21 days after operation and postoperative anesthesia in rats after orbital venous plexus at the blood testosterone in serum of the radioactive immune method (T) (FSH), follicle stimulating hormone, luteinizing hormone (LH), prolactin (PRL) and estradiol (E2) concentrations. Understand the change of sex hormone secretion.
4) to establish EDS cell specific Leydig knockout model, different dose of EDS intraperitoneal injection, 3 days 7,14 days after injection, the rats were sacrificed, removal of testicular tissue by HE staining, light microscope observation and P450scc immunohistochemical observation and gray analysis. Observation of Leydig cell specific knockout situation.
Result
1) PGP9.5 peptidergic nerves and CPON peptidergic nerves are mainly distributed in the testicular interstitium, and seminiferous tubules adjacent to CPON positive fibers are found in the blood vessels around the density of less than PGP9.5; NPY peptidergic nerves in human testicular parenchyma mainly in the testicular vessels around; VIP peptide was not found in the testicular parenchyma of nerve.
2) 1,4,8 days after operation, spermatogonia and stromal cells all had significant apoptosis. The apoptosis peak of spermatogonia appeared on the first day after operation. There was no significant difference in the apoptosis of interstitial cells at these 3 points.
3) determination of testosterone in serum: to spermatic nerve group after testosterone declined compared with before surgery, there was significant difference (P < 0.05), to the cord nerve group than in denervation group and denervation nerve cord testosterone group decreased more significantly, in 28 days when there were significantly differences (P < 0.05); luteinizing hormone determination in serum: spermatic nerve group after LH continued to rise, compared with before surgery, there was significant difference (P < 0.05), to the cord nerve group than in denervation group and denervation nerve cord group increased more significantly, in 28 the day when there are significant differences (P < 0.05); serum levels of FSH were measured: 1 days after surgery increased rapidly (P < 0.05), then decreased gradually to the preoperative level of serum hormone determination; lactation: go to the spermatic nerves of serum prolactin after operation gradually increased. After 28 the highest in Tianda, among them Go to the spermatic nerve group after 21 days and 28 days after surgery and preoperative ratio had significant difference (P < 0.05), go to the spermatic nerve group after 14 days, after 21 days and 28 days after surgery and preoperative ratio had significant difference (P < 0.05), to the cord the nerve group after 1 days, 7 days after operation, 14 days after the operation, after 21 days and 28 days after surgery and preoperative ratio had significant difference (P < 0.05); Determination of estradiol in serum: serum estradiol in the spermatic nerves were on the 1 postoperative day and decreased dramatically on the 14 day after surgery to the lowest (P < 0.05), and then recovered slightly and remained steady, but still lower than that before operation (P < 0.05).
4) EDS after 3 days of treatment, were observed in Leydig cells decreased, the 40mg/kg dose reduction degree is not obvious; 7 days after EDS treatment, the seminiferous tubules spermatogenic cells disordered phenomenon in 90mg/kg dosage group was more obvious; 14 days after treatment with EDS, a nuclear round appearance 60mg/kg dose group, nuclear staining, a larger volume of newly formed Leydig cells, while in the 75mg/kg group did not find this.P450scc cells by immunohistochemistry and light microscopy results. The gray analysis results showed that EDS treatment after 3 days and 7 days, each dose group and normal controls were significantly different (P < 0.05); 60mg/kg group 14 days and 3 days, there was significant difference between the 7 days (P < 0.05), and no significant difference with normal control (P > 0.05).
conclusion
1) PGP9.5, CPON and NPY peptide can provide a morphological basis for the spermatogenesis of the spermatic cord.
2) in the early stage of testicular denervation, there is a large number of apoptosis in spermatogonia and interstitial cells, which may be an important reason for pathological changes in testicular tissue at the late stage of denervation.
3) testicular denervation of spermatic cord has an obvious effect on the production of sex hormones in rats. The spermatic nerve plays an important role in regulating the occurrence of sperm.
4) EDS can specifically knock out Leydig cells in adult rats. The dose of 60mg/kg can better simulate the proliferation and differentiation process of Leydig cells in puberty.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R321.1

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 張冰;瑪依拉·阿布拉克;王新星;買(mǎi)爾江·艾合買(mǎi)提;;單側(cè)睪丸動(dòng)脈結(jié)扎/再通對(duì)小鼠生精上皮、血清抗精子抗體的影響[J];黑龍江動(dòng)物繁殖;2009年03期

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本文編號(hào)：1380758