本文選題：環(huán)境內(nèi)分泌干擾物　切入點(diǎn)：壬基酚同分異構(gòu)體　出處：《南京大學(xué)》2014年博士論文

【摘要】：壬基酚(NP)是壬基酚聚氧乙烯醚(NPE)的降解產(chǎn)物,已被證實(shí)是一種環(huán)境內(nèi)分泌干擾物(EDCs).研究表明圍產(chǎn)期或成年后的NP暴露對(duì)男性生殖系統(tǒng)有顯著的不利影響,尤其是對(duì)雄激素水平。事實(shí)上,環(huán)境中壬基酚是由不同異構(gòu)體組成的混合物,不同結(jié)構(gòu)壬基酚同分異構(gòu)體的雌激素活性存在差異。然而,關(guān)于壬基酚同分異構(gòu)體單體的研究卻少之又少。本課題檢測(cè)了壬基酚經(jīng)口攝入后在睪丸組織的分布、清除與富集,并成功分離睪丸間質(zhì)細(xì)胞,系統(tǒng)地研究了壬基酚對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞分泌睪酮的影響；同時(shí),進(jìn)一步對(duì)比研究了高純度壬基酚同分異構(gòu)體對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞分泌睪酮的影響差異。 第一部分：壬基酚經(jīng)口攝入后在雄性大鼠體內(nèi)的分布與富集 一、目的 了解壬基酚經(jīng)口攝入后在雄性大鼠體內(nèi)的分布與富集。 二、方法 1.對(duì)自由進(jìn)食大鼠一次灌胃14C-4-NP111(500mg/kg)后,不同時(shí)間點(diǎn)(0、0.5、1、2、4、8、12、24、48、72、96h)處死動(dòng)物,取血、肝、腎、睪丸和腦組織測(cè)定壬基酚含量。 2.對(duì)自由進(jìn)食大鼠灌胃14C-4-NP111(50mg/kg),分別持續(xù)一天、一周、一月后才處死動(dòng)物,取血、肝、腎、睪丸和腦組織測(cè)定壬基酚含量。 三、結(jié)果 1.自由攝食大鼠灌胃14C-4-NP111(500mg/kg)后,在血、肝臟、腎臟、睪丸和腦組織中的清除符合一室模型,其平均滯留時(shí)間分別為42.65、44.32、37.58、49.67和43.51h。 2.自由進(jìn)食大鼠持續(xù)灌胃14C-4-NP111(50mg/kg)一天、一周、一月后,血、肝臟、腎臟和腦組織中壬基酚的含量隨時(shí)間雖有所增加,但增幅不大,而睪丸中壬基酚的含量隨時(shí)間增加明顯。 四、結(jié)論 壬基酚經(jīng)口攝入后,在大鼠睪丸組織有明顯分布,滯留時(shí)間較長(zhǎng),且有明顯的富集效應(yīng)。 第二部分大鼠睪丸間質(zhì)細(xì)胞的原代培養(yǎng)、鑒定與功能監(jiān)測(cè) 一、目的 采用改良方法對(duì)大鼠睪丸間質(zhì)細(xì)胞進(jìn)行分離、.純化和原代培養(yǎng),以得到更高純度和穩(wěn)定的間質(zhì)細(xì)胞原代培養(yǎng)體系。 二、方法 1.采用酶消化法分離大鼠睪丸間質(zhì)細(xì)胞,再用Percoll連續(xù)密度梯度離心除去生精細(xì)胞、支持細(xì)胞等雜細(xì)胞,實(shí)現(xiàn)進(jìn)一步純化； 2.用3β-HSD特異性染色法鑒定間質(zhì)細(xì)胞純度； 3.采用放射免疫法測(cè)定間質(zhì)細(xì)胞睪酮分泌的活性。 三、結(jié)果 1.培養(yǎng)的間質(zhì)細(xì)胞純度達(dá)到95%以上,每個(gè)睪丸可獲取間質(zhì)細(xì)胞約1×106個(gè)； 2.通過(guò)3β-HSD特異性染色鑒定,該間質(zhì)細(xì)胞胞質(zhì)呈藍(lán)黑色,而且細(xì)胞具有分泌睪酮的活性。 四、結(jié)論 1.酶消化后以Percoll連續(xù)密度梯度離心可分離得到高純度的睪丸間質(zhì)細(xì)胞； 2.用該法分離得到的間質(zhì)細(xì)胞具有較好的睪酮分泌活性。 第三部分壬基酚對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞的影響 一、目的 探討壬基酚(NP)對(duì)大鼠睪丸間質(zhì)細(xì)胞分泌雄激素的影響。 二、方法 1.體外分離、培養(yǎng)睪丸間質(zhì)細(xì)胞,并采用3β-HSD染色對(duì)間質(zhì)細(xì)胞進(jìn)行鑒定； 2.以不同濃度的NP(分別為較低濃度0.005、0.015、0.025。0.05。0.1及稍高濃度0.5、5.0、10.0、15.0、25.0μmol/L)作用于間質(zhì)細(xì)胞48h； 3.采用3β-HSD染色觀察間質(zhì)細(xì)胞形態(tài)的變化,并檢測(cè)間質(zhì)細(xì)胞分泌睪酮含量的變化。 三、結(jié)果 1.間質(zhì)細(xì)胞經(jīng)NP處理后,細(xì)胞形態(tài)發(fā)生變化,細(xì)胞密度降低,NP作用濃度達(dá)到25μmol/L時(shí),細(xì)胞發(fā)生裂解。 2.0.005及0.015μmol/L NP處理后,間質(zhì)細(xì)胞睪酮分泌量增加,與對(duì)照組比較差異有顯著性(P0.05)。NP濃度達(dá)到0.5μmol/L后,睪酮分泌量與對(duì)照組相比顯著下降(P0.05),且當(dāng)NP濃度為5、10、15、25μmol/L時(shí)睪酮分泌量下降極為明顯(P0.01)。 四、結(jié)論 低濃度NP能促進(jìn)間質(zhì)細(xì)胞分泌睪酮,而高濃度NP抑制睪酮分泌,且高濃度NP能誘導(dǎo)間質(zhì)細(xì)胞壞死。 第四部分不同壬基酚同分異構(gòu)體對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞的影響 一、目的 作為分布廣泛的環(huán)境內(nèi)分泌干擾物,NP在一定濃度下對(duì)野生動(dòng)物和人類的生殖功能具有明確的負(fù)面影響。工業(yè)壬基酚是由不同異構(gòu)體組成的混合物,不同結(jié)構(gòu)壬基酚同分異構(gòu)體的雌激素活性存在差異。然而,以往進(jìn)行的壬基酚雌激素效應(yīng)研究多以工業(yè)壬基酚為研究對(duì)象,很少以單一的壬基酚同分異構(gòu)體作為受試化合物。本研究目的是評(píng)價(jià)不同壬基酚同分異構(gòu)體對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞分泌睪酮的影響。 二、方法 1.體外分離、培養(yǎng)睪丸間質(zhì)細(xì)胞,并采用3β-HSD染色對(duì)間質(zhì)細(xì)胞進(jìn)行鑒定； 2.篩選最適的藥物作用濃度：NP濃度達(dá)到0.5后,NP對(duì)睪酮分泌有明顯抑制作用。以不同濃度(分別為1.0、5.0、10.0、20.0μmol/L)的雌二醇(E2)、工業(yè)壬基酚(t-NP)、直鏈壬基酚(4n-NP)及壬基酚同分異構(gòu)體(p33-NP、262-NP. p353-NP、363-NP)作用于原代培養(yǎng)的睪丸間質(zhì)細(xì)胞6h,檢測(cè)間質(zhì)細(xì)胞分泌睪酮量的變化及細(xì)胞活力來(lái)篩選最適的藥物作用濃度。 3.最適的藥物濃度下,E2、t-NP、4n-NP、p33-NP及p363-NP作用于原代培養(yǎng)的睪丸間質(zhì)細(xì)胞6h,通過(guò)RT-PCR、Western blot技術(shù)分別從mRNA和蛋白水平檢測(cè)睪酮合成過(guò)程關(guān)鍵蛋白3β-HSD、Cyp11a1、Star的表達(dá)水平,通過(guò)TUNEL技術(shù)檢測(cè)暴露不同NP同分異構(gòu)體對(duì)原代培養(yǎng)間質(zhì)細(xì)胞凋亡水平的影響。 三、結(jié)果 1.篩選最適的藥物作用濃度：濃度≥5.0μmol/L時(shí),工業(yè)壬基酚(t-NP)、直鏈壬基酚(4n-NP)及壬基酚同分異構(gòu)體(p33-NP、p262-NP、353-NP、p363-NP)對(duì)間質(zhì)細(xì)胞睪酮分泌開始表現(xiàn)出有統(tǒng)計(jì)學(xué)意義的抑制作用；然而,濃度≥10.0μmol/L時(shí),間質(zhì)細(xì)胞活力會(huì)受到明顯影響。為此,選擇5.0μmol/L作為最適的藥物作用濃度。 2.5.0μmol/L E2、t-NP、4n-NP、p33-NP、p262-NP、p353-NP及p363-NP作用下,間質(zhì)細(xì)胞睪酮分泌均受到明顯抑制(P0.01),而不同NP同分異構(gòu)體的抑制程度彼此間有統(tǒng)計(jì)學(xué)差異,其中p363-NP的抑制作用最強(qiáng)。 3.5.0μmol/LE2、t-NP、4n-NP、353-NP及363-NP作用下,間質(zhì)細(xì)胞3β-HSD、 Cypllal、Star的表達(dá)在mRNA水平上均有不同程度下降,蛋白水平上與之一致。同時(shí),不同NP同分異構(gòu)體的對(duì)3β-HSD、Cyplla1、Star表達(dá)的抑制程度彼此不同,且更有偏重。 4.5.0μmol/L E2、t-NP、4n-NP、p33-NP、p262-NP、p353-NP及p363-NP作用下,間質(zhì)細(xì)胞凋亡水平上升。不同NP同分異構(gòu)體作用程度彼此間存在差異,p353-NP作用最弱,p33-NP、p262-NP、p363-NP作用相當(dāng)。 四、結(jié)論 1.較高濃度(≥5.0μmol/L)的NP同分異構(gòu)體對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞分泌睪酮有抑制作用。然而,不同NP同分異構(gòu)體的抑制程度彼此間存在差異,這種差異可能源于不同NP同分異構(gòu)體對(duì)3β-HSD、Cyplla1、Star表達(dá)水平和間質(zhì)細(xì)胞凋亡水平的影響程度不同。 2.NP同分異構(gòu)體對(duì)睪丸間質(zhì)細(xì)胞分泌睪酮的抑制程度與其體外雌激素活性并不完全一致,這提示可能還存在其他機(jī)制參與這一抑制作用。本研究的特色與創(chuàng)新之處： 1.本課題檢測(cè)了NP經(jīng)口攝入后,首次證明了其在睪丸組織內(nèi)存在,并探討了NP在動(dòng)物體內(nèi)不同器官的分布,了解了對(duì)NP在體內(nèi)的清除及富集過(guò)程。 2、成功分離、純化、培養(yǎng)了大鼠原代睪丸間質(zhì)細(xì)胞,系統(tǒng)地研究了壬基酚對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞分泌睪酮的影響； 2.率先探討了NP的不同同分異構(gòu)體對(duì)原代培養(yǎng)大鼠睪丸間質(zhì)細(xì)胞分泌睪酮的影響差異。
[Abstract]:Nonylphenol (NP) (NPE) is nonylphenol degradation products, which has been proven to be an environmental endocrine disruptors (EDCs). The research showed that perinatal or adult NP exposure had significant negative effects on the male reproductive system, especially on androgen levels. In fact, the environment is composed of a mixture of different isomers of nonylphenol the existence of estrogenic activity of different structural isomers of nonylphenol difference. However, research on nonylphenol isomers monomer is less and less. We detected the distribution in testicular tissue after oral intake of nonylphenol, removal and enrichment, and successfully isolated Leydig cells, systematic study the effect of nonylphenol in rat Leydig cell testosterone production in primary cultured; at the same time, further comparative study of the high purity of nonylphenol isomers in rat Leydig cells in primary culture The difference in the effect of testosterone.
Part 1: distribution and enrichment of nonylphenol in male rats after oral intake
First, the purpose
To understand the distribution and enrichment of nonylphenol in male rats after oral intake.
Two, method
1. the rats were fed with 14C-4-NP111 (500mg/kg) once a day for free. The animals were sacrificed at different time points (0,0.5,1,2,4,8,12,24,48,72,96h), and the contents of nonylphenol in blood, liver, kidney, testis and brain were determined.
2., the rats were fed 14C-4-NP111 (50mg/kg) for free for a day. After a week and a month later, the animals were killed. The contents of nonylphenol in blood, liver, kidney, testis and brain were determined.
Three, the result
After clearance of 14C-4-NP111 (500mg/kg) in 1. free feeding rats, the clearance in blood, liver, kidney, testis and brain accorded with one compartment model. The average residence time was 42.65,44.32,37.58,49.67 and 43.51h. respectively.
2. free feeding rats continued to intragastric administration of 14C-4-NP111 (50mg/kg) for one day. After a week and a month, the contents of nonylphenol in blood, liver, kidney and brain increased with time, but increased little. The content of nonylphenol in testis increased significantly with time.
Four. Conclusion
After oral intake of nonylphenol, the testicular tissue of rats was obviously distributed, and the retention time was longer, and there was an obvious enrichment effect.
Primary culture, identification and functional monitoring of the second part of rat Leydig cells
First, the purpose
The improved method was used to isolate the Leydig cells of the rat testis. The purification and primary culture were used to obtain the higher purity and stability of the primary culture system of interstitial cells.
Two, method
1., the rat Leydig cells were isolated by enzyme digestion. Then, Percoll and continuous density gradient centrifugation were used to remove spermatogenic cells, supporting cells and other heterozygous cells, and further purify them.
2. the purity of stromal cells was identified by 3 beta -HSD specific staining.
3. radioimmunoassay was used to determine the activity of testosterone secretion in interstitial cells.
Three, the result
1. the purity of cultured stromal cells was more than 95%, and about 1 * 106 of the stromal cells in each testis were obtained.
2. through 3 beta -HSD specific staining, the cytoplasm of Leydig cells and cells with blue black, with testosterone secretion activity.
Four. Conclusion
After 1. enzyme digestion, high purity Leydig cells were separated by Percoll continuous density gradient centrifugation.
2. the stromal cells isolated by this method have good testosterone secretion activity.
The effect of third nonylphenol on the primary cultured rat Leydig cells
First, the purpose
To investigate the effect of nonylphenol (NP) on the secretion of androgens in the Leydig cells of rat testis.
Two, method
1. the Leydig cells were isolated and cultured in vitro, and the interstitial cells were identified by 3 beta -HSD staining.
2. with different concentrations of NP (low concentration 0.005,0.015,0.025.0.05.0.1 and slightly high concentration 0.5,5.0,10.0,15.0,25.0 mol/L respectively), the interstitial cells (48h) were used.
3. the changes in the morphology of stromal cells were observed by 3 beta -HSD staining and the levels of testosterone secreted by interstitial cells were detected.
Three, the result
After the 1. stromal cells were treated with NP, the cell morphology changed and the cell density decreased. When the concentration of NP reached 25 mol/L, the cells lysed.
2.0.005 and 0.015 mol/L after NP treatment, the testosterone secretion of Leydig cells increased, there was significant difference compared with the control group (P0.05).NP concentration reached 0.5 mol/L, testosterone secretion decreased significantly compared to the control group (P0.05), and when the concentration of NP was 5,10,15,25 mol/L when testosterone secretion decreased extremely obviously (P0.01).
Four. Conclusion
Low concentration of NP can promote the secretion of testosterone in stromal cells, while high concentration of NP inhibits the secretion of testosterone, and high concentration of NP can induce interstitial cell necrosis.
The effect of fourth different nonylphenol isomers on the primary cultured rat Leydig cells
First, the purpose
As environmental endocrine disruptors are widely distributed, NP has a clear negative impact on wild animal and human reproductive function in a certain concentration. Industrial nonylphenol is composed of a mixture of different isomers, the estrogenic activity of different structure of nonylphenol isomers are different. However, the effect of nonylphenol estrogen to industry nonylphenol as the research object, rarely with a single nonylphenol isomers as test compounds. The purpose of this study is to evaluate the different effects of nonylphenol isomers in rat Leydig cell testosterone secretion of primary culture.
Two, method
1. the Leydig cells were isolated and cultured in vitro, and the interstitial cells were identified by 3 beta -HSD staining.
The optimal concentration of 2. drug screening: NP concentration reached 0.5, NP had significant inhibitory effect on testosterone secretion. At different concentrations (1.0,5.0,10.0,20.0 mol/L) of estradiol (E2), nonylphenol (t-NP), industrial chain nonylphenol (4n-NP) and nonylphenol isomers (p33-NP, 262-NP., p353-NP, 363-NP) in primary cultured Leydig cells of 6h, detection of interstitial cell secretion and cell viability of testosterone to drug screening effect of the optimal concentration.
The most suitable drug concentration 3., E2, t-NP, 4n-NP, p33-NP and p363-NP in primary cultured Leydig cells by 6h, RT-PCR, Western blot respectively from the key protein mRNA and protein levels of testosterone synthesis process of 3 beta -HSD, Cyp11a1, the expression level of Star, NP with different exposure the level of quality isomers on apoptosis in primary cultured between through TUNEL detection.
Three, the result
The optimal concentration of 1. drug screening: concentration of more than 5 mol/L, nonylphenol (t-NP), industrial chain nonylphenol (4n-NP) and nonylphenol isomers (p33-NP, p262-NP, 353-NP, p363-NP) on testosterone secretion of Leydig cells showed inhibitory effect was statistically significant; however, the concentration of more than 10 mol/L, interstitial cell viability will be significantly affected. Therefore, choosing 5 mol/L as the optimal concentration of drug action.
Under the action of 2.5.0, mol/L, E2, t-NP, 4n-NP, p33-NP, p262-NP, p353-NP and p363-NP, the secretion of testosterone in interstitial cells was significantly inhibited (P0.01), and the inhibition degree of different NP isomers was statistically different from each other, among which the inhibition of the strongest was the inhibition of p363-NP.
3.5.0 mol/LE2, t-NP, 4n-NP, 353-NP and 363-NP under the action of interstitial cells of 3 beta -HSD, Cypllal, Star and mRNA expressed in different degrees of decline, the protein level was consistent with that of 3. At the same time, beta -HSD, Cyplla1 different NP isomers, the degree of inhibition of Star expression with each other different, and more emphasis.
4.5.0, mol/L, E2, t-NP, 4n-NP, p33-NP, p262-NP, p353-NP and p363-NP increased the level of interstitial cell apoptosis. The degree of action of different NP isomers was different from each other, and the effect of p353-NP was the weakest, and the action of p353-NP, p363-NP and p353-NP was the same.
Four. Conclusion
1. high concentration (more than 5 mol/L) of the NP isoforms in rat Leydig cells secreting testosterone has inhibitory effect on primary culture. However, the degree of inhibition of different NP isomers exist differences, these differences may be due to different NP isomers of -HSD beta 3, Cyplla1 the degree of influence, the expression level of Star and interstitial cell apoptosis at different levels.
The inhibitory effect of 2.NP isomers on testosterone secretion in Leydig cells is not identical with estrogenic activity in vitro, suggesting that there may be other mechanisms involved in this inhibition.
1., after testing the oral intake of NP, it was first demonstrated that it was present in the testicular tissue, and the distribution of NP in different organs of animals was discussed. The clearance and accumulation process of NP in vivo was also understood.
2, we successfully isolated, purified and cultured rat primary Leydig cells, and systematically studied the effects of nonylphenol on testosterone secretion in primary rat Leydig cells.
2. the difference in the effect of different isomers of NP on the secretion of testosterone in primary cultured rat Leydig cells was investigated.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R114

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