壬基酚经口摄入后在雄性大鼠体内分布及其对大鼠睾丸间质细胞影响的研究

发布时间：2018-03-30 07:44

本文选题：环境内分泌干扰物　切入点：壬基酚同分异构体　出处：《南京大学》2014年博士论文

【摘要】：壬基酚(NP)是壬基酚聚氧乙烯醚(NPE)的降解产物,已被证实是一种环境内分泌干扰物(EDCs).研究表明围产期或成年后的NP暴露对男性生殖系统有显著的不利影响,尤其是对雄激素水平。事实上,环境中壬基酚是由不同异构体组成的混合物,不同结构壬基酚同分异构体的雌激素活性存在差异。然而,关于壬基酚同分异构体单体的研究却少之又少。本课题检测了壬基酚经口摄入后在睾丸组织的分布、清除与富集,并成功分离睾丸间质细胞,系统地研究了壬基酚对原代培养大鼠睾丸间质细胞分泌睾酮的影响；同时,进一步对比研究了高纯度壬基酚同分异构体对原代培养大鼠睾丸间质细胞分泌睾酮的影响差异。第一部分：壬基酚经口摄入后在雄性大鼠体内的分布与富集一、目的了解壬基酚经口摄入后在雄性大鼠体内的分布与富集。二、方法 1.对自由进食大鼠一次灌胃14C-4-NP111(500mg/kg)后,不同时间点(0、0.5、1、2、4、8、12、24、48、72、96h)处死动物,取血、肝、肾、睾丸和脑组织测定壬基酚含量。 2.对自由进食大鼠灌胃14C-4-NP111(50mg/kg),分别持续一天、一周、一月后才处死动物,取血、肝、肾、睾丸和脑组织测定壬基酚含量。三、结果 1.自由摄食大鼠灌胃14C-4-NP111(500mg/kg)后,在血、肝脏、肾脏、睾丸和脑组织中的清除符合一室模型,其平均滞留时间分别为42.65、44.32、37.58、49.67和43.51h。 2.自由进食大鼠持续灌胃14C-4-NP111(50mg/kg)一天、一周、一月后,血、肝脏、肾脏和脑组织中壬基酚的含量随时间虽有所增加,但增幅不大,而睾丸中壬基酚的含量随时间增加明显。四、结论壬基酚经口摄入后,在大鼠睾丸组织有明显分布,滞留时间较长,且有明显的富集效应。第二部分大鼠睾丸间质细胞的原代培养、鉴定与功能监测一、目的采用改良方法对大鼠睾丸间质细胞进行分离、.纯化和原代培养,以得到更高纯度和稳定的间质细胞原代培养体系。二、方法 1.采用酶消化法分离大鼠睾丸间质细胞,再用Percoll连续密度梯度离心除去生精细胞、支持细胞等杂细胞,实现进一步纯化； 2.用3β-HSD特异性染色法鉴定间质细胞纯度； 3.采用放射免疫法测定间质细胞睾酮分泌的活性。三、结果 1.培养的间质细胞纯度达到95%以上,每个睾丸可获取间质细胞约1×106个； 2.通过3β-HSD特异性染色鉴定,该间质细胞胞质呈蓝黑色,而且细胞具有分泌睾酮的活性。四、结论 1.酶消化后以Percoll连续密度梯度离心可分离得到高纯度的睾丸间质细胞； 2.用该法分离得到的间质细胞具有较好的睾酮分泌活性。第三部分壬基酚对原代培养大鼠睾丸间质细胞的影响一、目的探讨壬基酚(NP)对大鼠睾丸间质细胞分泌雄激素的影响。二、方法 1.体外分离、培养睾丸间质细胞,并采用3β-HSD染色对间质细胞进行鉴定； 2.以不同浓度的NP(分别为较低浓度0.005、0.015、0.025。0.05。0.1及稍高浓度0.5、5.0、10.0、15.0、25.0μmol/L)作用于间质细胞48h； 3.采用3β-HSD染色观察间质细胞形态的变化,并检测间质细胞分泌睾酮含量的变化。三、结果 1.间质细胞经NP处理后,细胞形态发生变化,细胞密度降低,NP作用浓度达到25μmol/L时,细胞发生裂解。 2.0.005及0.015μmol/L NP处理后,间质细胞睾酮分泌量增加,与对照组比较差异有显著性(P0.05)。NP浓度达到0.5μmol/L后,睾酮分泌量与对照组相比显著下降(P0.05),且当NP浓度为5、10、15、25μmol/L时睾酮分泌量下降极为明显(P0.01)。四、结论低浓度NP能促进间质细胞分泌睾酮,而高浓度NP抑制睾酮分泌,且高浓度NP能诱导间质细胞坏死。第四部分不同壬基酚同分异构体对原代培养大鼠睾丸间质细胞的影响一、目的作为分布广泛的环境内分泌干扰物,NP在一定浓度下对野生动物和人类的生殖功能具有明确的负面影响。工业壬基酚是由不同异构体组成的混合物,不同结构壬基酚同分异构体的雌激素活性存在差异。然而,以往进行的壬基酚雌激素效应研究多以工业壬基酚为研究对象,很少以单一的壬基酚同分异构体作为受试化合物。本研究目的是评价不同壬基酚同分异构体对原代培养大鼠睾丸间质细胞分泌睾酮的影响。二、方法 1.体外分离、培养睾丸间质细胞,并采用3β-HSD染色对间质细胞进行鉴定； 2.筛选最适的药物作用浓度：NP浓度达到0.5后,NP对睾酮分泌有明显抑制作用。以不同浓度(分别为1.0、5.0、10.0、20.0μmol/L)的雌二醇(E2)、工业壬基酚(t-NP)、直链壬基酚(4n-NP)及壬基酚同分异构体(p33-NP、262-NP. p353-NP、363-NP)作用于原代培养的睾丸间质细胞6h,检测间质细胞分泌睾酮量的变化及细胞活力来筛选最适的药物作用浓度。 3.最适的药物浓度下,E2、t-NP、4n-NP、p33-NP及p363-NP作用于原代培养的睾丸间质细胞6h,通过RT-PCR、Western blot技术分别从mRNA和蛋白水平检测睾酮合成过程关键蛋白3β-HSD、Cyp11a1、Star的表达水平,通过TUNEL技术检测暴露不同NP同分异构体对原代培养间质细胞凋亡水平的影响。三、结果 1.筛选最适的药物作用浓度：浓度≥5.0μmol/L时,工业壬基酚(t-NP)、直链壬基酚(4n-NP)及壬基酚同分异构体(p33-NP、p262-NP、353-NP、p363-NP)对间质细胞睾酮分泌开始表现出有统计学意义的抑制作用；然而,浓度≥10.0μmol/L时,间质细胞活力会受到明显影响。为此,选择5.0μmol/L作为最适的药物作用浓度。 2.5.0μmol/L E2、t-NP、4n-NP、p33-NP、p262-NP、p353-NP及p363-NP作用下,间质细胞睾酮分泌均受到明显抑制(P0.01),而不同NP同分异构体的抑制程度彼此间有统计学差异,其中p363-NP的抑制作用最强。 3.5.0μmol/LE2、t-NP、4n-NP、353-NP及363-NP作用下,间质细胞3β-HSD、 Cypllal、Star的表达在mRNA水平上均有不同程度下降,蛋白水平上与之一致。同时,不同NP同分异构体的对3β-HSD、Cyplla1、Star表达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,间质细胞凋亡水平上升。不同NP同分异构体作用程度彼此间存在差异,p353-NP作用最弱,p33-NP、p262-NP、p363-NP作用相当。四、结论 1.较高浓度(≥5.0μmol/L)的NP同分异构体对原代培养大鼠睾丸间质细胞分泌睾酮有抑制作用。然而,不同NP同分异构体的抑制程度彼此间存在差异,这种差异可能源于不同NP同分异构体对3β-HSD、Cyplla1、Star表达水平和间质细胞凋亡水平的影响程度不同。 2.NP同分异构体对睾丸间质细胞分泌睾酮的抑制程度与其体外雌激素活性并不完全一致,这提示可能还存在其他机制参与这一抑制作用。本研究的特色与创新之处： 1.本课题检测了NP经口摄入后,首次证明了其在睾丸组织内存在,并探讨了NP在动物体内不同器官的分布,了解了对NP在体内的清除及富集过程。 2、成功分离、纯化、培养了大鼠原代睾丸间质细胞,系统地研究了壬基酚对原代培养大鼠睾丸间质细胞分泌睾酮的影响； 2.率先探讨了NP的不同同分异构体对原代培养大鼠睾丸间质细胞分泌睾酮的影响差异。
[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.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R114

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