环境激素丙烯酰胺对SD大鼠生精细胞端粒酶活性及增殖的表达影响

发布时间：2018-07-08 16:13

本文选题：丙烯酰胺 + 生殖毒性　；参考：《河北医科大学》2013年硕士论文

【摘要】：目的：丙烯酰胺（Acrylamide AA）是一种白色晶体化学物质，，为生产聚丙烯酰胺的原料，属于环境雌激素的一种，广泛应用于水溶性聚合物和有机化学药品的制造。日常生活中，高温（120℃）烹调下，淀粉类食品很容易产生丙烯酰胺。人体可通过多种途径吸收丙烯酰胺，如消化道，呼吸道及皮肤黏膜等[1]，其中消化道吸收最快。胎儿和婴幼儿通过胎盘和乳汁也可吸收丙烯酰胺。早在20世纪80年代，科学家就已经对丙烯酰胺的毒性有了充分的认识，丙烯酰胺可引起神经毒性，引起嗜睡、记忆减退、失眠、幻觉等，急性中毒可出现迟发性的周围神经改变，如步态不稳、手套样改变、无力等。随着2002年4月，瑞典科学家首次报道高温加热淀粉类食品中发现丙烯酰胺后，丙烯酰胺再一次引起了全世界各国科学家的关注。丙烯酰胺除上述神经毒性外，还存在致癌性、胚胎发育毒性以及生殖毒性[2]。丙烯酰胺已被国际癌症研究机构（IARC）列入2A组“人类可能致癌物质”[3]。丙烯酰胺的生殖毒性体现在许多方面，有人认为丙烯酰胺可能会诱导生殖细胞发生染色体突变；也有人研究认为，丙烯酰胺可能会影响睾丸的基因表达，致使类固醇激素的转运以及精子的发生出现异常，同时，丙烯酰胺也会降低体内的睾酮含量。虽然科学家对丙烯酰胺的生殖毒性提出了很多假设，但仍然不能完整的描述其发生机制，因此，丙烯酰胺的生殖毒性毒理研究仍有待进一步的阐明。端粒酶是一种核糖核蛋白逆转录酶，能以自身的内源性RNA序列作为模板从头合成染色体末端端粒的DNA序列。端粒酶活性与细胞的增殖潜能、细胞周期、细胞分化、凋亡和死亡息息相关。在人体的大多数细胞中并没有端粒酶活性，但在干细胞、生殖细胞以及90%的肿瘤细胞中都可检测到端粒酶活性，因此，端粒酶已作为一种细胞增殖的指标被广泛应用。在生殖细胞当中，端粒酶活性保证端粒的DNA序列始终如一的保存，这样才能保证生殖细胞中DNA有序、稳定的分裂并遗传至下一代。作为DNA多聚酶δ辅助蛋白的PCNA是DNA复制过程中所必需的，具有维持DNA多聚酶高活性的作用，出现于G1早期，S期达到高峰，G2期开始减弱，M期消失,已被公认为反映细胞增殖状态的标记物。本研究希望通过观察丙烯酰胺对生精细胞端粒酶活性以及PCNA的表达影响为进一步探讨丙烯酰胺的生殖毒性提供一定的科学依据。材料和方法： 1分组：健康雄性SD大鼠40只，体重150~200g，随机分为4组，低剂量组(20mg/kg)、中剂量组(40mg/kg)、高剂量组(60mg/kg)，阴性对照组(蒸馏水)。每日一次，每周连续应用5d，连续2周。 2染毒方法及检测指标：各组动物均采用经口灌胃染毒，灌胃体积为0.01ml/g。于首次染毒后第14d脱颈椎处死，取睾丸和附睾。采用免疫组化方法测定大鼠生精细胞中的端粒酶逆转录酶及PCNA的表达。 3各项指标的统计学处理结果： 1睾丸组织病理学检测结果：对照组睾丸生精上皮细胞排列规则，低剂量组睾丸生精上皮细胞排列基本规则，中、高剂量组睾丸生精上皮细胞层数减少，各级生精细胞均见减少，管腔内成熟精子减少，管腔变大，成熟精子数量减少，组织结构变得疏松。 2附睾中精子密度测定结果：大鼠附睾中的精子密度随着丙烯酰胺剂量的增加而降低，低、中、高剂量组均明显下降，且差异有统计学意义.（P㩳0.05） 3生精细胞端粒酶表达测定结果：对照组端粒酶逆转录酶（TERT）主要表达于精原细胞及精母细胞，表达较高。低剂量组大鼠与对照组比较端粒酶逆转录酶（TERT）表达有所减少，但阳性产物平均吸光度比较差异无统计学意义（P＞0.05）。中、高剂量组大鼠生精细胞TERT阳性产物明显减少，阳性产物平均吸光度与对照组比较差异有统计学意义（P＜0.05）。 4生精细胞PCNA表达测定结果：对照组增殖细胞核抗原（PCNA）活性主要表达于精原细胞及精母细胞，表达较高。低剂量组大鼠与对照组比较PCNA表达有所减少，但阳性产物平均吸光度比较差异无统计学意义（P＞0.05）。中、高剂量组大鼠生精细胞PCNA阳性产物明显减少，阳性产物平均吸光度与对照组比较差异有统计学意义（P＜0.05）。结论： 1丙烯酰胺可造成SD大鼠附睾中精子密度降低。 2丙烯酰胺可造成SD大鼠生精细胞TERT的表达降低。 3丙烯酰胺可造成SD大鼠生精细胞PCNA的表达降低。 4丙烯酰胺可能通过降低SD大鼠生精细胞TERT、PCNA的表达，造成生殖细胞增殖-凋亡平衡的破坏，进而引起精子的生成障碍，导致生殖毒性。
[Abstract]:Objective: Acrylamide AA is a white crystal chemical, which is a raw material for the production of polyacrylamide and is one of the environmental estrogens. It is widely used in the manufacture of water-soluble and organic chemicals. In daily life, at high temperature (120 degrees C), the starch foods are easy to produce acrylamide. Human body can be used in the daily life. A variety of ways to absorb acrylamide, such as the digestive tract, respiratory tract and skin and mucous membrane, such as [1], the digestive tract is the fastest. The fetus and infant can also absorb acrylamide through the placenta and milk. Early in 1980s, scientists have fully understood the toxicity of acrylamide. Sleep, memory loss, insomnia, hallucination, and so on. Acute poisoning can lead to delayed peripheral nerve changes, such as gait instability, glove change, and weakness. As Swedish scientists reported the discovery of acrylamide in high temperature starchy foods for the first time in April 2002, acrylamide again caused attention to scientists all over the world. In addition to the neurotoxicity mentioned above, there are carcinogenicity, embryogenesis toxicity and reproductive toxicity of [2]. acrylamide, which has been included in the 2A group (IARC) for the reproductive toxicity of the "human possible carcinogenic substance" [3]. acrylamide, and some people think that propanamide may induce chromosomal processes in germ cells. It is also considered that acrylamide may affect the gene expression of the testicles, the transshipment of steroid hormones and the occurrence of abnormal sperm, and acrylamide also reduces the content of testosterone in the body. Although scientists have put forward a lot of hypotheses about the reproductive toxicity of acrylamide, it is still unable to describe its hair completely. Therefore, the toxicological study of acrylamide toxicity remains to be further elucidated.
Telomerase is a ribonucleoprotein reverse transcriptase, which can synthesize the DNA sequence of the terminal telomere of chromosomes with its own endogenous RNA sequence. Telomerase activity is closely related to cell proliferation potential, cell cycle, cell differentiation, apoptosis and death. In most of the human cells, there is no telomerase activity, but it is dry and thin. Telomerase activity can be detected in cells, germ cells and 90% tumor cells. Therefore, telomerase has been widely used as an indicator of cell proliferation. In reproductive cells, telomerase activity ensures that the DNA sequence of telomere is consistently preserved, so as to ensure the orderly, stable division and inheritance of DNA in the germ cells. PCNA, a DNA polymerase delta protein, is necessary in the process of DNA replication. It has the function of maintaining the high activity of DNA polymerase, appearing in the early G1, the peak of the S phase, the weakening of the G2 phase and the disappearance of the M phase, which have been recognized as a marker to reflect the cell proliferation state. The effects of enzyme activity and PCNA expression provide a scientific basis for further study on the reproductive toxicity of acrylamide.
Materials and methods:
1 groups: 40 healthy male SD rats and weight 150~200g were randomly divided into 4 groups, low dose group (20mg/kg), middle dose group (40mg/kg), high dose group (60mg/kg), negative control group (distilled water). Once a day, 5D was used continuously every week for 2 weeks.
2 the methods of exposure and detection: all the animals were treated with oral administration of the stomach. The volume of the gavage was 0.01ml/g. after the first exposure to 14d, and the testicle and epididymis were taken. The expression of telomerase reverse transcriptase and PCNA in the spermatogenic cells of rats was determined by immunohistochemical method.
3 statistical processing of various indexes
Result:
1 the pathological examination results of testicular histology: the testicular spermatogenic epithelial cells in the control group arranged rules, the testis epithelial cells in the low dose group arranged the basic rules, in the high dose group, the number of spermatogenic epithelial cells in the high dose group decreased, the spermatogenic cells at all levels decreased, the mature sperm in the lumen decreased, the lumen became larger, the number of mature spermatozoa decreased, and the tissue knot was reduced. The structure becomes loose.
2 sperm density in the epididymis: the sperm density in the epididymis of the rat decreased with the increase of the dosage of acrylamide. The low, middle, high dose group decreased obviously, and the difference was statistically significant. (P? 0.05)
Telomerase expression in 3 spermatogonial cells: telomerase reverse transcriptase (TERT) was mainly expressed in spermatogonial cells and spermatocytes. The expression of telomerase reverse transcriptase (TERT) in the low dose group was less than that in the control group, but there was no significant difference in the average absorbance difference between the positive products (P > 0.05). The TERT positive products in spermatogenic cells of the rats in the dose group were significantly reduced, and the average absorbance of the positive products was significantly different from that of the control group (P < 0.05).
The results of PCNA expression of 4 spermatogonial cells: the activity of proliferating cell nuclear antigen (PCNA) in the control group was mainly expressed in spermatogonial cells and spermatocytes. The expression of PCNA in the low dose group was less than that in the control group, but the difference of the average absorbance of the positive products was not statistically significant (P > 0.05). The PCNA positive products decreased significantly, and the average absorbance of the positive products was significantly different from that of the control group (P < 0.05).
Conclusion:
1 acrylamide can cause spermatozoa density decrease in epididymis of SD rats.
2 acrylamide can reduce the expression of TERT in spermatogenic cells of SD rats.
3 acrylamide can reduce the expression of PCNA in spermatogenic cells of SD rats.
4 acrylamide may be induced by reducing the expression of TERT and PCNA in the spermatogenic cells of SD rats, resulting in the destruction of the reproductive cell proliferation and apoptosis balance, thus causing the disturbance of spermatogenesis and causing reproductive toxicity.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】