本文選題：丙烯酰胺 + 生殖毒性��； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的：丙烯酰胺（Acrylamide AA）是一種白色晶體化學(xué)物質(zhì)，，為生產(chǎn)聚丙烯酰胺的原料，屬于環(huán)境雌激素的一種，廣泛應(yīng)用于水溶性聚合物和有機(jī)化學(xué)藥品的制造。日常生活中，高溫（120℃）烹調(diào)下，淀粉類食品很容易產(chǎn)生丙烯酰胺。人體可通過多種途徑吸收丙烯酰胺，如消化道，呼吸道及皮膚黏膜等[1]，其中消化道吸收最快。胎兒和嬰幼兒通過胎盤和乳汁也可吸收丙烯酰胺。早在20世紀(jì)80年代，科學(xué)家就已經(jīng)對丙烯酰胺的毒性有了充分的認(rèn)識，丙烯酰胺可引起神經(jīng)毒性，引起嗜睡、記憶減退、失眠、幻覺等，急性中毒可出現(xiàn)遲發(fā)性的周圍神經(jīng)改變，如步態(tài)不穩(wěn)、手套樣改變、無力等。隨著2002年4月，瑞典科學(xué)家首次報(bào)道高溫加熱淀粉類食品中發(fā)現(xiàn)丙烯酰胺后，丙烯酰胺再一次引起了全世界各國科學(xué)家的關(guān)注。丙烯酰胺除上述神經(jīng)毒性外，還存在致癌性、胚胎發(fā)育毒性以及生殖毒性[2]。丙烯酰胺已被國際癌癥研究機(jī)構(gòu)（IARC）列入2A組“人類可能致癌物質(zhì)”[3]。丙烯酰胺的生殖毒性體現(xiàn)在許多方面，有人認(rèn)為丙烯酰胺可能會誘導(dǎo)生殖細(xì)胞發(fā)生染色體突變；也有人研究認(rèn)為，丙烯酰胺可能會影響睪丸的基因表達(dá)，致使類固醇激素的轉(zhuǎn)運(yùn)以及精子的發(fā)生出現(xiàn)異常，同時(shí)，丙烯酰胺也會降低體內(nèi)的睪酮含量。雖然科學(xué)家對丙烯酰胺的生殖毒性提出了很多假設(shè)，但仍然不能完整的描述其發(fā)生機(jī)制，因此，丙烯酰胺的生殖毒性毒理研究仍有待進(jìn)一步的闡明。 端粒酶是一種核糖核蛋白逆轉(zhuǎn)錄酶，能以自身的內(nèi)源性RNA序列作為模板從頭合成染色體末端端粒的DNA序列。端粒酶活性與細(xì)胞的增殖潛能、細(xì)胞周期、細(xì)胞分化、凋亡和死亡息息相關(guān)。在人體的大多數(shù)細(xì)胞中并沒有端粒酶活性，但在干細(xì)胞、生殖細(xì)胞以及90%的腫瘤細(xì)胞中都可檢測到端粒酶活性，因此，端粒酶已作為一種細(xì)胞增殖的指標(biāo)被廣泛應(yīng)用。在生殖細(xì)胞當(dāng)中，端粒酶活性保證端粒的DNA序列始終如一的保存，這樣才能保證生殖細(xì)胞中DNA有序、穩(wěn)定的分裂并遺傳至下一代。作為DNA多聚酶δ輔助蛋白的PCNA是DNA復(fù)制過程中所必需的，具有維持DNA多聚酶高活性的作用，出現(xiàn)于G1早期，S期達(dá)到高峰，G2期開始減弱，M期消失,已被公認(rèn)為反映細(xì)胞增殖狀態(tài)的標(biāo)記物。本研究希望通過觀察丙烯酰胺對生精細(xì)胞端粒酶活性以及PCNA的表達(dá)影響為進(jìn)一步探討丙烯酰胺的生殖毒性提供一定的科學(xué)依據(jù)。 材料和方法： 1分組：健康雄性SD大鼠40只，體重150~200g，隨機(jī)分為4組，低劑量組(20mg/kg)、中劑量組(40mg/kg)、高劑量組(60mg/kg)，陰性對照組(蒸餾水)。每日一次，每周連續(xù)應(yīng)用5d，連續(xù)2周。 2染毒方法及檢測指標(biāo)：各組動物均采用經(jīng)口灌胃染毒，灌胃體積為0.01ml/g。于首次染毒后第14d脫頸椎處死，取睪丸和附睪。采用免疫組化方法測定大鼠生精細(xì)胞中的端粒酶逆轉(zhuǎn)錄酶及PCNA的表達(dá)。 3各項(xiàng)指標(biāo)的統(tǒng)計(jì)學(xué)處理 結(jié)果： 1睪丸組織病理學(xué)檢測結(jié)果：對照組睪丸生精上皮細(xì)胞排列規(guī)則，低劑量組睪丸生精上皮細(xì)胞排列基本規(guī)則，中、高劑量組睪丸生精上皮細(xì)胞層數(shù)減少，各級生精細(xì)胞均見減少，管腔內(nèi)成熟精子減少，管腔變大，成熟精子數(shù)量減少，組織結(jié)構(gòu)變得疏松。 2附睪中精子密度測定結(jié)果：大鼠附睪中的精子密度隨著丙烯酰胺劑量的增加而降低，低、中、高劑量組均明顯下降，且差異有統(tǒng)計(jì)學(xué)意義.（P㩳0.05） 3生精細(xì)胞端粒酶表達(dá)測定結(jié)果：對照組端粒酶逆轉(zhuǎn)錄酶（TERT）主要表達(dá)于精原細(xì)胞及精母細(xì)胞，表達(dá)較高。低劑量組大鼠與對照組比較端粒酶逆轉(zhuǎn)錄酶（TERT）表達(dá)有所減少，但陽性產(chǎn)物平均吸光度比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。中、高劑量組大鼠生精細(xì)胞TERT陽性產(chǎn)物明顯減少，陽性產(chǎn)物平均吸光度與對照組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 4生精細(xì)胞PCNA表達(dá)測定結(jié)果：對照組增殖細(xì)胞核抗原（PCNA）活性主要表達(dá)于精原細(xì)胞及精母細(xì)胞，表達(dá)較高。低劑量組大鼠與對照組比較PCNA表達(dá)有所減少，但陽性產(chǎn)物平均吸光度比較差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。中、高劑量組大鼠生精細(xì)胞PCNA陽性產(chǎn)物明顯減少，陽性產(chǎn)物平均吸光度與對照組比較差異有統(tǒng)計(jì)學(xué)意義（P＜0.05）。 結(jié)論： 1丙烯酰胺可造成SD大鼠附睪中精子密度降低。 2丙烯酰胺可造成SD大鼠生精細(xì)胞TERT的表達(dá)降低。 3丙烯酰胺可造成SD大鼠生精細(xì)胞PCNA的表達(dá)降低。 4丙烯酰胺可能通過降低SD大鼠生精細(xì)胞TERT、PCNA的表達(dá)，造成生殖細(xì)胞增殖-凋亡平衡的破壞，進(jìn)而引起精子的生成障礙，導(dǎo)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R114

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