本文選題：羧基化多壁碳納米管 + 大鼠　； 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：[目的]本文采用QRT-PCR,透射電鏡,組織病理學(xué)等方法,綜合分析羧基化多壁碳納米管對(duì)雄性大鼠睪丸組織中MAPKs蛋白在基因水平上的影響,從而揭示羧基化多壁碳納米管不同劑量暴露對(duì)大鼠生殖系統(tǒng)的毒理效應(yīng)。[方法]試驗(yàn)前期通過(guò)TEM、SEM、紅外線和拉曼光譜等技術(shù)對(duì)羧基化修飾多壁碳納米管進(jìn)行表征檢測(cè)。建立試驗(yàn)大鼠模型,將40只SD雄性大鼠隨機(jī)分成4組：對(duì)照組、低劑量組、中劑量組和高劑量組。自由飲水和采食,每隔4天稱重。具體劑量為：配制含0.5%Tween-20的PBS緩沖液,對(duì)照組給予不含MWCNT-COOH的緩沖液,低、中、高劑量組分別給予濃度為2.5,5,10mg/kg MWCNT-COOH的緩沖液。灌胃期間觀察并記錄各小鼠的生長(zhǎng)狀況。連續(xù)灌胃64天后,剖殺大鼠,取附睪、精子、睪丸進(jìn)行相關(guān)試驗(yàn)；通過(guò)光鏡、TEM觀察睪丸生精小管形態(tài)、結(jié)構(gòu)及超微結(jié)構(gòu),重點(diǎn)觀察各級(jí)生精細(xì)胞的變化及納米材料的存在方式;并采用熒光定量PCR檢測(cè)MAPKs信號(hào)通路(ERK通路,JNK通路和P38通路)30個(gè)相關(guān)基因的mRNA表達(dá)水平,綜合評(píng)估MWCNT-COOH對(duì)雄性大鼠的生殖影響。[結(jié)果]1.在60-64天間,各MWCNT-COOH暴露組大鼠體重有所下降,但與對(duì)照組相比均無(wú)顯著差異。各劑量組附睪重和睪丸重與對(duì)照組相比差異均不顯著。2.與對(duì)照組相比,各試驗(yàn)組大鼠精子密度呈下降趨勢(shì),且與對(duì)照組相比,10mg/kg組差異顯著。各試驗(yàn)組大鼠精子畸形率呈上升趨勢(shì),且與對(duì)照組相比,10mg/kg組差異顯著。與對(duì)照組相比,各試驗(yàn)組大鼠精子存活率呈下降趨勢(shì),其中2.5mg/kg和5mg/kg組差異顯著(P0.05),10mg/kg組差異非常顯著(P0.001)。3.睪丸HE染色結(jié)果顯示,對(duì)照組大鼠睪丸間質(zhì)細(xì)胞排列整齊,細(xì)胞核圓,胞質(zhì)紅染;曲細(xì)精管管壁結(jié)構(gòu)完整,支持細(xì)胞和各級(jí)生精細(xì)胞層次清晰,排列整齊,且管腔內(nèi)可見(jiàn)大量成熟精子。與對(duì)照組相比,試驗(yàn)組大鼠睪丸組織中均看見(jiàn)生精小管、支持細(xì)胞、各級(jí)生精細(xì)胞不同程度的損傷。且2.5mg/kg組,精原細(xì)胞數(shù)量減少,精子落入管腔；5mg/kg組和10mg/kg組生精細(xì)胞數(shù)量減少且排列不整齊,生精小管有一定擴(kuò)張和膨脹且排列松散,生精小管管腔內(nèi)精子數(shù)量減少。利用透射電鏡觀察顯示,5mg/kg組和10mg/kg組細(xì)胞中均出現(xiàn)了線粒體空泡化、核膜受損及納米管在細(xì)胞中聚集的現(xiàn)象。4.應(yīng)用QRT-PCR對(duì)大鼠睪丸組織中MAPKs通路上下游相關(guān)基因的mRNA表達(dá)量進(jìn)行分析。結(jié)果顯示,ERK通路中的ERK2和ERK5有一定變化趨勢(shì),但與對(duì)照組相比差異不顯著,而ERK1呈下降趨勢(shì),且差異顯著。JNK通路中JNK1和JNK3的mRNA表達(dá)均呈下降趨勢(shì),且與對(duì)照組相比,差異顯著;JNK2的mRNA表達(dá)呈上升趨勢(shì),且差異極顯著。而JNK通路上游的CASP、TRAF2、 MEKK1、MLK3、MKK4和MKK7的mRNA表達(dá)均呈下降趨勢(shì),且與對(duì)照組相比,TRAF2和MKK7的mRNA表達(dá)差異極顯著,剩余基因mRNA表達(dá)差異顯著。.JNK通路下游的EIK-1和JunD的mRNA表達(dá)均呈下降趨勢(shì),且與對(duì)照組相比,差異顯著；c-Jun和ATF-2的mRNA表達(dá)趨勢(shì)不穩(wěn)定,且與對(duì)照組相比,c-Jun差異非常顯著,ATF-2無(wú)顯著差異。P38通路中P38的mRNA表達(dá)呈下降趨勢(shì),且與對(duì)照組相比,差異顯著。而P38通路上游的DAXX、ASK1、MKK3和MKK6的mRNA表達(dá)均呈下降趨勢(shì),且與對(duì)照組相比,DAXX差異極顯著;ASK1、MKK3和MKK6無(wú)顯著差異。P38通路下游的Sapla、 Max、MEF2C和P53均呈下降趨勢(shì),GADD153呈上升趨勢(shì),且與對(duì)照組相比,Sapla和GADD153差異顯著;MEF2C和P53差異極顯著;Max無(wú)顯著差異。而與睪丸炎癥相關(guān)的細(xì)胞因子TNF-α、IL-1β、FAS和TGF-β的mRNA表達(dá)與對(duì)照組相比差異均不顯著。[結(jié)論]不同劑量MWCNT-COOH引起大鼠睪丸組織不同程度的損傷,通過(guò)基因結(jié)果發(fā)現(xiàn),MWCNT-COOH可能主要通過(guò)影響睪丸組織MAPKs通路中的P38和JNK信號(hào)通路而對(duì)生殖產(chǎn)生影響。
[Abstract]:[Objective] the effects of carboxyl multi wall carbon nanotubes on the gene level of MAPKs in the testis of male rats were synthetically analyzed by QRT-PCR, transmission electron microscopy and histopathology. The toxicological effects of different doses of carboxyl multi wall carbon nanotubes on the reproductive system of rats were revealed. [Methods] through TEM, S EM, infrared and Raman spectroscopy were used to characterize the carboxyl modified multi walled carbon nanotubes. The experimental rat model was established and 40 SD male rats were randomly divided into 4 groups: the control group, the low dose group, the middle dose group and the high dose group. The free drinking water and the food intake were weighed every 4 days. The specific dose was the preparation of the PBS buffer containing 0.5%Tween-20. In the control group, the control group was given a buffer without MWCNT-COOH. The group was given a buffer solution of 2.5,5,10mg/kg MWCNT-COOH in the low, middle and high dose groups. During the gavage, the mice were observed and recorded. After 64 days of continuous gavage, the rats were killed and the epididymis, spermatozoa and testicles were tested. The testicular seminiferous tubules were observed by light microscopy and TEM. State, structure and ultrastructure, focusing on the changes in spermatogenic cells at all levels and the existence of nanomaterials, and using fluorescence quantitative PCR to detect the mRNA expression levels of 30 related genes in the MAPKs signaling pathway (ERK pathway, JNK pathway and P38 pathway), and to evaluate the reproductive effects of MWCNT-COOH on male rats. [results]1. at 60-64 days, MWCNT The body weight of -COOH exposed rats decreased, but there was no significant difference compared with the control group. Compared with the control group, the difference of epididymal weight and testicular weight in each group was not significant compared with the control group. Compared with the control group, the sperm density decreased in the experimental group, and the difference in the 10mg/kg group was significant compared with the control group. The sperm malformation rate of the rats in the experimental group was present. Compared with the control group, the difference of the 10mg/kg group was significant. Compared with the control group, the sperm survival rate of the rats in each test group showed a decreasing trend, in which the difference of 2.5mg/kg and 5mg/kg groups was significant (P0.05). The difference of 10mg/kg group was very significant (P0.001).3. testis HE staining results showed that the Leydig cells of the testicular cells in the control group were arranged neatly and the nucleus round, The wall structure of the tubules was complete. The support cells and all levels of spermatogenic cells were clear and orderly, and a large number of mature spermatozoa were seen in the tube. Compared with the control group, the spermatogenic tubules were seen in the testis tissue of the experimental group, and the cells were damaged in different degrees. And the number of spermatogonial cells in group 2.5mg/kg and spermatogonial cells. The sperm fall into the lumen; the number of spermatogenic cells in the 5mg/kg and 10mg/kg groups is reduced and the arrangement is irregular. The spermatogenic tubule is dilated and expanded and the number of spermatozoa is loosely arranged. The number of spermatozoa in the cavities of the spermatogenic tubule is reduced. By transmission electron microscopy, the mitochondria vacuolization, the damage of nuclear membrane and the nanoscale in the 5mg/kg and 10mg/kg groups have been observed. The phenomenon of the aggregation of tube in the cell.4. was used to analyze the mRNA expression of the upstream and downstream related genes in the MAPKs pathway of the rat testicular tissue. The results showed that the ERK2 and ERK5 in the ERK pathway had a certain trend of change, but the difference was not significant compared with the control group, but the ERK1 showed a decreasing trend, and the difference of JNK1 and JNK3 mRNA in the.JNK pathway was significant. The expression of mRNA was significantly different from the control group, and the expression of JNK2 was on the rise, and the difference was very significant. The expressions of CASP, TRAF2, MEKK1, MLK3, MKK4 and MKK7 in the upstream of the JNK pathway were all decreasing, and the difference of the expression of TRAF2 and MKK7 was significant compared with the control group, and the difference of the remaining gene expression was significant. The mRNA expression of EIK-1 and JunD in the downstream of the NK pathway decreased, and the difference was significant compared with the control group. The mRNA expression trend of c-Jun and ATF-2 was unstable, and the c-Jun difference was very significant compared with the control group, and ATF-2 had no significant difference in the.P38 pathway, and the P38 mRNA expression in the.P38 pathway was decreasing, and the difference was significant compared with the control group. The mRNA expression of the upstream DAXX, ASK1, MKK3 and MKK6 showed a downward trend, and the DAXX difference was very significant compared with the control group; ASK1, MKK3 and MKK6 had no significant difference in Sapla in the downstream of the.P38 pathway. There was no significant difference. The mRNA expression of cytokine TNF- alpha, IL-1 beta, FAS and TGF- beta associated with orchitis was not significantly different from that of the control group. [Conclusion] different doses of MWCNT-COOH caused different degrees of damage to the testicular tissue of rats. Through the gene results, MWCNT-COOH could be mainly affected by P38 in the MAPKs pathway of testicular tissue. And the effect of JNK signaling pathway on reproduction.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.91;X503.22

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王森;楊婉身;劉陽(yáng);杜宇;;納米技術(shù)在動(dòng)物免疫和疾病檢測(cè)中的應(yīng)用[J];中國(guó)畜牧獸醫(yī);2007年10期

相關(guān)重要報(bào)紙文章 前1條

1 郭良宏 中國(guó)科學(xué)院生態(tài)環(huán)境研究中心研究員 江桂斌 中國(guó)科學(xué)院院士;[N];中國(guó)社會(huì)科學(xué)報(bào);2010年

相關(guān)碩士學(xué)位論文 前2條

1 賈芳;納米二氧化鈦對(duì)青春期小鼠睪酮生成的影響及其機(jī)制研究[D];山西農(nóng)業(yè)大學(xué);2013年

2 高迪;口服暴露碳納米管對(duì)小鼠胃腸道的影響[D];吉林大學(xué);2014年

，

本文編號(hào)：1932031