羧基化多壁碳纳米管对大鼠睾丸MAPKs通路的影响

发布时间：2018-05-25 04:13

本文选题：羧基化多壁碳纳米管 + 大鼠　；参考：《山西农业大学》2015年硕士论文

【摘要】：[目的]本文采用QRT-PCR,透射电镜,组织病理学等方法,综合分析羧基化多壁碳纳米管对雄性大鼠睾丸组织中MAPKs蛋白在基因水平上的影响,从而揭示羧基化多壁碳纳米管不同剂量暴露对大鼠生殖系统的毒理效应。[方法]试验前期通过TEM、SEM、红外线和拉曼光谱等技术对羧基化修饰多壁碳纳米管进行表征检测。建立试验大鼠模型,将40只SD雄性大鼠随机分成4组：对照组、低剂量组、中剂量组和高剂量组。自由饮水和采食,每隔4天称重。具体剂量为：配制含0.5%Tween-20的PBS缓冲液,对照组给予不含MWCNT-COOH的缓冲液,低、中、高剂量组分别给予浓度为2.5,5,10mg/kg MWCNT-COOH的缓冲液。灌胃期间观察并记录各小鼠的生长状况。连续灌胃64天后,剖杀大鼠,取附睾、精子、睾丸进行相关试验；通过光镜、TEM观察睾丸生精小管形态、结构及超微结构,重点观察各级生精细胞的变化及纳米材料的存在方式;并采用荧光定量PCR检测MAPKs信号通路(ERK通路,JNK通路和P38通路)30个相关基因的mRNA表达水平,综合评估MWCNT-COOH对雄性大鼠的生殖影响。[结果]1.在60-64天间,各MWCNT-COOH暴露组大鼠体重有所下降,但与对照组相比均无显著差异。各剂量组附睾重和睾丸重与对照组相比差异均不显著。2.与对照组相比,各试验组大鼠精子密度呈下降趋势,且与对照组相比,10mg/kg组差异显著。各试验组大鼠精子畸形率呈上升趋势,且与对照组相比,10mg/kg组差异显著。与对照组相比,各试验组大鼠精子存活率呈下降趋势,其中2.5mg/kg和5mg/kg组差异显著(P0.05),10mg/kg组差异非常显著(P0.001)。3.睾丸HE染色结果显示,对照组大鼠睾丸间质细胞排列整齐,细胞核圆,胞质红染;曲细精管管壁结构完整,支持细胞和各级生精细胞层次清晰,排列整齐,且管腔内可见大量成熟精子。与对照组相比,试验组大鼠睾丸组织中均看见生精小管、支持细胞、各级生精细胞不同程度的损伤。且2.5mg/kg组,精原细胞数量减少,精子落入管腔；5mg/kg组和10mg/kg组生精细胞数量减少且排列不整齐,生精小管有一定扩张和膨胀且排列松散,生精小管管腔内精子数量减少。利用透射电镜观察显示,5mg/kg组和10mg/kg组细胞中均出现了线粒体空泡化、核膜受损及纳米管在细胞中聚集的现象。4.应用QRT-PCR对大鼠睾丸组织中MAPKs通路上下游相关基因的mRNA表达量进行分析。结果显示,ERK通路中的ERK2和ERK5有一定变化趋势,但与对照组相比差异不显著,而ERK1呈下降趋势,且差异显著。JNK通路中JNK1和JNK3的mRNA表达均呈下降趋势,且与对照组相比,差异显著;JNK2的mRNA表达呈上升趋势,且差异极显著。而JNK通路上游的CASP、TRAF2、 MEKK1、MLK3、MKK4和MKK7的mRNA表达均呈下降趋势,且与对照组相比,TRAF2和MKK7的mRNA表达差异极显著,剩余基因mRNA表达差异显著。.JNK通路下游的EIK-1和JunD的mRNA表达均呈下降趋势,且与对照组相比,差异显著；c-Jun和ATF-2的mRNA表达趋势不稳定,且与对照组相比,c-Jun差异非常显著,ATF-2无显著差异。P38通路中P38的mRNA表达呈下降趋势,且与对照组相比,差异显著。而P38通路上游的DAXX、ASK1、MKK3和MKK6的mRNA表达均呈下降趋势,且与对照组相比,DAXX差异极显著;ASK1、MKK3和MKK6无显著差异。P38通路下游的Sapla、 Max、MEF2C和P53均呈下降趋势,GADD153呈上升趋势,且与对照组相比,Sapla和GADD153差异显著;MEF2C和P53差异极显著;Max无显著差异。而与睾丸炎症相关的细胞因子TNF-α、IL-1β、FAS和TGF-β的mRNA表达与对照组相比差异均不显著。[结论]不同剂量MWCNT-COOH引起大鼠睾丸组织不同程度的损伤,通过基因结果发现,MWCNT-COOH可能主要通过影响睾丸组织MAPKs通路中的P38和JNK信号通路而对生殖产生影响。
[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.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.91;X503.22

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