纳米二氧化钛对青春期小鼠睾酮生成的影响及其机制研究

发布时间：2018-06-03 04:48

本文选题：纳米二氧化钛 + 睾酮　；参考：《山西农业大学》2013年硕士论文

【摘要】：[目的]纳米二氧化钛作为一种遮光剂,在各种化妆品涂料中广泛应用。近来研究表明,纳米颗粒的摄入会导致人睾酮水平发生改变。本文利用RT-PCR, Western-blot,透射电镜,组织切片等方法,结合精子活率,畸形率,血清睾酮含量的变化等情况,综合分析纳米二氧化钛对青春期小鼠雄性生殖的影响,并对其可能机制进行研究。 [方法]4周龄雄性昆明小鼠40只,随机分为四组：对照组,低剂量组,中剂量组和高剂量组。自由饮水和采食,每天称重灌胃。具体剂量如下：配置含0.5%Tween80的PBS缓冲液。对照组灌服不含Nano-TiO2的缓冲液,低剂量组灌服含10mg/kgNano-TiO2的缓冲液,中剂量组灌服含50mg/kgNano-TiO2的缓冲液,高剂量组灌服含250mg/kgNano-TiO2的缓冲液。灌胃期间观察记录各组小鼠的活动,被毛及体重等变化情况。42天后,眼球采血,取睾丸组织分别进行RT-PCR, Western-blot,以及组织病理学切片等试验；并结合精子密度,畸形率等变化情况,综合评估纳米二氧化钛对小鼠生殖系统的影响。 [结果]1.与对照组相比,各纳米二氧化钛暴露组的小鼠体重均有所下降,高剂量组的体增重与对照组相比明显降低,差异显著。各攻毒组睾丸、附睾的脏器指数与对照组相比,无显著性差异。 2.各组小鼠精子密度相比,无显著性差异。与对照组相比,各实验组小鼠精子活率和血清睾酮呈降低趋势,中高剂量组差异显著。与此相反,各攻毒组小鼠畸形率与对照组相比有所上升,其中,中高剂量组差异显著。 3.睾丸组织病理学切片结果显示,对照组睾丸组织的曲细精管结构完整,各级生精细胞排列整齐,可见到大量的成熟精子。低剂量组精子细胞有所减少,曲细精管间隙增大。中高剂量组各级生精细胞明显减少,细胞排列不规则,管腔塌陷,曲细精管间有空泡形成。透射电镜结果显示：纳米二氧化钛可导致睾丸线粒体膨大,内部嵴消失。 4.应用RT-PCR对小鼠睾丸中StAR、P450-17α、17β-HSD、3p-HSD、Cyp19、P450scc、AR和GATA-4的nRNA表达水平分析得知：与对照组相比,三个剂量组睾丸GATA-4、17β-HSD的mRNA表达均显著降低,中、高剂量组睾丸P450-17a的mRNA表达显著降低；高剂量组Cyp19mRNA表达量显著升高；其它基因mRNA表达无显著性差异。 5.用Western-blot对各组小鼠睾丸组织的P450-17α、17β-HSD和Cyp19的蛋白表达量进行分析可知：与对照组相比,高剂量组P450-17a的蛋白表达量显著降低；中、高剂量组17β-HSD的蛋白表达量显著降低。与此相反,Cyp19的蛋白表达量则呈升高的趋势,且中、高剂量组与对照组相比,差异显著。 [结论]实验中,睾酮合成过程中的关键基因P450-17α、17β-HSD表达水平在基因和蛋白水平有不同程度的下降；同时,Cyp19的表达显著升高。说明纳米二氧化钛通过两方面的作用影响雄性小鼠的睾酮含量进而导致其生殖功能的下降：一方面,Nano-TiO2抑制了小鼠睾酮合成通路上P450-17a和17β-HSD基因表达来降低睾酮的合成；另一方面,Nano-TiO2刺激了小鼠睾丸中雌激素的生成,导致了雌雄激素比例失调。
[Abstract]:[Objective] nanoscale titanium dioxide is widely used in various cosmetic coatings as a kind of light shading agent. Recent studies have shown that the intake of nanoparticles will lead to changes in the level of human testosterone. This paper uses RT-PCR, Western-blot, transmission electron microscopy, tissue section and other methods, combined with the survival rate, deformity rate, and serum testosterone content, and so on. The effects of nano titanium dioxide on male reproduction in puberty mice were comprehensively analyzed and its possible mechanism was studied.
[Methods] 40 male Kunming mice were randomly divided into four groups: the control group, the low dose group, the middle dose group and the high dose group. The free drinking water and the high dose group were weighed every day. The specific dosage was as follows: the PBS buffer containing 0.5%Tween80 was assigned to the control group. The control group was filled with the gentle punching solution without Nano-TiO2, and the low dose group was given the buffer of 10mg/kgNano-TiO2 containing 10mg/kgNano-TiO2 In the medium dose group, the buffer solution containing 50mg/kgNano-TiO2 was perfused, and the high dose group was given the buffer solution containing 250mg/kgNano-TiO2. During the period of gavage, the activities of the mice were recorded and the eyeball was collected after.42 days after the changes of hair and weight. The testis tissues were taken RT-PCR, Western-blot, and histopathological sections respectively. The effects of nano titanium dioxide on reproductive system of mice were comprehensively evaluated.
[results compared with the control group, the weight of mice in the nanoscale titanium dioxide exposure group decreased and the body weight gain of the high dose group decreased significantly compared with the control group. There was no significant difference in the visceral index of the testis and epididymis of the attack group compared with the control group.
2. the sperm density of mice in each group had no significant difference. Compared with the control group, the sperm motility and serum testosterone of the mice in the experimental groups were decreased, and the difference in the middle and high dose groups was significant.
3. the pathological sections of the testicular tissue showed that the structure of the seminiferous tubules in the testicular tissue of the control group was complete, the spermatogenic cells at all levels were arranged neatly, and a large number of mature spermatozoa were found. The low dose group was reduced and the space of the fine tubule increased. The fine cells in the middle and high dose groups decreased obviously, the cell arrangement was irregular, the lumen collapsed, and the curve was collapsed. There was vacuolization between seminiferous tubules. Transmission electron microscopy showed that nano-TiO2 could cause testicular mitochondria to expand and internal cristae disappear.
4. the expression level of StAR, P450-17 alpha, 17 beta -HSD, 3p-HSD, Cyp19, P450scc, AR and GATA-4 in mice testis was analyzed. The expression of GATA-4,17 beta -HSD in the three doses group decreased significantly compared with the control group, and the expression of the testis in the high dose group was significantly lower than that in the control group. The expression of mRNA was not significantly different.
5. the protein expression of P450-17 alpha, 17 beta -HSD and Cyp19 in each group of mice was analyzed with Western-blot, and the protein expression of P450-17a in high dose group decreased significantly compared with the control group; in the high dose group, the protein expression of 17 beta -HSD decreased significantly. In contrast, the expression of Cyp19 protein showed an increasing trend. The difference between the high dose group and the control group was significant.
[Conclusion] in the experiment, the key gene P450-17 alpha and 17 beta -HSD in the process of testosterone synthesis decreased in varying degrees in genes and protein levels; meanwhile, the expression of Cyp19 was significantly increased. It indicated that the effect of nano titanium dioxide on the testosterone content of male mice and the decrease of reproductive function in male mice, on the one hand, was on the one hand. Nano-TiO2 inhibits the expression of P450-17a and 17 beta -HSD gene in the mouse testosterone synthesis pathway to reduce the synthesis of testosterone; on the other hand, Nano-TiO2 stimulates the formation of estrogen in the testis of mice and leads to the imbalance of male and female hormones.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【共引文献】