孔雀石绿对成年小鼠睾丸结构与功能的影响
发布时间：2018-09-01 18:15
【摘要】： 目的 观察孔雀石绿(malachite green，MG)急性和慢性染毒对雄性成年小鼠睾丸组织学结构及超微结构的影响，以了解MG对小鼠睾丸组织及细胞的损伤特点；研究MG对睾丸内凋亡诱导蛋白Fas／Fas-L表达的影响，以明确MG对小鼠睾丸生精上皮的损伤特点；分析染毒后睾丸基因组随机扩增多态性DNA分子标记扩增图谱的变化，以揭示MG对睾丸遗传物质的损伤。上述研究有助于深入地了解MG对雄性生殖系统的损伤及其特点，增加环境毒物与生殖健康相互关系的认识，为雄(男)性不育症的病因学提供参考资料。 材料与方法 1．实验动物及分组 实验动物为6-8周龄的雄性BALB／c清洁级小鼠，体重18-22g。用于急性实验的小鼠共32只，随机均分为4组，按照MG浓度设为：A组(0 mg／L)、B组(100 mg／L)、C组(400 mg／L)、D组(800 mg／L)；用于慢性实验的小鼠共16只，随机均分为4组后按照MG浓度设为A’组(0 mg／L)、B’组(100 mg／L)、C’组(300 mg／L)、D’组(600 mg／L)。急性实验组连续灌胃给药30d，慢性实验组灌胃给药持续90d，急、慢性实验组中的MG均溶于生理盐水，A和A’组则以等量生理盐水代替MG。 2．检测方法 各实验组均检测小鼠体重、睾丸及附睾重量；应用HE染色及透射电镜观察睾丸组织结构及超微结构的变化；用免疫组化法检测凋亡诱导蛋白Fas／Fas-L的表达部位及表达量的改变；用随机扩增多态性DNA分子标记对睾丸基因组进行PCR扩增，通过比较扩增图谱的差异来鉴定睾丸基因组的损伤。 结果 1．小鼠体重、睾丸与附睾重量的变化 急、慢性染毒后，各实验组小鼠体重均比对照组减轻，D和D’组的体重减轻具显著性(P＜0.05)；各实验组小鼠睾丸重量均比对照组有所降低，其中C’、D、D’组的睾丸重量降低具显著性(P＜0.05，P＜0.01)；附睾重量仅在D’组明显降低(P＜0.01)，在其它组无显著变化(P＞0.05)。 2．小鼠睾丸组织学结构的变化 急、慢性染毒均能造成睾丸的组织的病理学改变，急性染毒可使生精上皮细胞层数减少、生精细胞排列紊乱或脱落入管腔、精子生成减少等。慢性实验组损伤比急性实验组更加严重，随剂量增加，急、慢性实验组的病理变化均更为明显。 3．小鼠睾丸超微结构的改变 各实验组小鼠的超微结构均与对照组的有明显差异，可见基膜皱褶，精原细胞和支持细胞胞浆空泡化，支持细胞与生精细胞间连接消失，线粒体肿胀、空泡化，精子形态异常等。慢性实验组变化比急性实验组更加明显，而急、慢性实验组的病理变化都随剂量增大而加重。 4．小鼠睾丸内Fas／Fas-L的表达 Fas的阳性表达集中于支持细胞胞浆以及精子和其残余胞浆，Fas-L的阳性表达定位于支持细胞的胞浆及管腔中集合在一起的絮状精子尾。Fas在除B组外的其它各组精子及支持细胞的表达均比对照组显著增强(P＜0.05)；Fas-L在各实验组支持细胞的表达均显著强于对照组(P＜0.05)，而其在精子尾的表达则无明显变化(P＞0.05)。各实验组支持细胞内Fas／Fas-L的表达水平均随剂量增加而升高，急、慢性实验组间无明显差别。 5．小鼠睾丸基因组RAPD分子标记的改变 各实验组睾丸基因组DNA的RAPD分子标记扩增图谱发生改变，包括扩增条带明亮的变化，扩增条带数目的改变和差异条带的产生。各实验组与对照组的差异条带的比例随染毒剂量增大而增大，急、慢性实验组间无明显差别。 结论 1．MG对小鼠的急、慢性染毒均能导致小鼠体重、睾丸重量的减轻，且减轻程度随剂量增大而加大。提示MG能够对睾丸造成损伤，且损伤呈剂量效应。 2．MG的急、慢性染毒均能造成睾丸的组织学结构及超微结构的病理损伤，慢性染毒组的病理损伤更严重，且急、慢性染毒后睾丸的损伤程度都随剂量增加而加重。提示MG能够对睾丸内多种细胞造成损伤，且损伤呈剂量效应，，长期低剂量的MG染毒比短期高剂量的染毒更能导致睾丸损伤。 3．MG急、慢性染毒均可诱导睾丸内的凋亡蛋白Fas／Fas-L在支持细胞的表达上调，且上调水平随剂量增加而升高。提示MG可能通过Fas／Fas-L通路使生精细胞凋亡增加，从而对睾丸造成损伤，使精子生成减少，生育力降低。 4．MG急、慢性染毒均使睾丸基因组DNA的RAPD分子标记扩增图谱改变，提示MG能对睾丸基因组DNA造成损伤，DNA的损伤可进一步诱发睾丸癌，或者精子DNA的损伤致使受精后的胚胎异常。
[Abstract]:objective
To observe the effects of malachite green (MG) on histology and ultrastructure of testis in male adult mice, to understand the damage characteristics of MG on testis tissue and cells, to study the effect of MG on the expression of apoptosis-inducing protein Fas/Fas-L in testis, and to clarify the damage of MG on spermatogenic epithelium of testis in mice. The above research will help us to understand the damage of MG to male reproductive system and its characteristics, and increase the understanding of the relationship between environmental toxicity and reproductive health, so as to be male (male) infertility. Provide references for etiology.
Materials and methods
1. experimental animals and groups
Thirty-two male BALB/c clean-grade mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into four groups according to MG concentration: group A (0 mg/L), group B (100 mg/L), group C (400 mg/L), and group D (800 mg/L); and 16 mice for chronic experiment were randomly divided into four groups and then set to A'according to MG concentration. Group A (100 mg / L), group B (100 mg / L), group C (300 mg / L) and group D (600 mg / L). The acute group was given continuous intragastric administration for 30 days, the chronic group for 90 days, the acute group and the chronic group were all dissolved in normal saline, and group A and A were given the same amount of normal saline instead of MG.
2. detection methods
The weight of mice, testis and epididymis were measured in each experimental group; the histological and ultrastructural changes of testis were observed by HE staining and transmission electron microscopy; the expression of apoptosis-inducing protein Fas/Fas-L was detected by immunohistochemistry; the genome of testis was amplified by random amplified polymorphic DNA molecular markers by PCR The difference in amplification map was used to identify testicular genome damage.
Result
1. body weight, testicular and epididymal weight in mice
After acute and chronic exposure, the weight of mice in each experimental group was lower than that in the control group, and the weight of mice in D and D'groups was significantly lower than that in the control group (P < 0.05). There was no significant change in other groups (P > 0.05).
2. histological changes of testis in mice
Acute and chronic exposure can cause pathological changes in testis. Acute exposure can reduce the number of spermatogenic epithelial cells, disorder of spermatogenic cells arrangement or fall into the lumen, and decrease spermatogenesis.
3. changes in the ultrastructure of testis in mice
The ultrastructure of the mice in each experimental group was obviously different from that in the control group. There were basement membrane folds, vacuolation of spermatogonia and Sertoli cells, disappearance of junction between Sertoli cells and spermatogenic cells, mitochondrial swelling, vacuolation, abnormal sperm morphology and so on. Pathological changes were aggravated with the increase of dose.
4. expression of Fas / Fas-L in testis of mice
The positive expression of Fas was concentrated in the cytoplasm of Sertoli cells, sperm and their residual cytoplasm. The positive expression of Fas-L was located in the cytoplasm of Sertoli cells and the flocculent sperm tail in lumen. The expression of Fas in sperm and Sertoli cells of all groups except group B was significantly higher than that of control group (P < 0.05). The expression of Fas/Fas-L in Sertoli cells was significantly higher than that in control group (P < 0.05), but there was no significant change in the expression of Fas/Fas-L in sperm tail (P > 0.05).
5. the change of RAPD molecular marker in mouse testis genome
The RAPD molecular marker amplification profiles of testicular genomic DNA in each experimental group changed, including the brightness of amplified bands, the number of amplified bands and the generation of differential bands.
conclusion
1. Both acute and chronic exposure to MG can result in weight loss and testicular weight loss in mice, and the degree of reduction increases with the increase of dose.
2. Both acute and chronic exposure to MG can cause histological and ultrastructural pathological damage of the testis. The pathological damage of the chronic exposure group is more serious, and the degree of the damage of the testis is aggravated with the increase of dosage. Exposure to poison is more likely to cause testicular injury than short-term high-dose exposure.
3. Both acute and chronic exposure to MG can induce the expression of apoptotic protein Fas/Fas-L in Sertoli cells to be up-regulated, and the up-regulated level increases with the increase of dosage.
4. Both acute and chronic exposure to MG can alter the RAPD molecular marker amplification profiles of testicular genomic DNA, suggesting that MG can cause damage to testicular genomic DNA, DNA damage can further induce testicular cancer, or sperm DNA damage can cause abnormal embryos after fertilization.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R698.2;R363
本文编号：2217969
[Abstract]:objective
To observe the effects of malachite green (MG) on histology and ultrastructure of testis in male adult mice, to understand the damage characteristics of MG on testis tissue and cells, to study the effect of MG on the expression of apoptosis-inducing protein Fas/Fas-L in testis, and to clarify the damage of MG on spermatogenic epithelium of testis in mice. The above research will help us to understand the damage of MG to male reproductive system and its characteristics, and increase the understanding of the relationship between environmental toxicity and reproductive health, so as to be male (male) infertility. Provide references for etiology.
Materials and methods
1. experimental animals and groups
Thirty-two male BALB/c clean-grade mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into four groups according to MG concentration: group A (0 mg/L), group B (100 mg/L), group C (400 mg/L), and group D (800 mg/L); and 16 mice for chronic experiment were randomly divided into four groups and then set to A'according to MG concentration. Group A (100 mg / L), group B (100 mg / L), group C (300 mg / L) and group D (600 mg / L). The acute group was given continuous intragastric administration for 30 days, the chronic group for 90 days, the acute group and the chronic group were all dissolved in normal saline, and group A and A were given the same amount of normal saline instead of MG.
2. detection methods
The weight of mice, testis and epididymis were measured in each experimental group; the histological and ultrastructural changes of testis were observed by HE staining and transmission electron microscopy; the expression of apoptosis-inducing protein Fas/Fas-L was detected by immunohistochemistry; the genome of testis was amplified by random amplified polymorphic DNA molecular markers by PCR The difference in amplification map was used to identify testicular genome damage.
Result
1. body weight, testicular and epididymal weight in mice
After acute and chronic exposure, the weight of mice in each experimental group was lower than that in the control group, and the weight of mice in D and D'groups was significantly lower than that in the control group (P < 0.05). There was no significant change in other groups (P > 0.05).
2. histological changes of testis in mice
Acute and chronic exposure can cause pathological changes in testis. Acute exposure can reduce the number of spermatogenic epithelial cells, disorder of spermatogenic cells arrangement or fall into the lumen, and decrease spermatogenesis.
3. changes in the ultrastructure of testis in mice
The ultrastructure of the mice in each experimental group was obviously different from that in the control group. There were basement membrane folds, vacuolation of spermatogonia and Sertoli cells, disappearance of junction between Sertoli cells and spermatogenic cells, mitochondrial swelling, vacuolation, abnormal sperm morphology and so on. Pathological changes were aggravated with the increase of dose.
4. expression of Fas / Fas-L in testis of mice
The positive expression of Fas was concentrated in the cytoplasm of Sertoli cells, sperm and their residual cytoplasm. The positive expression of Fas-L was located in the cytoplasm of Sertoli cells and the flocculent sperm tail in lumen. The expression of Fas in sperm and Sertoli cells of all groups except group B was significantly higher than that of control group (P < 0.05). The expression of Fas/Fas-L in Sertoli cells was significantly higher than that in control group (P < 0.05), but there was no significant change in the expression of Fas/Fas-L in sperm tail (P > 0.05).
5. the change of RAPD molecular marker in mouse testis genome
The RAPD molecular marker amplification profiles of testicular genomic DNA in each experimental group changed, including the brightness of amplified bands, the number of amplified bands and the generation of differential bands.
conclusion
1. Both acute and chronic exposure to MG can result in weight loss and testicular weight loss in mice, and the degree of reduction increases with the increase of dose.
2. Both acute and chronic exposure to MG can cause histological and ultrastructural pathological damage of the testis. The pathological damage of the chronic exposure group is more serious, and the degree of the damage of the testis is aggravated with the increase of dosage. Exposure to poison is more likely to cause testicular injury than short-term high-dose exposure.
3. Both acute and chronic exposure to MG can induce the expression of apoptotic protein Fas/Fas-L in Sertoli cells to be up-regulated, and the up-regulated level increases with the increase of dosage.
4. Both acute and chronic exposure to MG can alter the RAPD molecular marker amplification profiles of testicular genomic DNA, suggesting that MG can cause damage to testicular genomic DNA, DNA damage can further induce testicular cancer, or sperm DNA damage can cause abnormal embryos after fertilization.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R698.2;R363
【参考文献】
相关期刊论文 前3条
1 周立红,陈学豪,徐长安;用微核技术研究孔雀石绿对鱼的诱变作用[J];集美大学学报(自然科学版);1997年02期
2 邱绪建;林洪;王联珠;江洁;王建华;;孔雀石绿及其代谢物无色孔雀石绿在鲫鱼肌肉中的代谢规律[J];中国海洋大学学报(自然科学版);2006年05期
3 黄英,柯才焕,周时强;几种药物对波部东风螺早期发育的影响[J];厦门大学学报(自然科学版);2001年03期
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