【作者】 王伟；

【导师】 钟秀会；

【作者基本信息】 河北农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 主要研究银杏叶提取物及其单体成分槲皮素对己烯雌酚（DES）致成年小鼠睾丸损伤的保护作用。通过用光镜观察睾丸组织的形态学改变,检测睾丸组织及体外培养的生精细胞培养液中超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性、乳酸脱氢酶（LDH）及丙二醛（MDA）含量,及用免疫组化的方法检测睾丸生精细胞增殖细胞抗原（PCNA）的表达等方法,探讨银杏叶提取物（EGb）及单体成分槲皮素（Que）保护睾丸的生精作用及可能的作用机制,寻找预防环境雌激素对雄性动物生殖系统损伤的措施。试验分为5组,A组为对照组,B组为DES损伤模型组,C-E组分别为不同银杏叶的药物预防组,EGb组采用腹腔注射的方法、Que组采用灌胃法,模型组和药物预防组均皮下给予DES 40 mg/kg·d,于给药7d后剖杀。结果表明各DES组小鼠的睾丸及精子密度显著下降（P＜0.01）,睾丸组织中SOD,GSH-Px活力随DES剂量增加而逐渐减少,与对照组差异显著（P＜0.05）;MDA含量升高,与对照组差异显著（P＜0.05）。光镜下DES组小鼠睾丸生精细胞出现细胞变性,核固缩,且损伤程度与DES剂量明显相关。DES对体外培养的成年仓鼠生精细胞也有直接的损伤作用,在浓度10^-7～10^-4M范围内,其损伤作用随浓度的增大而增大,随培养时间的延长,测得培养液中MDA和LDH含量均显著升高（P＜0.01,P＜0.05）。因此DES适合作为研究雌激素对成年雄性动物睾丸损伤的模型。预先给予不同银杏叶成分后,睾丸组织中SOD,GSH-Px活力增加,LDH活性和MDA含量减少,PCNA表达增加。其中以EGb组的效果最为明显,睾丸系数升高,差异显著;睾丸匀浆液中SOD、GSH-Px活性均高于DES模型组,有显著性差异（P＜0.01）;相反,LDH活性和MDA含量均低于DES组,有显著性差异（P＜0.01,P＜0.05）。免疫组化结果表明,DES组睾丸PCNA阳性细胞密度略有下降,但与对照组相比差异不显著;但应用中药后均升高,与DES模型组相比差异显著（P＜0.01,P＜0.05）。所以,DES可以引起成年雄性小鼠睾丸氧化损伤。EGb和Que均能够清除自由基,抑制脂质过氧化反应,可作为一种预防性的抗氧化剂减轻这种氧化损伤,可以提高DES引起的睾丸生精细胞PCNA表达水平,其保护作用可能与促进生精上皮的增殖有关。更多还原

【Abstract】 The study was carried out to evaluate the effects of the extract of gingko biloba（EGb）and its monomer components-Quecretin（Que）on testicle injuries induced by diethylstilbestrol in mouse testes.The pathological changes of the testicle tissue were observed under light microscope.The enzyme activities of SOD,LDH and GSH-Px,and the malonaldehyde （MDA）contents of the testes and spermatogenic cell culture medium in vitro culture were analyzed using commercial kits according to the manufacturer’s instructions.The expression of proliferating cell nuclear antigen（PCNA）were detected by immunohistochemical method.To protect the testis spermatogenic effects,explore the possible mechanism of EGb and Que,and look for the prevention of male reproductive system injuries induced by environmental estrogens in animals measures.In this study, mice were divided into 5 groups,group A as control,group B as DES-induced model, group C to E were EGb and its components-Que anti DES-induced injury mice respectively,the EGb group peritoneally injected,the Que group treated with a method of stomach-lavaging,the B to E injected hypodermically with DES at 40 mg/kg BW per day 4 hours.All these mice were sacrificed after seven days treatment.The results as follows,as the dose of DES increasesd,the weight of testis and the sperm counts decreased significantly（P＜0.01）.The activities of SOD and GSH-Px in three DES groups decreased significantly than the control group（P＜0.05）;the malonaldehyde（MDA）content of the testis were raised in comparison with those of the control group（P＜0.05）.Light microscopic evaluation of testes revealed spermatogenic cells degeneration and increased sloughed cellular element in tubular lumen of DES-treated mice.The histological structure of the testes became more and more disordered.DES had a direct role in the damage on cultured adult hamsters spermatogenic cells.The cultured spermatogenic cells were treated with different doses of DES(10^-7to 10^-4M)for different times.With the extension of cultured time,the MDA content and LDH activities of the medium were significantly increased（P＜0.01,P＜0.05）in accordance with the characteristics of the model of testicle injuries induced by diethylstilbestrol in mice.When Chinese herbal medicine was used to prevent DES-induced tesis injury,the enzyme activities of SOD and GSH-Px significantly elevated in testes,LDH activities and MDA contents decreased,and PCNA expression increased.EGb could get the best effect in this group.Compared with DES group,the testis coefficients increased in the three EGb treated groups（P＜0.05）.The enzyme activities of SOD and GSH-Px significantly elevated in testes of EGb treated groups respectively（P＜0.05）.LDH activities and MDA contents decreased in testes of the three EGb groups significantly（P＜0.05）.The PCNA expression in testes deseased significantly after injecting DES compared with normal mice;but the index all increased obviously after taking EGb compared with DES group（P＜0.05,P＜0.01）.Therefore,DES could induce the oxidative injury of the male mice testes.Both EGb and Que could scavenge free radicals and inhibit lipid peroxidation.It is suggested that ginkgo biloba as a preventive antioxidant can protect against diethylstilbestrol-induced testicular injury.EGb and Que as a preventive antioxidant can protect against diethylstilbestrol-induced testicular injury. Spermatogenic cells PCNA expression of the DES-induced testis increased.Protective effects may be related to the promotion of spermatogenic epithelium proliferation.更多还原