【作者】 张晶璇；

【导师】 杨继红；

【作者基本信息】 上海医药工业研究院 ， 药理学， 2006， 硕士

【摘要】 目的：探索中药对雄性动物生殖毒性的试验方法 方法：体外试验：建立体外SD大鼠原代睾丸支持细胞／生精细胞的混合培养和支持细胞的培养系统，选择从临床已发现有男性生殖毒性的昆明山海棠分离出来的两种主要有效成分——雷公藤甲素（Triptoliede，TL）和雷公藤次碱（Wilforine，WR）作为受试物，应用MTT法检测不同浓度的TL和WR对培养细胞增殖的影响，应用免疫细胞化学技术检测两种受试物对培养细胞广谱钙粘素（pan-cadherin）和卵泡刺激素受体（fshr）表达的影响，验证体外试验方法。 体内试验：选择临床已发现有男性生殖毒性的骨风宁（主要成份为昆明山海棠）给予SD大鼠经口灌胃3个月，进行睾丸切片的HE和PAS染色以及免疫组织化学技术等病理学方法研究其雄性生殖毒性。 结果：TL对体外混合培养细胞的最低有毒浓度为10 μg／L，IC₅₀为1.22 mg／L；TL对培养支持细胞的最低有毒浓度为10³ μg／L，IC₅₀为28.15 mg／L；并均呈剂量依赖性。WR对培养支持细胞的最低有毒浓度为10⁵ μg／L，IC₅₀=139.26 mg／L；WR对混合培养细胞无毒性作用。 在TL 0.1、1和10 μg／L浓度水平时混合培养细胞pan-cadherin的表达上调；TL各组与溶剂对照组相比对培养支持细胞中pan-cadherin和fshr的表达无统计学差异；WR各组与溶剂对照组相比对混合培养细胞和培养支持细胞中的pan-cadherin和fshr的表达无统计学差异。 骨风宁给药组SD大鼠睾丸生精细胞减少或消失，可见多核巨细胞，受损的睾丸中生精上皮处于Ⅰ～Ⅲ期的曲细精管比例增多。给药期末给药组动物睾丸组织中pan-cadherin的表达与对照组无统计学差异，给药期末骨风宁高剂量组动物的睾丸组织中fshr的表达降低。恢复期末骨风宁中、高剂量组动物睾丸组织中pan-cadherin的表达分别升高和降低，恢复期末给药组动物睾丸组织中fshr的表达与对照组无统计学差异。 结论：中药的雄性生殖毒性研究可以联合应用体外试验和体内试验。体外试验可以用来进行生殖毒物的初步筛选；体内动物实验提供毒性证据。探讨毒性作用机更多还原

【Abstract】 OBJECTIVE: To investigate methods of male reproductive toxicity induced by Chinesetraditional medicine.METHOD: In vitro tests: Spermatogenic cells and sertoli cells from SD rat testis wereisolated and cultured. The effects of Triptolide （TL） and Wilforine （WR）, activecomponents extracted from Gufengning which possesses male reproductive toxicityclinically, at different concentrations on cell proliferation were examined using MTTmethod.The expression of pan-cadherin and fshr in the cultured cells was examined usingimmunocytochemistry.In vivo test: SD rats were given daily gavage doses of Gufengning for 3-months. Testes slideswere treated with HE, PAS staining and immunohistochemical technology to examinemale reproductive toxicity.RESULT:In vitro tests: The lowest toxic concentration of TL was 10 μg/L in mix culturedspermatogenic cells and sertoli cells, its IC50 was 1.22 mg/L; the lowest toxicconcentration of TL was 10³ μg/L in cultured sertoli cells, its IC₅₀ was 28.15 mg/L; andthe inhibition rates were related to doses. The lowest toxic concentration of WR was 10⁵μg/L in cultured sertoli cells, its IC₅₀ was 139.26 mg/L. WR showed no toxicity in mixcultured cells.Pan-cadherin expression increased in mix cultured cells at TL concentration levels of 0.1,1 and 10 μg/L. No statistical significant difference in pan-cadherin and fshr expressionwas observed in cultured sertoli cells between TL treated groups and vehicle controlgroup. No statistical significant difference in pan-cadherin and fshr expression wasobserved in mix cultured cells and cultured sertoli cells between WR treated groups andvehicle control group.In vivo tests: Decreased quantity or disappearance of spermatogenic cells, multinucleargiant cells, increasing proportion of stage Ⅰ ～ Ⅲ seminiferoustubuli cycle wereobserved in SD rats treated with Gufengning for 3 months. No statistical significantdifference in pan-cadherin expression was observed in testes of control and dosinggroups after termination of dosing. Fshr expression was increased in testes of high dosegroup after termination of dosing. Pan-cadherin expression was increased in mid dose group,but decreased in high dose group after recovery period. No statistical significant differencein fshr expression was observed in testes of control and dosing groups after recovery period.更多还原