睾丸性不育动物模型的建立

Making of the Animal Model with Sterilized Testes

目的:探讨制作睾丸性不育动物模型的方法。方法:①X-射线局部照射:取8～10周龄的BALB/c雄鼠70只,分实验组1、2、3、4、5、6和对照组各10只。实验组1、2、3、4、5、6分别用1000、1200、1400、1600、1800、2000cGy6种不同的剂量照射睾丸局部10min;对照组不做任何处理。做受孕试验。②环磷酰胺腹腔注射:取4～5周龄的雄鼠40只,分实验组1、2、3和对照组各10只。实验组分别以30、5070mg/kg体重剂量的环磷酰胺腹腔注射,每周2次,连续注射5周;对照组腹腔注射生理盐水。做受孕试验。③达菲林腹腔注射:取8～10周龄的雄鼠20只,分实验组和对照组各10只。实验组腹腔注射0.375mg/ml达菲林0.4m,l对照组腹腔注射生理盐水0.4ml。做受孕试验。④通过脱氧核苷酸末端转移酶介导的缺口末端标记法(TUNEL)、苏木精-伊红(HE)染色及免疫组化等病理学检查鉴定。未使雌鼠受孕的雄性小鼠定为睾丸性不育,共30只。结果:①X-射线局部照射:实验组1、2的雄鼠分别于X-射线照射后10、15d使雌鼠受孕;实验组3、4的雄鼠,经观察3个月也未能使雌鼠受孕;实验组5、6的雄鼠,于X-射线照射后第5d和第2d死亡;对照组雄鼠皆于3d内使雌鼠受孕。②环磷酰胺腹腔注射:实验组1雄鼠体重增加7g左右,停药后9～14d恢复生育能力,使雌鼠受孕;实验组2雄鼠体重增加4g左右,停药后观察3个月未使雌鼠受孕;实验组3雄鼠体重不增,呈消瘦状,分别于用药第3、4、5周相继死亡;对照组雄鼠皆于3d内使雌鼠受孕。③达菲林腹腔注射:实验组雄鼠于停药后3周左右使雌鼠受孕;对照组雄鼠皆于3d内使雌鼠受孕。④TUNEL结果:对照组睾丸组织偶见凋亡细胞,占(0.71±0.12)%;睾丸性不育组睾丸组织凋亡细胞数量明显增加,占(10.36±1.48)%,两者比较差异有显著性(P<0.05)。HE染色:对照组睾丸组织正常,生精小管呈饱满椭圆形,各级生精细胞排列有序,内含大量精子细胞;睾丸性不育组生精小管萎缩,细胞排列疏松,未见各级生精细胞,只有支持细胞,各生精小管间腔隙扩大,间质细胞减少。免疫组化染色:CD29、Hsp90α、CD117的阳性表达率对照组分别为(50.3±5.2)%、(41.6±3.5)%、(73.6±3.7)%,而睾丸性不育组明显降低,分别为(1.3±0.2)%、0%、(1.6±0.3)%(P<0.01)。p53阳性表达率对照组为(19.7±0.8)%,而睾丸性不育组明显增高,为(39.4±2.9)%(P<0.01)。结论:X-射线睾丸局部照射、环磷酰胺腹腔注射法均可制作睾丸性不育动物模型。

Objective： To explore the methods of making an animal model with sterilized testes. Methods： （1） X-ray local irradiation. Seventy 8 - 10-week old male mice were equally divided into 6 experiment groups and a control group. The testes of the mice in the 6 experiment groups were irradiated sequentially by 1 000, 1 200, 1 400, 1 600, 1 800 and 2 000 cGy X-ray for 10 minutes, while those in the control group remained untreated. And then the pregnancy test was performed. （2） Cyclophosphamide injection. Forty 4 -5-week old male mice were divided into 3 experiment groups and a control group, the former treated with different doses of Cyclophosphamide via ip and the latter Natiichloridi Saline （ N. S. ） via ip, followed by the pregnancy test. （3） Diphereline injection. Twenty 8 - 10-week old male mice were equally divided into an experiment group and a control group, the former treated with Diphereline via ip and the latter N. S. via ip, followed by the pregnancy test. （4） Identification by such pathologic examinations as TUNEL technology, HE staining and immunohistochemical staining. Results ： （1）X-ray local irradiation. The male mice of Group 1 and 2 made their female partners pregnant respectively 10 and 15 days after the X-ray irradiation, but not those of Group 3 and 4 in our 3-month observation, and those of Group 5 and 6 died respectively 2 and 5 days after the X-ray irradiation. By comparison, the controls got their female partners pregnant within 3 days after placed together. （2） Cyclophosphamide injection. The male mice of Group 1 gained weight about 7 g and achieved pregnancy 9 - 14 days after drug termination, those of Group 2 gained around 4g but failed to effect pregnancy, and those in Group 3 lost weight and died respective at 3,4 and 5 weeks during the medication, while the controls all got their female partners pregnant within 3 days after put together. （3）Diphereline injection. The 10 male mice of the experiment group effected pregnancy 3 weeks after drug termination, while the 10 controls achieved the same result with 3 days after placed together. （4）Pathologic identification： TUNEL technology showed that apoptotic cells were occasionally seen （0.71 ±0.12） % in the testis tissue of the control group and remarkably increased （ 10.36 ± 1.48） % in the model group, with significant difference between the two groups （P 〈 0.05 ）. HE staining revealed normal testis tissues and convoluted seminiferous tubules with large numbers of germ cells in the control group, but atrophied convoluted seminiferous tubules and estranged cell linage with only Ledig＇s cells but no germ cells in the model group. Immunohistochemical staining showed that the positive expression rates of CD29, Hsp9Oct and CD117 were respectively （50.30 ± 5.2 ）%, （41.6 ± 3.5 ） % and （73.6 ± 3.7） % in the control group, as compared with （ 1.3 ± 0.2） %, 0 % and （ 1.6 ± 0.3 ） % in the model group, with significant difference （P 〈0.01 ）. The positive expression rate of p53 was （19.7 ± 0.8 ）% in the control group, significantly different from that of the model group, which was （39.4 ± 2.9） % （P 〈 0.01 ）. Conclusion ： The animal model with sterilized testes can be made either by X-ray local irradiation of the testis or by Cyclophosphamide injection via ip. Natl J Androl,2007, 13 （2） ：125-129