小鼠尿道下裂动物模型的建立
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摘要
尿道下裂是小儿泌尿生殖系统最常见的畸形之一，国外发病率约0.3％，国内发病率约0.1％，近年来发病率呈上升趋势。尿道下裂的病因至今不明，但环境雌激素对尿道下裂的发生起了一定的作用。为了解环境雌激素导致尿道下裂发生的作用机制以及对尿道下裂治疗提供指导，有必要建立雌激素和抗雄激素物质诱导的尿道下裂动物模型。
第一部分 苯甲酸雌二醇诱导小鼠尿道下裂模型的建立
目的 建立苯甲酸雌二醇诱导的小鼠尿道下裂模型，为研究雌激素致尿道下裂的分子作用机制提供进一步研究的基础。方法 50只孕鼠随机分成五组A、B、C、D、E，在孕12～16天连续五天各组每只皮下分别注射苯甲酸雌二醇0 mg·kg~(-1)·d~(-1)、0.2 mg·kg~(-1)·d~(-1)、1 mg·kg~(-1)·d~(-1)、5 mg·kg~(-1)·d~(-1)、25 mg·kg~(-1)·d~(-1)，出生时观察小鼠死亡率，出生时每组取两只母鼠的仔鼠解剖，观察小鼠睾丸位置、前列腺发育情况。出生后四周观察，有无尿道下裂和隐睾。结果 A、B、C、D、E各组死亡率分别为21.6％、21.5％、41.4％、56.6％、75％。C、D、E组子代尿道下裂发生率分别为3.3％(1／30)、20％(6／20)、23％(3／13)；隐睾发生率分别为6.6％(2／30)、30％(9／30)、61.6％(8／13)。结论 E组死亡率较高，D组苯甲酸雌二醇用药量为小鼠尿道下裂造模的适宜剂量。
第二部分 氟他胺诱导小鼠尿道下裂模型的建立
目的 采用抗雄性激素受体药物氟他胺，诱导建立小鼠尿道下裂动物模型，为研究尿道下裂发病的分子作用机制，提供进一步研究的基础，为尿道下裂的治疗提供研究平台。方法 80只孕鼠随机分成四组A、B、C、D，在孕12～16天连续五天各组每只皮下分别注射氟他胺0 mg·kg(-1)·d(-1)、25 mg·kg(-1)·d(-1)、50 mg·kg(-1)·d(-1)、100 mg·kg(-1)·d(-1)。
南京医科大学硕士学位论文
出生时每组取两只母鼠的仔鼠解剖观察小鼠争九位置、前列腺发育情
况。出生后四周观察，有无尿道下裂、隐争，解剖观察前列腺发育情
况。结果A、B、C、D组子代尿道下裂发生率分别为0%、44.2%、
92.7%、100%;隐肇发生率分别为。%、4.8%、23.2%、32.4%。C、
D组前列腺均不发育，B、C、D组肛门至尿生殖结节的距离(AGD)
均缩短。结论用氟他胺可以诱导出稳定的尿道下裂模型，适宜推广。
Hypospadias is one of the most common congenital abnormalities of the genitalia , seen in about 3 per 1000 male births abroad and 1 per 1000 male births domestic, and the birth rate of hypospadias has increased during the last few decades. Although the etiology of hypospadias is elusive, the environmental estrogens accout for parts. In order to elucidate the mechanism of environmental estrogens of hypospadias and to instruct treatment, it is necessary to establish a mouse model of hypospadias induced by estrogen or antiandrogen.
Part one A Mouse Model of Hypospadias Induced by Estradiol Benzoate
Objective To establish a mouse model of hypospadias by estradiol benzoate to further studying molecular mechanisms of hypospadias. Method Fifty timed pregnant mouse randomly divided into five groups A, B, C, D, E. Estradiol benzoate was injected subcutaneously(sc) with mixture sesame oil at 0 mg-kg-1-d-1, 0.2 mg-kg-1-d-1, 1 mg-kg-1-d-1, 5 mg-kg-1-d-1 , 25 mg-kg-1-d-1 from GD 12 to 16, respectively. On the day of delivery mortality was recorded. The fetus of two pregnants from each group were anatomied to observe the positions of testes and prostates agenesis on the day of delivery. Urethras and the position of testes were examined on postnatal day 28 . Result Mortality of group A, B, C, D, E was 21.6%, 21.5%, 41.4%, 56.6%, 75% on the day of delivery, respectively. Hypospadias was seen in the group C 3.3%(1/30), D 20% (4/20), E 23% (3/13) and cryptorchidism in the group C 6.6% (2/30), D 30% (6/20), E 61.6% (8/13) on postnatal day 28. Conclusion The experimental model of hypospadias induced by estradiol benzoa
te in
group D was more steady considering high mortality of group E. The dose of group D was suitable for establishing mouse model of hypospa-dias.
Part two A Mouse Model of Hypospadias Induced by
Flutamide
Objective To establish a mouse model of hypospadias induced by fluta-mide to further studying molecular mechanisms of hypospadias etiology. Method Eighty timed pregnant mouse randomly divided into four groups A, B, C, D. Flutamide was injected subcutaneously(sc) with mixture sesame oil at 0 mg-kg-1 d-1 25 ing-kg-1-d-1, 50 mg-kg-1 d-1, 100 mg-kg-1-d-1 from GD 12 to 16, respectively. The fetus of two pregnants from each group were anatomied to observe the position of testes and prostates agenesis on the day of delivery. Urethras and the position of testes and prostates agenesis were examined on postnatal day 28. Result Hypospadias were seen in the group A 0%, B 44.2%, C 92.7%, D 100% and cryptorchidism in the group A 0%, B 4.8%, C 23.2%, D 32.4% on postnatal day 28, respectively. Flutamide caused 100% incidence of prostate agenesis in groups C, D and 19.2% in the group B and 100% incidence of female-like anogenital distance in groups B, C, D. Conclusion The experimental model of hypospadias induced by flutamid
e was more steady and suitable for spread.
第一部分 苯甲酸雌二醇诱导小鼠尿道下裂模型的建立
目的 建立苯甲酸雌二醇诱导的小鼠尿道下裂模型，为研究雌激素致尿道下裂的分子作用机制提供进一步研究的基础。方法 50只孕鼠随机分成五组A、B、C、D、E，在孕12～16天连续五天各组每只皮下分别注射苯甲酸雌二醇0 mg·kg~(-1)·d~(-1)、0.2 mg·kg~(-1)·d~(-1)、1 mg·kg~(-1)·d~(-1)、5 mg·kg~(-1)·d~(-1)、25 mg·kg~(-1)·d~(-1)，出生时观察小鼠死亡率，出生时每组取两只母鼠的仔鼠解剖，观察小鼠睾丸位置、前列腺发育情况。出生后四周观察，有无尿道下裂和隐睾。结果 A、B、C、D、E各组死亡率分别为21.6％、21.5％、41.4％、56.6％、75％。C、D、E组子代尿道下裂发生率分别为3.3％(1／30)、20％(6／20)、23％(3／13)；隐睾发生率分别为6.6％(2／30)、30％(9／30)、61.6％(8／13)。结论 E组死亡率较高，D组苯甲酸雌二醇用药量为小鼠尿道下裂造模的适宜剂量。
第二部分 氟他胺诱导小鼠尿道下裂模型的建立
目的 采用抗雄性激素受体药物氟他胺，诱导建立小鼠尿道下裂动物模型，为研究尿道下裂发病的分子作用机制，提供进一步研究的基础，为尿道下裂的治疗提供研究平台。方法 80只孕鼠随机分成四组A、B、C、D，在孕12～16天连续五天各组每只皮下分别注射氟他胺0 mg·kg(-1)·d(-1)、25 mg·kg(-1)·d(-1)、50 mg·kg(-1)·d(-1)、100 mg·kg(-1)·d(-1)。
南京医科大学硕士学位论文
出生时每组取两只母鼠的仔鼠解剖观察小鼠争九位置、前列腺发育情
况。出生后四周观察，有无尿道下裂、隐争，解剖观察前列腺发育情
况。结果A、B、C、D组子代尿道下裂发生率分别为0%、44.2%、
92.7%、100%;隐肇发生率分别为。%、4.8%、23.2%、32.4%。C、
D组前列腺均不发育，B、C、D组肛门至尿生殖结节的距离(AGD)
均缩短。结论用氟他胺可以诱导出稳定的尿道下裂模型，适宜推广。
Hypospadias is one of the most common congenital abnormalities of the genitalia , seen in about 3 per 1000 male births abroad and 1 per 1000 male births domestic, and the birth rate of hypospadias has increased during the last few decades. Although the etiology of hypospadias is elusive, the environmental estrogens accout for parts. In order to elucidate the mechanism of environmental estrogens of hypospadias and to instruct treatment, it is necessary to establish a mouse model of hypospadias induced by estrogen or antiandrogen.
Part one A Mouse Model of Hypospadias Induced by Estradiol Benzoate
Objective To establish a mouse model of hypospadias by estradiol benzoate to further studying molecular mechanisms of hypospadias. Method Fifty timed pregnant mouse randomly divided into five groups A, B, C, D, E. Estradiol benzoate was injected subcutaneously(sc) with mixture sesame oil at 0 mg-kg-1-d-1, 0.2 mg-kg-1-d-1, 1 mg-kg-1-d-1, 5 mg-kg-1-d-1 , 25 mg-kg-1-d-1 from GD 12 to 16, respectively. On the day of delivery mortality was recorded. The fetus of two pregnants from each group were anatomied to observe the positions of testes and prostates agenesis on the day of delivery. Urethras and the position of testes were examined on postnatal day 28 . Result Mortality of group A, B, C, D, E was 21.6%, 21.5%, 41.4%, 56.6%, 75% on the day of delivery, respectively. Hypospadias was seen in the group C 3.3%(1/30), D 20% (4/20), E 23% (3/13) and cryptorchidism in the group C 6.6% (2/30), D 30% (6/20), E 61.6% (8/13) on postnatal day 28. Conclusion The experimental model of hypospadias induced by estradiol benzoa
te in
group D was more steady considering high mortality of group E. The dose of group D was suitable for establishing mouse model of hypospa-dias.
Part two A Mouse Model of Hypospadias Induced by
Flutamide
Objective To establish a mouse model of hypospadias induced by fluta-mide to further studying molecular mechanisms of hypospadias etiology. Method Eighty timed pregnant mouse randomly divided into four groups A, B, C, D. Flutamide was injected subcutaneously(sc) with mixture sesame oil at 0 mg-kg-1 d-1 25 ing-kg-1-d-1, 50 mg-kg-1 d-1, 100 mg-kg-1-d-1 from GD 12 to 16, respectively. The fetus of two pregnants from each group were anatomied to observe the position of testes and prostates agenesis on the day of delivery. Urethras and the position of testes and prostates agenesis were examined on postnatal day 28. Result Hypospadias were seen in the group A 0%, B 44.2%, C 92.7%, D 100% and cryptorchidism in the group A 0%, B 4.8%, C 23.2%, D 32.4% on postnatal day 28, respectively. Flutamide caused 100% incidence of prostate agenesis in groups C, D and 19.2% in the group B and 100% incidence of female-like anogenital distance in groups B, C, D. Conclusion The experimental model of hypospadias induced by flutamid
e was more steady and suitable for spread.
引文
[1] Baskin LS. Hypospadias and urethral development. Urol, 2000, 163 (3): 951-956.
[2] 吴艳乔，王艳萍，朱军，等。国人尿道下裂的流行病学特征。中华泌尿外科杂志，1997，18(3)：753-757。
[3] Paulozzi LJ, Enckson JD, Jackson RJ. Hypospadias trends in two US Surveillance systems. Pediatrics, 1997,100(5): 831-837.
[4] Mylchreest E, Cattley RC, Foster PM. Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: an antiandrogenic mechanism?. Toxicol Sci, 1998, 43(1): 47-60.
[5] Mclntyre BS, Barlow NJ, Foster PM. Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen dependent tissues. Toxicol Sci, 2001, 62 (2): 236-249.
[6] Hernandez DS. Iatrogenic legacy from diethylstilbestrol exposure. J Lancet, 2002, 359(9312): 1081-1082.
[7] Yamada G, Satoh Y, Baskin LS, et al. Cellular and molecular mechanisms of development of the external genitalia. Differentiation, 2003, 71(8): 445-460.
[8] Kojima Y, Hayashi Y, Mizuno K, et al. Spermatogenesis, fertility and sexual behavior in a hypospadiac mouse model. J Urol, 2002,167(3): 1532-1537.
[9] Shono T, Ramm-Anderson S, Gob DW, et al. The effect of flutamide on testicular descent in rats examined by scanning electron microscopy. Journal of Pediatric Surgery, 1994, 29(6): 839-844.
[10] Silversides DW, Price CA, Cooke GM. Effects of short-term exposure to hydroxyflutamide in utero on the development of the re-
productive tract in male mice. Can J Physiol Pharmacol. 1995,73(11): 1582-1588
[2] 吴艳乔，王艳萍，朱军，等。国人尿道下裂的流行病学特征。中华泌尿外科杂志，1997，18(3)：753-757。
[3] Paulozzi LJ, Enckson JD, Jackson RJ. Hypospadias trends in two US Surveillance systems. Pediatrics, 1997,100(5): 831-837.
[4] Mylchreest E, Cattley RC, Foster PM. Male reproductive tract malformations in rats following gestational and lactational exposure to Di(n-butyl) phthalate: an antiandrogenic mechanism?. Toxicol Sci, 1998, 43(1): 47-60.
[5] Mclntyre BS, Barlow NJ, Foster PM. Androgen-mediated development in male rat offspring exposed to flutamide in utero: permanence and correlation of early postnatal changes in anogenital distance and nipple retention with malformations in androgen dependent tissues. Toxicol Sci, 2001, 62 (2): 236-249.
[6] Hernandez DS. Iatrogenic legacy from diethylstilbestrol exposure. J Lancet, 2002, 359(9312): 1081-1082.
[7] Yamada G, Satoh Y, Baskin LS, et al. Cellular and molecular mechanisms of development of the external genitalia. Differentiation, 2003, 71(8): 445-460.
[8] Kojima Y, Hayashi Y, Mizuno K, et al. Spermatogenesis, fertility and sexual behavior in a hypospadiac mouse model. J Urol, 2002,167(3): 1532-1537.
[9] Shono T, Ramm-Anderson S, Gob DW, et al. The effect of flutamide on testicular descent in rats examined by scanning electron microscopy. Journal of Pediatric Surgery, 1994, 29(6): 839-844.
[10] Silversides DW, Price CA, Cooke GM. Effects of short-term exposure to hydroxyflutamide in utero on the development of the re-
productive tract in male mice. Can J Physiol Pharmacol. 1995,73(11): 1582-1588