抗早颗粒对雌性性早熟大鼠Kiss-1/GPR54mRNA表达的影响及临床研究
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摘要
目的：
本文以达那唑诱导的雌性性早熟大鼠为模型,通过观察调控青春期发育的关键分子下丘脑Kiss-1基因衍生肽kisspeptin及其受体GPR54 (G-protein couped receptor 54)在青春期发育不同时期的表达规律,探讨kisspeptin/GPR54在青春启动时的作用；观察中药抗早颗粒对kisspeptin/GPR54表达的影响以及kisspeptin/GPR54在抗早颗粒治疗性早熟中的作用,进一步认识抗早颗粒的作用机制；抗早颗粒治疗特发性中枢性性早熟女童临床疗效观察,研究抗早颗粒对性早熟女童雌二醇(E2)、促黄体生成素(LH)、促卵泡生成素(FSH)、胰岛素样生长因子(IGF-1)的影响,以及抗早颗粒改善性早熟女童的中医证候优势,探讨抗早颗粒的作用机制;评价抗早颗粒治疗本病的临床经济学意义。
方法：
(1)出生后3天的SD雌性大鼠,随母鼠共同饲养,随机分为正常对照A组(青春前期)、正常对照B组(青春早期)、实验A组(青春早期)、实验B组(青春中期)。实验组大鼠在出生后5日龄给予皮下注射300ug达那唑。采用荧光定量PCR技术分别观察青春期启动前(出生后23日龄)、青春期启动(阴门开启为标志)、青春期启动后(建立2个规则的性周期)大鼠下丘脑Kiss-l/GPR54mRNA的表达规律。
(2)出生后3天的SD雌性大鼠,随母鼠共同饲养,随机分为正常组、模型组、曲普瑞林组、中药组(大剂量组、中剂量组、小剂量组),每组6只。模型组、曲普瑞林组和中药组大鼠在出生后5日龄给予皮下注射300ug达那唑。曲普瑞林组大鼠15日开始用曲普瑞林100ug/kg,皮下注射,1次/日,同时中药组灌胃中药,1次/日,大剂量60m1/kg,中剂量30ml/kg,小剂量15ml/kg。同时模型组给予生理盐水1ml/只灌胃。模型组大鼠建立第一个发情期后于动情前期将该动物处死,其余各组按1：1比例同时处死。计算卵巢指数和子宫指数,观察卵巢组织切片黄体的出现情况,ELISA法测定血清E2、LH、IGF-1水平,荧光定量PCR技术测定大鼠下丘脑GnRH/GnRH-RmRNA、Kiss-1/GPR54mRNA的表达变化,探讨抗早颗粒对Kiss-1/GPR54mRNA表达的影响及抗早颗粒治疗性早熟的作用机制。
(3)临床观察将特发性中枢性性早熟女童随机分2组,中药组62例,西药组31例。中药组口服抗早颗粒每日1剂,温水冲100ml,分2次口服,连服6个月；西药组肌肉注射盐酸曲普瑞林(达菲林),按60-100ug/kg,28天1次,连续6个月。观察治疗前后两组的疗效,研究抗早颗粒改善性早熟女童的中医证候的优势,分析抗早颗粒对性早熟女童LH、FSH、E2、IGF-1的影响,以及性早熟女童体重指数(BMI)与E2、IGF-1的相关性,探讨抗早颗粒的作用机制；评价抗早颗粒治疗本病的临床经济学意义。
结果：
(1)实验组5日龄雌性大鼠皮下注射达那唑后,阴门开启和建立两个规则的性周期时间明显提前,卵巢和子宫的脏器指数较正常对照A组大鼠明显增加；青春期启动时下丘脑GnRHmRNA的表达水平较正常对照A组显著升高。正常对照A组(正常青春前期)、模型A组(青春期早期)、模型B组(青春期中期)的Kiss-1mRNA和GPR54mRNA的表达逐渐增高,提示Kiss-1/GPR54mRNA的表达与生长发育的各个时期密切相关。同时观察到青春期启动时正常大鼠下丘脑Kiss-1/GPR54mRNA表达增加,与GnRH的增加趋于同步。
(2)观察到抗早颗粒可以降低性早熟模型大鼠血清E2、LH、IGF-1水平,治疗前后有非常显著性差异(P<0.01)。荧光定量PCR技术观察到中药高、中、低剂量组、曲普瑞林组大鼠下丘脑GnRH、Kiss-1及GPR54 mRNA含量明显降低,与模型组比较,有显著性差异(P<0.01)。表明抗早颗粒可下调性早熟模型大鼠下丘脑Kiss-1、GPR54及GnRHmRNA的表达。
(3)临床观察中药组有效率达83.87%,西药组有效率87.09%,两组疗效无显著性差异(P>0.05),中药组与西药组疗效相当；两组治疗后显著降低患儿血清FSH, LH及E2水平,子宫卵巢明显回缩,第二性征消退。中药组可显著改善性早熟女童形体偏胖、面赤口渴、肢体困倦、大便干结等中医证候,与西药组比较,有显著性差异(P<0.05)。
(4)临床经济学评价,成本-效价比中药组显著优于西药组。
结论：
(1)性早熟大鼠青春期启动时kisspeptin/GPR54的表达异常,下丘脑Kiss-1/GPR54 mRNA的表达水平增高,可能参与并促进大鼠下丘脑-垂体-性腺轴(HPGA)的提前启动,导致大鼠性早熟的发生。
(2)抗早颗粒可通过下调下丘脑Kiss-1/GPR54mRNA的表达,抑制GnRH的释放,延缓性早熟大鼠下丘脑-垂体-性腺轴的启动。
(3)抗早颗粒临床与曲普瑞林疗效相当,可能对性早熟患儿下丘脑-垂体-性腺轴功能有抑制作用。成本-效价比中药组显著优于西药组,显示抗早颗粒中药有进一步研究开发推广的价值。
Objective:
In this paper, we use female precocious puberty rats model which induced danazol, to observe the key molecular Kiss-1 gene derived peptide kisspeptin which regulate the puberty development and its receptor GPR54 (G-protein couped receptor 54) expression at different stages, to discuss the role of kisspeptin/GPR54 in youth start, to observe the effects of kangzao on kisspeptin/GPR54 expression and the function of kisspeptin/GPR54 granules in the treatment of precocious puberty, to make a further understanding of the role of kangzao granules.
Kangzao granules are used to treat idiopathic central precocious puberty in girls of 62 cases. We study the relationship of E2, IGF-1 and BMI in precocious puberty girls, as well as kangzao granule regulated Chinese medicine symptoms of precocious puberty girls, to explore the function of kangzao granule. In the mean while, we evaluate the meaning of kangzao granules to treat this disease in clinical economics.
Method:
(1) The female rats of SD 3 days old who co-feeding with their mother were randomly divided into normal control group A(prepubertal), normal control group B (early youth), experimental group A (early youth), experimental group B (youth medium). Experimental group rats of 5 days old were given subcutaneous injection of 300ug danazol.We used fluorescence quantitative PCR technology to observe Kiss-1/GPR54 mRNA Expression pattern before puberty (23 days after birth), puberty (vaginal opening as a symbol), after puberty (create two rules of the cycle) in hypothalamus of rats..
(2) The female rats after 3 days birth who co-feeding with their mother were randomly divided into normal group, model group, triptorelin group, Chinese medicine group (high dose group, middle dose group, low dose group), each group contains 6 rats only. Rats of 5 days birth in model group, triptorelin group and TCM group were given subcutaneous injection of 300ug danazol. The rats of 15days old in triptorelin group begun to use triptorelin 100ug/kg, subcutaneous injection,1 times/day; at the same time, the rats in Chinese medicine group were given 60ml/kg dose one time per day,30ml/kg mid dose,15ml/kg low-dose.
While model group rats received normal saline lml/fed only. In Model group, after established the first estrus, the rats were killed during proestrus early process. The rats in other groups as 1:1 ratio at the same time., To calculated ovarian index and uterine index and observed the occurrence of ovarian tissue sections of luteal, we use ELISA method to measure blood E2, LH, IGF-1 levels, and measured Kiss-1, GPR54, GnRH mRNA, GnRH-R mRNA expression changes in hypothalamus by quantitative PCR technology. To discuss the effect of kangzao granules which affect Kiss-1/GPR54mRNA expression and the function of kangzao granules in treatment of precocious puberty.
(3) Clinical treatment of idiopathic central precocious puberty girls were randomly divided into 2 groups,62 cases contained in traditional Chinese medicine group,31 cases in western medicine group. Chinese medicine group were treated with kangzao granules one dose per day, flush 100ml in 2 times and served for 6 months continuously; western medicine group were given hydrochloride intramuscular triptorelin, by 60-100ug/kg, for 6 months. To observe the efficacy and advantage before and after treatment in these two groups, and to analyzed the effect of kangzao granules in curing girls'LH、FSH、E2、IGF-1 as well as the relationship of precocious puberty in girls (body mass index) with BMI and E2 (estradiol), IGF-1 (insulin-like growth factor), and kangzao granule to regulate the symptoms of precocious puberty girls with Chinese medicine.To explore the function mechanism of kangzao granules. We evaluate the meaning of kangzao granules to treat this disease in clinical economics.
Results:
(1) After given subcutaneous injection of danazol to the female rats of 5 days old, the vaginal opening and sexual cycle time are significantly advanced. Ovarian and uterine organ index was significantly increased when comparing with the rats in normal control group A; Puberty of hypothalamic GnRH mRNA expression levels was significantly higher than normal control group A. The expression of Kiss-1mRNA in normal control group A(normal prepubertal) . model group A (early puberty). model group B (mid puberty) were increased gradually,which indicate Kiss-1 mRNA and GPR54mRNA expressions are closely related to growth and development period. At the same time, we observed that when puberty started, the Kiss-1,/GPR54mRNA expression in hypothalamus increased with GnRH simultaneously.
(2) We observed that kangzao can be reduced on blood E2, LH (luteinizing hormone), IGF-1 levels on precocious puberty rat model. We observed there was triptorelin group hypothalamus GnRH, Kiss-1 and GPR54 mRNA were significantly decreased by fluorescence quantitative PCR, in Chinese high, medium and low dose group. And there is a significant difference when compared with model group. (P<0.01) It shows that kangzao can regulate expression of Kiss-1, GPR54 and GnRH mRNA in hypothalamic precocious puberty model rats by quantitative PCR technology
(3) The TCM clinical efficacy is 83.87%, the western medicine group is 87.09%,there is no significant difference in there two group. The patients serum levels of FSH, LH and E2 levels were decreased significantly, ovary and uterus was retracted, secondary sexual characteristics faded off. The Chinese group can significantly improve the precocious puberty girls body fat symptom, red face, thirst, physical drowsiness, dry stools and other traditional Chinese medicine symptoms.
(4) Clinical economics evaluated that Chinese medicine group is better than western medicine group in cost - effective value.
本文以达那唑诱导的雌性性早熟大鼠为模型,通过观察调控青春期发育的关键分子下丘脑Kiss-1基因衍生肽kisspeptin及其受体GPR54 (G-protein couped receptor 54)在青春期发育不同时期的表达规律,探讨kisspeptin/GPR54在青春启动时的作用；观察中药抗早颗粒对kisspeptin/GPR54表达的影响以及kisspeptin/GPR54在抗早颗粒治疗性早熟中的作用,进一步认识抗早颗粒的作用机制；抗早颗粒治疗特发性中枢性性早熟女童临床疗效观察,研究抗早颗粒对性早熟女童雌二醇(E2)、促黄体生成素(LH)、促卵泡生成素(FSH)、胰岛素样生长因子(IGF-1)的影响,以及抗早颗粒改善性早熟女童的中医证候优势,探讨抗早颗粒的作用机制;评价抗早颗粒治疗本病的临床经济学意义。
方法：
(1)出生后3天的SD雌性大鼠,随母鼠共同饲养,随机分为正常对照A组(青春前期)、正常对照B组(青春早期)、实验A组(青春早期)、实验B组(青春中期)。实验组大鼠在出生后5日龄给予皮下注射300ug达那唑。采用荧光定量PCR技术分别观察青春期启动前(出生后23日龄)、青春期启动(阴门开启为标志)、青春期启动后(建立2个规则的性周期)大鼠下丘脑Kiss-l/GPR54mRNA的表达规律。
(2)出生后3天的SD雌性大鼠,随母鼠共同饲养,随机分为正常组、模型组、曲普瑞林组、中药组(大剂量组、中剂量组、小剂量组),每组6只。模型组、曲普瑞林组和中药组大鼠在出生后5日龄给予皮下注射300ug达那唑。曲普瑞林组大鼠15日开始用曲普瑞林100ug/kg,皮下注射,1次/日,同时中药组灌胃中药,1次/日,大剂量60m1/kg,中剂量30ml/kg,小剂量15ml/kg。同时模型组给予生理盐水1ml/只灌胃。模型组大鼠建立第一个发情期后于动情前期将该动物处死,其余各组按1：1比例同时处死。计算卵巢指数和子宫指数,观察卵巢组织切片黄体的出现情况,ELISA法测定血清E2、LH、IGF-1水平,荧光定量PCR技术测定大鼠下丘脑GnRH/GnRH-RmRNA、Kiss-1/GPR54mRNA的表达变化,探讨抗早颗粒对Kiss-1/GPR54mRNA表达的影响及抗早颗粒治疗性早熟的作用机制。
(3)临床观察将特发性中枢性性早熟女童随机分2组,中药组62例,西药组31例。中药组口服抗早颗粒每日1剂,温水冲100ml,分2次口服,连服6个月；西药组肌肉注射盐酸曲普瑞林(达菲林),按60-100ug/kg,28天1次,连续6个月。观察治疗前后两组的疗效,研究抗早颗粒改善性早熟女童的中医证候的优势,分析抗早颗粒对性早熟女童LH、FSH、E2、IGF-1的影响,以及性早熟女童体重指数(BMI)与E2、IGF-1的相关性,探讨抗早颗粒的作用机制；评价抗早颗粒治疗本病的临床经济学意义。
结果：
(1)实验组5日龄雌性大鼠皮下注射达那唑后,阴门开启和建立两个规则的性周期时间明显提前,卵巢和子宫的脏器指数较正常对照A组大鼠明显增加；青春期启动时下丘脑GnRHmRNA的表达水平较正常对照A组显著升高。正常对照A组(正常青春前期)、模型A组(青春期早期)、模型B组(青春期中期)的Kiss-1mRNA和GPR54mRNA的表达逐渐增高,提示Kiss-1/GPR54mRNA的表达与生长发育的各个时期密切相关。同时观察到青春期启动时正常大鼠下丘脑Kiss-1/GPR54mRNA表达增加,与GnRH的增加趋于同步。
(2)观察到抗早颗粒可以降低性早熟模型大鼠血清E2、LH、IGF-1水平,治疗前后有非常显著性差异(P<0.01)。荧光定量PCR技术观察到中药高、中、低剂量组、曲普瑞林组大鼠下丘脑GnRH、Kiss-1及GPR54 mRNA含量明显降低,与模型组比较,有显著性差异(P<0.01)。表明抗早颗粒可下调性早熟模型大鼠下丘脑Kiss-1、GPR54及GnRHmRNA的表达。
(3)临床观察中药组有效率达83.87%,西药组有效率87.09%,两组疗效无显著性差异(P>0.05),中药组与西药组疗效相当；两组治疗后显著降低患儿血清FSH, LH及E2水平,子宫卵巢明显回缩,第二性征消退。中药组可显著改善性早熟女童形体偏胖、面赤口渴、肢体困倦、大便干结等中医证候,与西药组比较,有显著性差异(P<0.05)。
(4)临床经济学评价,成本-效价比中药组显著优于西药组。
结论：
(1)性早熟大鼠青春期启动时kisspeptin/GPR54的表达异常,下丘脑Kiss-1/GPR54 mRNA的表达水平增高,可能参与并促进大鼠下丘脑-垂体-性腺轴(HPGA)的提前启动,导致大鼠性早熟的发生。
(2)抗早颗粒可通过下调下丘脑Kiss-1/GPR54mRNA的表达,抑制GnRH的释放,延缓性早熟大鼠下丘脑-垂体-性腺轴的启动。
(3)抗早颗粒临床与曲普瑞林疗效相当,可能对性早熟患儿下丘脑-垂体-性腺轴功能有抑制作用。成本-效价比中药组显著优于西药组,显示抗早颗粒中药有进一步研究开发推广的价值。
Objective:
In this paper, we use female precocious puberty rats model which induced danazol, to observe the key molecular Kiss-1 gene derived peptide kisspeptin which regulate the puberty development and its receptor GPR54 (G-protein couped receptor 54) expression at different stages, to discuss the role of kisspeptin/GPR54 in youth start, to observe the effects of kangzao on kisspeptin/GPR54 expression and the function of kisspeptin/GPR54 granules in the treatment of precocious puberty, to make a further understanding of the role of kangzao granules.
Kangzao granules are used to treat idiopathic central precocious puberty in girls of 62 cases. We study the relationship of E2, IGF-1 and BMI in precocious puberty girls, as well as kangzao granule regulated Chinese medicine symptoms of precocious puberty girls, to explore the function of kangzao granule. In the mean while, we evaluate the meaning of kangzao granules to treat this disease in clinical economics.
Method:
(1) The female rats of SD 3 days old who co-feeding with their mother were randomly divided into normal control group A(prepubertal), normal control group B (early youth), experimental group A (early youth), experimental group B (youth medium). Experimental group rats of 5 days old were given subcutaneous injection of 300ug danazol.We used fluorescence quantitative PCR technology to observe Kiss-1/GPR54 mRNA Expression pattern before puberty (23 days after birth), puberty (vaginal opening as a symbol), after puberty (create two rules of the cycle) in hypothalamus of rats..
(2) The female rats after 3 days birth who co-feeding with their mother were randomly divided into normal group, model group, triptorelin group, Chinese medicine group (high dose group, middle dose group, low dose group), each group contains 6 rats only. Rats of 5 days birth in model group, triptorelin group and TCM group were given subcutaneous injection of 300ug danazol. The rats of 15days old in triptorelin group begun to use triptorelin 100ug/kg, subcutaneous injection,1 times/day; at the same time, the rats in Chinese medicine group were given 60ml/kg dose one time per day,30ml/kg mid dose,15ml/kg low-dose.
While model group rats received normal saline lml/fed only. In Model group, after established the first estrus, the rats were killed during proestrus early process. The rats in other groups as 1:1 ratio at the same time., To calculated ovarian index and uterine index and observed the occurrence of ovarian tissue sections of luteal, we use ELISA method to measure blood E2, LH, IGF-1 levels, and measured Kiss-1, GPR54, GnRH mRNA, GnRH-R mRNA expression changes in hypothalamus by quantitative PCR technology. To discuss the effect of kangzao granules which affect Kiss-1/GPR54mRNA expression and the function of kangzao granules in treatment of precocious puberty.
(3) Clinical treatment of idiopathic central precocious puberty girls were randomly divided into 2 groups,62 cases contained in traditional Chinese medicine group,31 cases in western medicine group. Chinese medicine group were treated with kangzao granules one dose per day, flush 100ml in 2 times and served for 6 months continuously; western medicine group were given hydrochloride intramuscular triptorelin, by 60-100ug/kg, for 6 months. To observe the efficacy and advantage before and after treatment in these two groups, and to analyzed the effect of kangzao granules in curing girls'LH、FSH、E2、IGF-1 as well as the relationship of precocious puberty in girls (body mass index) with BMI and E2 (estradiol), IGF-1 (insulin-like growth factor), and kangzao granule to regulate the symptoms of precocious puberty girls with Chinese medicine.To explore the function mechanism of kangzao granules. We evaluate the meaning of kangzao granules to treat this disease in clinical economics.
Results:
(1) After given subcutaneous injection of danazol to the female rats of 5 days old, the vaginal opening and sexual cycle time are significantly advanced. Ovarian and uterine organ index was significantly increased when comparing with the rats in normal control group A; Puberty of hypothalamic GnRH mRNA expression levels was significantly higher than normal control group A. The expression of Kiss-1mRNA in normal control group A(normal prepubertal) . model group A (early puberty). model group B (mid puberty) were increased gradually,which indicate Kiss-1 mRNA and GPR54mRNA expressions are closely related to growth and development period. At the same time, we observed that when puberty started, the Kiss-1,/GPR54mRNA expression in hypothalamus increased with GnRH simultaneously.
(2) We observed that kangzao can be reduced on blood E2, LH (luteinizing hormone), IGF-1 levels on precocious puberty rat model. We observed there was triptorelin group hypothalamus GnRH, Kiss-1 and GPR54 mRNA were significantly decreased by fluorescence quantitative PCR, in Chinese high, medium and low dose group. And there is a significant difference when compared with model group. (P<0.01) It shows that kangzao can regulate expression of Kiss-1, GPR54 and GnRH mRNA in hypothalamic precocious puberty model rats by quantitative PCR technology
(3) The TCM clinical efficacy is 83.87%, the western medicine group is 87.09%,there is no significant difference in there two group. The patients serum levels of FSH, LH and E2 levels were decreased significantly, ovary and uterus was retracted, secondary sexual characteristics faded off. The Chinese group can significantly improve the precocious puberty girls body fat symptom, red face, thirst, physical drowsiness, dry stools and other traditional Chinese medicine symptoms.
(4) Clinical economics evaluated that Chinese medicine group is better than western medicine group in cost - effective value.
引文
[1]Kaiser UB, Kuohung W. KiSS-1 and GPR54 as new players in gonadotropin regulation and puberty[J]. Endocrine,2005,26 (3):277-284.
[2]Navarro VM, Castellano JM,, Garcia-Galiano D, et al Neuroendocrine factors in the initiation of puberty:the emergent role of kisspeptin[J]. Rev Endocr Metab Disord,2007,8(1):11-20-
[3]Smith JT, Clifton DK,Steiner RA.Regulation of the neuroendocrine reproductiveaxis by kisspeptin-GPR54 signaling[J]. Reproduction,2006,131 (4):623-30.
[4]Navarro VM, Fernandez R, Castellano J M, Roa J, Mayem A, Barreiro ML, et al. Advanced vaginal opening and precocious activation of the reproductive axis by KISS-1 peptide, the endogenous ligand of GPR54[J]. Physiol,2004,561 (2):379-386.
[5]杜敏联.性早熟的临床研究进展[J].新医学2007,38(2)：118-140.
[6]Choi JH, Shin YL, Yoo HW. Predictive factors for organic central precocious puberty and utility of simplified gonadotropin-releasing hormone tests[J]. Pediatr Int,2007,49 (6):806-810.
[7]李嫔.性早熟病因及发病机制的研究进展[J].实用儿科临床杂志,2006,20(6)：498-500.
[8]郭正霞,辛秀娟,肖君华.性早熟相关基因的研究进展[J].国外医学儿科学分册,2004,31(6)：300-302.
[9]Huhtaniemi I. Mutations affecting gonadotropin secretion and action[J]. Horm Res,2003, 60 (supp13):S21-S30.
[10]BritoVN, LatronicoAC, Arnhold IJ. et al Update on the etiology, diagnosis and therapeutic management of sexual precocity [J]. Arq BrasEndocrinol Metabol 2008,52 (1):18-31.
[11]Murphy KG. Kisspepetins. regulators of metastasis and the hypothalamic-pituitary-gonadal axis[J]. J Neuroendocrinol,2005,17 (8):519-525.
[l2]Gianetti E, Seminara S. Kisspeptin and KisslR:a critical pathway in the reproductive system[J]. Reproduction,2008,136,295-301.
[l3]Semple RK, Achermann JC, Ellery J. et al. Two novel missense mutations in G-protein-coupled receptor 54 in a patiant with hypogonadotropic hypogonadism[J]. Clin Endocrinol Metab,2005,90 (3):1849-55.
[14]Nicolas de Roux,Emmanuelle Genin, Jean-Claude Carel, et al. Hypogonadotropic hypogona-dism due to loss of function of the KiSSl-derived peptide receptor GPR54 [J]. Proc NatlAcad Sci USA,2003,100 (19):10972-10976.
[15]Gianetti E, Seminara S. Kisspeptin and KisslR:a critical pathway in the reproductive system[J]. Reproduction,2008,136,295-301.
[16]Semple RK, Achermann JC, Ellery J. et al. Two novel missense mutations in G-protein-coupled receptor 54 in a patiant with hypogonadotropic hypogonadism[J]. Clin Endocrinol Metab,2005,90 (3):1849-55.
[17]Nicolas de Roux,Emmanuelle Genin, Jean-Claude Carel, et al. Hypogonadotropic hypogona-dism due to loss of function of the KiSSl-derived peptide receptor GPR54 [J]. Proc Natl Acad Sci USA,2003,100 (19):10972-10976.
[18]Gianetti E, Seminara S. Kisspeptin and Kiss1R:a critical pathway in the reproductive system[J]. Reproduction,2008,136,295-301.
[19]曾畿生,王德芬主编.现代儿科内分泌学[M].上海科学技术文献出版社,2002,126.
[20]Toshiaki T, Hiroo N, Nobutake M, et al. Results of Long-Term Follow-Up after Treatment of Central Precocious Puberty with Leuprorelin Acetate:Evaluation of Effectiveness of Treatment and Recovery of Gonadal Function.The TAP-144-SR Japanese Study Group on Central Precocious Puberty[J]. J Clin Endocrinol Metab 2005,90:1371-1376.
[21]Roth C, Neu C, Jarry H, et al. Different effects of agonistic vs.antagonistic GnRH-analogues (Triptorelin vs.Cetrorelix)on bone modeling and remodeling in peripubertal female rats[J]. Exp Clin Endocrinol Diabetes,2005,113 (8):451-456.
[22]Roth CL, Brendel L, Ruckert C, et al. Antagonistic and agonistic GnRH analogue treatment of precocious puberty:Tracking gonadotrop in concentrations in urine[J]. Horm Res,2005,63 (5):257-262.
[23]Pucarelli I, Segni M, Ortore M, et al. Effects of combined gonadotropin-releasing hormone agonist and growth hormone therapy on adult height in precocious puberty:a further contribution[J]. J Pediatr Endocrinal Metab 2003,16 (7):1005-1010.
[24]李文京,朱逞,颜纯.达那唑对中枢性性早熟治疗的临床分析[J].北京医学,2005,27(9)：522-524.
[25]徐雯,邱志文.加减知柏地黄汤对特发性中枢性性早熟激素水平的影响[J].中医杂志,2007,48(4)：335-336.
[26]蔡德培.滋阴泻火中药为主对改善性早熟儿童骨骼发育作用及机理的探讨[J].中医杂志,1997,38(10)：615.
[27]赵均.小儿性早熟中医药治疗进展[J].陕西中医学院学报,2007,3(4)：56.
[28]曾畿生,王德芬主编.现代儿科内分泌学[M].上海科学技术文献出版社,2002,126.
[29]潘键,李海浪,郑意楠.促性腺激素拟似剂与儿童真性性早熟[J].国外医学儿科学分册2001,28(4),214-216.
[30]Tato L, et al. Use of combined Gn-RH agonist and hGH therapy for better attaining the goals in precocious puberty treatment[J]. Horm Res.1995,44:49-54.
[3l]Di Martino-Nardi J, et al. The effectof luteinizing hormone-releasing hormone analog for central precociouspuberty on growth hormone (GH) and GH-binding protein[J]. ClinEndocrinol Metab.1994,78:664-668.
[32]Harris DA, et al. Somatomedin-C in normalpuberty and in true precocious puberty before and after treatment witha potent luteinizing hormone-releasing hormone agonist[J]. Clin Endocrinol Metab.1985,61:152-159.
[33]Pasquino AM, et al. Adult height in girls with central precocious puberty treated with gonadotropin-releasing hormone analogues and growth hormone. [J]. Clin Endocrinol Metab.1999; 84:449-452.
[34]Gevers EF, Wit J M, Robinson ICAF. Effects of long-term gonadotropin releasing hormone analog treatment on growth,growth hormone (GH) secretion, GH receptors, and GH-binding protein in the rat[J]. International Pediatric Research Foundation 1998,43 (1):111-120.
[35]李文京,朱逞,颜纯.女性特发性中枢性性早熟GnRHa治疗探讨[J].临床儿科杂志2005,23(8)：530-533.
[36]Pucarelli I, Segni M, Ortore M, et al. Effects of combined gonadotropin-releasing hormone agonist and growth hormone therapy on adult height in precocious puberty:a further contribution[J]. J Pediatr Endocrinal Metab 2003,16(7):1005-1010.
[37]徐雯,邱志文.加减知柏地黄汤对特发性中枢性性早熟激素水平的影响[J].中医杂志,2007,48(4)：335-336.
[38]蔡德培.滋阴泻火中药为主对改善性早熟儿童骨骼发育作用及机理的探讨[J].中医杂志,1997,38(10)：615.
[39]赵均.小儿性早熟中医药治疗进展[J].陕西中医学院学报,2007,3(4)：56.
[40]刘百祥.逍遥散加味治疗女童性早熟34例[J].湖南中医杂志,2004,20(4)：6.
[41]项秀荷.柴胡疏肝散结汤治疗女童性早熟3例疗效观察[J].中医杂志,2001,42(1)36,61.
[42]赵鉴,虞坚尔.抗早2号方治疗女童真性性早熟的临床研究[J].中国中西医结合杂志,2003,23(3)：182-184.
[43]高华,刘云鹏.治疗女童特发性性早熟经验[J].湖北中医杂志,2004,26(12)：13-14.
[44]杨明亮.龙胆泻肝汤加减治疗女童性早熟38例[J].湖南中医杂志,2004,20(3)60.
[45]徐孝华,蔡迎庆,严靖.知柏地黄丸联合大补阴丸治疗儿童真性性早熟69例临床分析[J].南通大学学报,2005,25(2)：134-135.
[46]朱红,杨玉霞.中药治疗女童特发性早熟41例[J].江苏中医,2003,24(7)：32-33.
[47]夏龙汀,肖挹.三草汤治疗女童性早熟23例[J].实用医学临床杂志,2004,1(1)：92.
[48]秦萍.柴胡橘叶汤治疗女童特发性中枢性性早熟30例[J].上海中医药杂志,2004,38(4)：140-141.
[49]杨丽珍,郑杨,刘利.桅早汤治疗女童真性性早熟55例临床观察[J].中医药信息,2007,24(4)：48-50.
[50]叶进.抗早颗粒剂治疗女童性早熟46例[J].辽宁中医杂志,2007,34(4)：435-436.
[51]顾可钦,虞坚尔.消结合剂治疗女童性早熟62例疗效观察[J].福建医药志,1998,20(3)：88-89.
[52]陈祺.“小儿早熟1号”治疗女童性早熟69例[J].中国中西医结合杂志,2003,23(8)：632.
[53]蔡德培,陈伯英,张炜等.中药调整性早熟儿童青春发育进程的机制研究[J].中西医结合学报,2006,4(2)：166-174.
[54]曹守凤,李海浪,郑意楠等.儿科Ⅱ号合剂对真性性早熟大鼠骨生长的影响[J].江苏医药,2007,33(1)：65-67.
[55]陈祺,戴方伟,萨晓婴等.抗性早熟颗粒对性早熟SD大鼠模型雌二醇的调节作用[J].现代中西医结合杂志,2008,17(21)：3255-3258.
[56]孙青,李海浪,郑意楠等.滋阴泻火方对性早熟模型大鼠下丘脑-垂体-性腺轴的影响[J].现代生物医学进展,2007,17(1)：39-58.
[57]罗小平,王慕逖.性早熟诊断与治疗的现状和进展[J].临床儿科杂志,2002,20(9)：515-519.
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[65]骆聪哲.IGF-1和1GFBP3在Graves'病糖代谢中的作用.国外医学内分泌学分册,2001,21(4)：212-214
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[2]Navarro VM, Castellano JM,, Garcia-Galiano D, et al Neuroendocrine factors in the initiation of puberty:the emergent role of kisspeptin[J]. Rev Endocr Metab Disord,2007,8(1):11-20-
[3]Smith JT, Clifton DK,Steiner RA.Regulation of the neuroendocrine reproductiveaxis by kisspeptin-GPR54 signaling[J]. Reproduction,2006,131 (4):623-30.
[4]Navarro VM, Fernandez R, Castellano J M, Roa J, Mayem A, Barreiro ML, et al. Advanced vaginal opening and precocious activation of the reproductive axis by KISS-1 peptide, the endogenous ligand of GPR54[J]. Physiol,2004,561 (2):379-386.
[5]杜敏联.性早熟的临床研究进展[J].新医学2007,38(2)：118-140.
[6]Choi JH, Shin YL, Yoo HW. Predictive factors for organic central precocious puberty and utility of simplified gonadotropin-releasing hormone tests[J]. Pediatr Int,2007,49 (6):806-810.
[7]李嫔.性早熟病因及发病机制的研究进展[J].实用儿科临床杂志,2006,20(6)：498-500.
[8]郭正霞,辛秀娟,肖君华.性早熟相关基因的研究进展[J].国外医学儿科学分册,2004,31(6)：300-302.
[9]Huhtaniemi I. Mutations affecting gonadotropin secretion and action[J]. Horm Res,2003, 60 (supp13):S21-S30.
[10]BritoVN, LatronicoAC, Arnhold IJ. et al Update on the etiology, diagnosis and therapeutic management of sexual precocity [J]. Arq BrasEndocrinol Metabol 2008,52 (1):18-31.
[11]Murphy KG. Kisspepetins. regulators of metastasis and the hypothalamic-pituitary-gonadal axis[J]. J Neuroendocrinol,2005,17 (8):519-525.
[l2]Gianetti E, Seminara S. Kisspeptin and KisslR:a critical pathway in the reproductive system[J]. Reproduction,2008,136,295-301.
[l3]Semple RK, Achermann JC, Ellery J. et al. Two novel missense mutations in G-protein-coupled receptor 54 in a patiant with hypogonadotropic hypogonadism[J]. Clin Endocrinol Metab,2005,90 (3):1849-55.
[14]Nicolas de Roux,Emmanuelle Genin, Jean-Claude Carel, et al. Hypogonadotropic hypogona-dism due to loss of function of the KiSSl-derived peptide receptor GPR54 [J]. Proc NatlAcad Sci USA,2003,100 (19):10972-10976.
[15]Gianetti E, Seminara S. Kisspeptin and KisslR:a critical pathway in the reproductive system[J]. Reproduction,2008,136,295-301.
[16]Semple RK, Achermann JC, Ellery J. et al. Two novel missense mutations in G-protein-coupled receptor 54 in a patiant with hypogonadotropic hypogonadism[J]. Clin Endocrinol Metab,2005,90 (3):1849-55.
[17]Nicolas de Roux,Emmanuelle Genin, Jean-Claude Carel, et al. Hypogonadotropic hypogona-dism due to loss of function of the KiSSl-derived peptide receptor GPR54 [J]. Proc Natl Acad Sci USA,2003,100 (19):10972-10976.
[18]Gianetti E, Seminara S. Kisspeptin and Kiss1R:a critical pathway in the reproductive system[J]. Reproduction,2008,136,295-301.
[19]曾畿生,王德芬主编.现代儿科内分泌学[M].上海科学技术文献出版社,2002,126.
[20]Toshiaki T, Hiroo N, Nobutake M, et al. Results of Long-Term Follow-Up after Treatment of Central Precocious Puberty with Leuprorelin Acetate:Evaluation of Effectiveness of Treatment and Recovery of Gonadal Function.The TAP-144-SR Japanese Study Group on Central Precocious Puberty[J]. J Clin Endocrinol Metab 2005,90:1371-1376.
[21]Roth C, Neu C, Jarry H, et al. Different effects of agonistic vs.antagonistic GnRH-analogues (Triptorelin vs.Cetrorelix)on bone modeling and remodeling in peripubertal female rats[J]. Exp Clin Endocrinol Diabetes,2005,113 (8):451-456.
[22]Roth CL, Brendel L, Ruckert C, et al. Antagonistic and agonistic GnRH analogue treatment of precocious puberty:Tracking gonadotrop in concentrations in urine[J]. Horm Res,2005,63 (5):257-262.
[23]Pucarelli I, Segni M, Ortore M, et al. Effects of combined gonadotropin-releasing hormone agonist and growth hormone therapy on adult height in precocious puberty:a further contribution[J]. J Pediatr Endocrinal Metab 2003,16 (7):1005-1010.
[24]李文京,朱逞,颜纯.达那唑对中枢性性早熟治疗的临床分析[J].北京医学,2005,27(9)：522-524.
[25]徐雯,邱志文.加减知柏地黄汤对特发性中枢性性早熟激素水平的影响[J].中医杂志,2007,48(4)：335-336.
[26]蔡德培.滋阴泻火中药为主对改善性早熟儿童骨骼发育作用及机理的探讨[J].中医杂志,1997,38(10)：615.
[27]赵均.小儿性早熟中医药治疗进展[J].陕西中医学院学报,2007,3(4)：56.
[28]曾畿生,王德芬主编.现代儿科内分泌学[M].上海科学技术文献出版社,2002,126.
[29]潘键,李海浪,郑意楠.促性腺激素拟似剂与儿童真性性早熟[J].国外医学儿科学分册2001,28(4),214-216.
[30]Tato L, et al. Use of combined Gn-RH agonist and hGH therapy for better attaining the goals in precocious puberty treatment[J]. Horm Res.1995,44:49-54.
[3l]Di Martino-Nardi J, et al. The effectof luteinizing hormone-releasing hormone analog for central precociouspuberty on growth hormone (GH) and GH-binding protein[J]. ClinEndocrinol Metab.1994,78:664-668.
[32]Harris DA, et al. Somatomedin-C in normalpuberty and in true precocious puberty before and after treatment witha potent luteinizing hormone-releasing hormone agonist[J]. Clin Endocrinol Metab.1985,61:152-159.
[33]Pasquino AM, et al. Adult height in girls with central precocious puberty treated with gonadotropin-releasing hormone analogues and growth hormone. [J]. Clin Endocrinol Metab.1999; 84:449-452.
[34]Gevers EF, Wit J M, Robinson ICAF. Effects of long-term gonadotropin releasing hormone analog treatment on growth,growth hormone (GH) secretion, GH receptors, and GH-binding protein in the rat[J]. International Pediatric Research Foundation 1998,43 (1):111-120.
[35]李文京,朱逞,颜纯.女性特发性中枢性性早熟GnRHa治疗探讨[J].临床儿科杂志2005,23(8)：530-533.
[36]Pucarelli I, Segni M, Ortore M, et al. Effects of combined gonadotropin-releasing hormone agonist and growth hormone therapy on adult height in precocious puberty:a further contribution[J]. J Pediatr Endocrinal Metab 2003,16(7):1005-1010.
[37]徐雯,邱志文.加减知柏地黄汤对特发性中枢性性早熟激素水平的影响[J].中医杂志,2007,48(4)：335-336.
[38]蔡德培.滋阴泻火中药为主对改善性早熟儿童骨骼发育作用及机理的探讨[J].中医杂志,1997,38(10)：615.
[39]赵均.小儿性早熟中医药治疗进展[J].陕西中医学院学报,2007,3(4)：56.
[40]刘百祥.逍遥散加味治疗女童性早熟34例[J].湖南中医杂志,2004,20(4)：6.
[41]项秀荷.柴胡疏肝散结汤治疗女童性早熟3例疗效观察[J].中医杂志,2001,42(1)36,61.
[42]赵鉴,虞坚尔.抗早2号方治疗女童真性性早熟的临床研究[J].中国中西医结合杂志,2003,23(3)：182-184.
[43]高华,刘云鹏.治疗女童特发性性早熟经验[J].湖北中医杂志,2004,26(12)：13-14.
[44]杨明亮.龙胆泻肝汤加减治疗女童性早熟38例[J].湖南中医杂志,2004,20(3)60.
[45]徐孝华,蔡迎庆,严靖.知柏地黄丸联合大补阴丸治疗儿童真性性早熟69例临床分析[J].南通大学学报,2005,25(2)：134-135.
[46]朱红,杨玉霞.中药治疗女童特发性早熟41例[J].江苏中医,2003,24(7)：32-33.
[47]夏龙汀,肖挹.三草汤治疗女童性早熟23例[J].实用医学临床杂志,2004,1(1)：92.
[48]秦萍.柴胡橘叶汤治疗女童特发性中枢性性早熟30例[J].上海中医药杂志,2004,38(4)：140-141.
[49]杨丽珍,郑杨,刘利.桅早汤治疗女童真性性早熟55例临床观察[J].中医药信息,2007,24(4)：48-50.
[50]叶进.抗早颗粒剂治疗女童性早熟46例[J].辽宁中医杂志,2007,34(4)：435-436.
[51]顾可钦,虞坚尔.消结合剂治疗女童性早熟62例疗效观察[J].福建医药志,1998,20(3)：88-89.
[52]陈祺.“小儿早熟1号”治疗女童性早熟69例[J].中国中西医结合杂志,2003,23(8)：632.
[53]蔡德培,陈伯英,张炜等.中药调整性早熟儿童青春发育进程的机制研究[J].中西医结合学报,2006,4(2)：166-174.
[54]曹守凤,李海浪,郑意楠等.儿科Ⅱ号合剂对真性性早熟大鼠骨生长的影响[J].江苏医药,2007,33(1)：65-67.
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