子宫内膜异位症间质细胞离体培养及GnRH Ⅰ、GnRH Ⅱ对其增殖抑制作用的研究
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摘要
目的:通过对子宫内膜异位症(endometriosis,EMs)患者在位及异位子宫内膜间质细胞进行体外分离培养,并予鉴定;再以不同浓度的GnRHⅡ作用于离体培养的间质细胞,并以GnRHⅠ作对照,探讨GnRHⅡ、GnRHⅠ对离体培养间质细胞的直接作用,并进行分析比较。
方法:采用胰蛋白酶、胶原酶消化结合筛网过滤及离心分离法进行子宫内膜异位症在位、异位间质细胞的分离、培养及鉴定;然后在培养液中加入10~(-10)M、10~(-8)M、10~(-6)M的GnRHⅡ,继续培养24h、48h、72h,并以GnRHⅠ类似物(戈舍瑞林,Goserelin)作对比,同时设对照组(不加GnRH),采用四唑盐比色(MTT)试验法测定间质细胞的存活情况,计算抑制率并进行比较。
结果:1.采用胰蛋白酶、胶原酶、筛网过滤及离心法,在位内膜间质细胞的分离培养成活率为83.33%,异位间质细胞的分离培养成活率为66.67%。
2.用10~(-10)M、10~(-8)M、10~(-6)M的GnRHⅡ对EMs患者离体培养的在位子宫内膜间质细胞进行干预,培养24h后的抑制率(%)分别为(15.32±2.43、26.41±2.75、38.06±4.15),培养48h后的抑制率(%)分别为(30.41±3.50、41.78±4.49、53.34±5.83),培养72h后的抑制率(%)分别为(50.01±3.70、63.29±4.47、81.58±3.44)。对异位子宫内膜间质细胞培养24h后的抑制率(%)分别为(38.42±2.67,51.35±3.70,62.84±4.13),培养48h后的抑制率(%)分别为(53.41±3.79,64.47±4.78,76.39±5.71),培养72h后的抑制率(%)分别为(70.29±2.87,83.25±3.73,93.39±4.85)。发现:依次升高浓度的GnRHⅡ在相同时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),相同浓度的GnRHⅡ在相对长时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),提示:GnRHⅡ对离体培养间质细胞抑制率呈剂量时间依赖性,差别有统计学意义(P＜0.05),且对异位子宫内膜间质细胞增殖的抑制率高于在位,差别有统计学意义(P＜0.05)。
3.以10~(-10)M、10~(-8)M、10~(-6)M的GnRHⅠ类似物(戈舍瑞林)对EMs患者离体培养的在位子宫内膜间质细胞干预,培养24h后的抑制率(%)分别为(5.42±0.93,11.19±1.29,23.97±3.00),培养48h后的抑制率(%)分别为(18.00±2.02,28.98±3.91,41.98±4.86),培养72h后的抑制率(%)分别为(32.20±2.91,46.94±4.08,60.73±4.70);对异位子宫内膜间质细胞进行干预,培养24h后的抑制率(%)分别为(25.63±1.24,37.53±2.35,48.70±3.15),培养48h后的抑制率(%)分别为(38.54±2.77,50.70±3.46,60.65±4.25),培养72h后的抑制率(%)分别为(58.38±1.36,69.75±2.18,78.69±3.75)。发现:依次升高浓度的GnRHⅠ类似物在相同时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),相同浓度的GnRHⅠ类似物在相对长时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),提示:GnRHⅠ对离体培养间质细胞抑制率呈剂量时间依赖性,差别有统计学意义(P＜0.05),且对异位子宫内膜间质细胞增殖的抑制率高于在位(P＜0.05)。
4.GnRHⅡ对EMs患者离体培养的在位与异位子宫内膜间质细胞增殖的抑制率明显高于GnRHⅠ类似物(戈舍瑞林)(P＜0.05)。
结论
1.采用胰蛋白酶、胶原酶消化结合筛网过滤及离心分离法,成功建立了子宫内膜异位症间质细胞的体外模型,为研究EMs的发病机制和药物治疗提供了实验基础。
2.卵巢EMs间质细胞培养成功率与所取部位有关。
3.GnRHⅡ对EMs患者离体培养的子宫内膜间质细胞的增殖有明显的抑制作用,呈剂量时间依赖性,尤其是对异位内膜间质细胞,且作用明显强于GnRHⅠ类似物(戈舍瑞林),提示:在EMs药物治疗方面,GnRHⅡ可能较GnRHⅠ更有效,为寻找EMs新药开发提供新的理论依据。
Objective
To culture the eutopic and ectopic endometrial stromal cells from patients with endometriosis and identify them in vitro,then different concentrations of GnRHⅡwere added in the stromal cells in vitro culture, and compared to GnRHⅠ,to investigate the direct effect of GnRHⅡand GnRHⅠon the endometrial stromal cell in vitro,to compare and analyse them.
Methods
Eutopic and ectopic endometrium were obtained from thirty patients who underwent gynaecological surgery for endometriosis.They did not take hormone in past six months,and had no other gynecologic、endocrine secretion diseases、immune and metabolismic diseases. Endometrial tissues were collected by cerettage at the time of surgery in women with ovarian endometrioma,endometrial tissues were collected from the walls of endometriomas.The stromal cells were separated from the grandular epithelium and cultured、identificated in vitro,which were divided into three groups:1.treated by 10~(-10)、10~(-8)、10~(-6)M GnRHⅡ; 2.treated by 10~(-10)、10~(-8)、10~(-6)M GnRHⅠ;3.control group,not treated by GnRH,and then were continued to culture for 24 hours、48 hours、72hours.MTT was used to measure survival cell,and calculated to compare the rate of stromal cells' inhibition in vitro.
Results
1.Eutopic endometrial stromal cells of survival rate with the method of trypsin、collagenase、mesh filtration and centeifugation was 83.33%,ectopic endometrial stromal cells of survival rate is 66.67%.
2.The eutopic endometrial stromal cell was cultured and treated with graded concentrations of GnRHⅡ(10~(-10)M,10~(-8)M,10~(-6)M) for 24 hours,cell inhibition rate(%)were(15.32±2.43、26.41±2.75、38.06±4.15) respectively,for 48 hours(%)were(30.41±3.50、41.78±4.49、53.34±5.83) respectively,for 72 hours(%)were(50.01±3.70、63.29±4.47、81.58±3.44) respectively.On the ectopic endometrial stromal cell,for 24 hours(%)were (38.42±2.67,51.35±3.70,62.84±4.13)respectively,for 48 hours(%)were (53.41±3.79,64.47±4.78,76.39±5.71)respectively,for 72 hours(%)were (70.29±2.87,83.25±3.73,93.39±4.85)respectively,the cell inhibition rate of the same endometrial stromal cell with different graded concentrations of GnRHⅡat the same time was gradually increased,significant difference was observed(P＜0.05),the cell inhibition rate of the same endometrial stromal cell with same concentrations of GnRHⅡat different time was gradually increased,significant difference was observed(P＜0.05), in dose-and time-dependent manner,significant difference was observed (P＜0.05),the cell inhibition rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell,significant difference was observed(P＜0.05).
3.The eutopic endometrial stromal cell was cultured and treated with graded concentrations of GnRHⅠ(10~(-10)M,10~(-8)M,10~(-6)M) for 24 hours,cell inhibition rate(%)were(5.42±0.93,11.19±1.29,23.97±3.00)respectively, for 48 hours(%)were(18.00±2.02,28.98±3.91,41.98±4.86)respectively, for 72 hours(%)were(32.20±2.91,46.94±4.08,60.73±4.70)respectively. On ectopic endometrial stromal cell,for 24 hours(%)were(25.63±1.24, 37.53±2.35,48.70±3.15)respectively,for 48 hours(%)were(38.54±2.77, 50.70±3.46,60.65±4.25)respectively,for 72 hours(%)were(58.38±1.36, 69.75±2.18,78.69±3.75) respectively,the cell inhibition rate of the same endometrial stromal cell with different graded concentrations of GnRHⅠat the same time was gradually increased,significant difference was observed.(P＜0.05),the cell inhibition rate of the same endometrial stromal cell with same concentrations of GnRHⅠat different time was gradually increased,significant difference was observed(P＜0.05),in dose and time dependent manner,significant difference was observed(P＜0.05), the cell inhibition rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell,ignificant difference was observed(P＜0.05).
4.GnRHⅡhad higher cell inhibition rate on endometrial stromal cell in vitro than GnRHⅠ,significant difference was observed(P＜0.05).
Conclusion
1.Highly purified eutopic and ectopic endometrial stromal cells were seperated and cultured successfully with the method of trypsin、collagenase、mesh filtration and centeifugation,providing a successful cell model for EMs,providing expremental basis for the pathogenesis and drug therapy of EMs.
2.The successful culture of ovarian stromal cells for EMs was its site.
3.GnRHⅡhad more antiproliferative effects on endometrial stromal cell than GnRHⅠanalogues(goserelin) in vitro,especially on ectopic endometrial stromal cells,it suggested GnRHⅡwas more effective than GnRHⅠ,providing a new theory for finding new drug for EMs.
方法:采用胰蛋白酶、胶原酶消化结合筛网过滤及离心分离法进行子宫内膜异位症在位、异位间质细胞的分离、培养及鉴定;然后在培养液中加入10~(-10)M、10~(-8)M、10~(-6)M的GnRHⅡ,继续培养24h、48h、72h,并以GnRHⅠ类似物(戈舍瑞林,Goserelin)作对比,同时设对照组(不加GnRH),采用四唑盐比色(MTT)试验法测定间质细胞的存活情况,计算抑制率并进行比较。
结果:1.采用胰蛋白酶、胶原酶、筛网过滤及离心法,在位内膜间质细胞的分离培养成活率为83.33%,异位间质细胞的分离培养成活率为66.67%。
2.用10~(-10)M、10~(-8)M、10~(-6)M的GnRHⅡ对EMs患者离体培养的在位子宫内膜间质细胞进行干预,培养24h后的抑制率(%)分别为(15.32±2.43、26.41±2.75、38.06±4.15),培养48h后的抑制率(%)分别为(30.41±3.50、41.78±4.49、53.34±5.83),培养72h后的抑制率(%)分别为(50.01±3.70、63.29±4.47、81.58±3.44)。对异位子宫内膜间质细胞培养24h后的抑制率(%)分别为(38.42±2.67,51.35±3.70,62.84±4.13),培养48h后的抑制率(%)分别为(53.41±3.79,64.47±4.78,76.39±5.71),培养72h后的抑制率(%)分别为(70.29±2.87,83.25±3.73,93.39±4.85)。发现:依次升高浓度的GnRHⅡ在相同时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),相同浓度的GnRHⅡ在相对长时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),提示:GnRHⅡ对离体培养间质细胞抑制率呈剂量时间依赖性,差别有统计学意义(P＜0.05),且对异位子宫内膜间质细胞增殖的抑制率高于在位,差别有统计学意义(P＜0.05)。
3.以10~(-10)M、10~(-8)M、10~(-6)M的GnRHⅠ类似物(戈舍瑞林)对EMs患者离体培养的在位子宫内膜间质细胞干预,培养24h后的抑制率(%)分别为(5.42±0.93,11.19±1.29,23.97±3.00),培养48h后的抑制率(%)分别为(18.00±2.02,28.98±3.91,41.98±4.86),培养72h后的抑制率(%)分别为(32.20±2.91,46.94±4.08,60.73±4.70);对异位子宫内膜间质细胞进行干预,培养24h后的抑制率(%)分别为(25.63±1.24,37.53±2.35,48.70±3.15),培养48h后的抑制率(%)分别为(38.54±2.77,50.70±3.46,60.65±4.25),培养72h后的抑制率(%)分别为(58.38±1.36,69.75±2.18,78.69±3.75)。发现:依次升高浓度的GnRHⅠ类似物在相同时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),相同浓度的GnRHⅠ类似物在相对长时间对相同间质细胞增殖的抑制率逐渐升高,差别有统计学意义(P＜0.05),提示:GnRHⅠ对离体培养间质细胞抑制率呈剂量时间依赖性,差别有统计学意义(P＜0.05),且对异位子宫内膜间质细胞增殖的抑制率高于在位(P＜0.05)。
4.GnRHⅡ对EMs患者离体培养的在位与异位子宫内膜间质细胞增殖的抑制率明显高于GnRHⅠ类似物(戈舍瑞林)(P＜0.05)。
结论
1.采用胰蛋白酶、胶原酶消化结合筛网过滤及离心分离法,成功建立了子宫内膜异位症间质细胞的体外模型,为研究EMs的发病机制和药物治疗提供了实验基础。
2.卵巢EMs间质细胞培养成功率与所取部位有关。
3.GnRHⅡ对EMs患者离体培养的子宫内膜间质细胞的增殖有明显的抑制作用,呈剂量时间依赖性,尤其是对异位内膜间质细胞,且作用明显强于GnRHⅠ类似物(戈舍瑞林),提示:在EMs药物治疗方面,GnRHⅡ可能较GnRHⅠ更有效,为寻找EMs新药开发提供新的理论依据。
Objective
To culture the eutopic and ectopic endometrial stromal cells from patients with endometriosis and identify them in vitro,then different concentrations of GnRHⅡwere added in the stromal cells in vitro culture, and compared to GnRHⅠ,to investigate the direct effect of GnRHⅡand GnRHⅠon the endometrial stromal cell in vitro,to compare and analyse them.
Methods
Eutopic and ectopic endometrium were obtained from thirty patients who underwent gynaecological surgery for endometriosis.They did not take hormone in past six months,and had no other gynecologic、endocrine secretion diseases、immune and metabolismic diseases. Endometrial tissues were collected by cerettage at the time of surgery in women with ovarian endometrioma,endometrial tissues were collected from the walls of endometriomas.The stromal cells were separated from the grandular epithelium and cultured、identificated in vitro,which were divided into three groups:1.treated by 10~(-10)、10~(-8)、10~(-6)M GnRHⅡ; 2.treated by 10~(-10)、10~(-8)、10~(-6)M GnRHⅠ;3.control group,not treated by GnRH,and then were continued to culture for 24 hours、48 hours、72hours.MTT was used to measure survival cell,and calculated to compare the rate of stromal cells' inhibition in vitro.
Results
1.Eutopic endometrial stromal cells of survival rate with the method of trypsin、collagenase、mesh filtration and centeifugation was 83.33%,ectopic endometrial stromal cells of survival rate is 66.67%.
2.The eutopic endometrial stromal cell was cultured and treated with graded concentrations of GnRHⅡ(10~(-10)M,10~(-8)M,10~(-6)M) for 24 hours,cell inhibition rate(%)were(15.32±2.43、26.41±2.75、38.06±4.15) respectively,for 48 hours(%)were(30.41±3.50、41.78±4.49、53.34±5.83) respectively,for 72 hours(%)were(50.01±3.70、63.29±4.47、81.58±3.44) respectively.On the ectopic endometrial stromal cell,for 24 hours(%)were (38.42±2.67,51.35±3.70,62.84±4.13)respectively,for 48 hours(%)were (53.41±3.79,64.47±4.78,76.39±5.71)respectively,for 72 hours(%)were (70.29±2.87,83.25±3.73,93.39±4.85)respectively,the cell inhibition rate of the same endometrial stromal cell with different graded concentrations of GnRHⅡat the same time was gradually increased,significant difference was observed(P＜0.05),the cell inhibition rate of the same endometrial stromal cell with same concentrations of GnRHⅡat different time was gradually increased,significant difference was observed(P＜0.05), in dose-and time-dependent manner,significant difference was observed (P＜0.05),the cell inhibition rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell,significant difference was observed(P＜0.05).
3.The eutopic endometrial stromal cell was cultured and treated with graded concentrations of GnRHⅠ(10~(-10)M,10~(-8)M,10~(-6)M) for 24 hours,cell inhibition rate(%)were(5.42±0.93,11.19±1.29,23.97±3.00)respectively, for 48 hours(%)were(18.00±2.02,28.98±3.91,41.98±4.86)respectively, for 72 hours(%)were(32.20±2.91,46.94±4.08,60.73±4.70)respectively. On ectopic endometrial stromal cell,for 24 hours(%)were(25.63±1.24, 37.53±2.35,48.70±3.15)respectively,for 48 hours(%)were(38.54±2.77, 50.70±3.46,60.65±4.25)respectively,for 72 hours(%)were(58.38±1.36, 69.75±2.18,78.69±3.75) respectively,the cell inhibition rate of the same endometrial stromal cell with different graded concentrations of GnRHⅠat the same time was gradually increased,significant difference was observed.(P＜0.05),the cell inhibition rate of the same endometrial stromal cell with same concentrations of GnRHⅠat different time was gradually increased,significant difference was observed(P＜0.05),in dose and time dependent manner,significant difference was observed(P＜0.05), the cell inhibition rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell,ignificant difference was observed(P＜0.05).
4.GnRHⅡhad higher cell inhibition rate on endometrial stromal cell in vitro than GnRHⅠ,significant difference was observed(P＜0.05).
Conclusion
1.Highly purified eutopic and ectopic endometrial stromal cells were seperated and cultured successfully with the method of trypsin、collagenase、mesh filtration and centeifugation,providing a successful cell model for EMs,providing expremental basis for the pathogenesis and drug therapy of EMs.
2.The successful culture of ovarian stromal cells for EMs was its site.
3.GnRHⅡhad more antiproliferative effects on endometrial stromal cell than GnRHⅠanalogues(goserelin) in vitro,especially on ectopic endometrial stromal cells,it suggested GnRHⅡwas more effective than GnRHⅠ,providing a new theory for finding new drug for EMs.
引文
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[18]司徒镇强,吴军正。西安:世界图书出版公司,2003,67-68。
[19]Salamalekis E,Vasiliadis TX,Kairi P,etal.Perineal endometriosis.Int J Gynecol Obstet,1990,31:75.
[20]杨菠,杨冬梓。GnRHa治疗子宫内膜异位症机制的实验研究。2002.5
[21]Sehalty AV,Nair RM,Redding FW,et al.Isolation of the lute-inizing hormone and follicle-stimulating hormone-releasing hormone from porcine hypothahuni.J Biol Chem,1971,246(23):7230-7236.
[22]Chi Keung Cheng and Peter C.K.Leung.Molecular Biology of Gonadoteopin-releasing hormone GnRH Ⅰ,GnRH Ⅱ,and Their Receptors in Humans.Endocrine Society,2005,26(2):283-306.
[23]Xiao ping Luo,Li Ding,Nasser Chegini.Gonadotropin-releasing hormone and TGF-β activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells.Am J Physiol Edocrinol Metab,2004,287:991-1001.
[24]Luo X,Xu J,Chegini N.Gonadotropin receasing hormone analogue(GnRHa)alters the expression and activation of smad in human endometrial epithelial and stromal cells[J].Reprod Biol Ednocrinol:2003,1:215
[25]Enomoto M,Endo D,Kawashima S,et al.Human type Ⅱ GnRH receptor mediates effects of GnRH on cell proliferation.Zool Sci,2004,21(7):763-770.
[26]Borroni R,Di Blasio AM,Gaffuri B,et al.Expression of GnRH receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate.Mol Cell Endocrinol,2000,159(1-2):37-43.
[27]Imai A,Takagi A and Tamaya T.Gonadotropin-releasing hormone analog repairs reduced endometrial cell apoptosis in endometriosis in vitro.Am J Obstet Gynecol, 2000,182 (5) : 1142-1146.
[28]Raga F, Casan EM, Krussel JS,WenY, Huang HY, Nezhat C, Polan ML. Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod , 1998,59:661-669.
[29]Carsten Grundker,Lars Scholotawa,et al.Antiproliferative effects of the GnRH antagonist cetrorelix and of GnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor. European Journal of Endocrinology.2004,151:141-149.
[30]Meresman GF,B ilotasM , BuquetR A,etal. Gonadotorpin-releasing hormone agonist induces apoptosis and rduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertil Steril, 2003, 80: 702-707.
[31]Chou CS,Tai CJ,et al. Dose-dependent effects of gondotopin releasing hormone on matrix metalloproproteinase (MMP)-2,and MMP-9 and tissue specific inhibitor of metallo-proteinases-1 nmessenger ribonucleic acid levels in human decidual stromal cells in vitro.Cli Endocrinol Metab [J],2003,88(2): 680-688
[32]Gazvania MR,Chistmas S.Quenby S,Kirwan J,Johnson PM and Kingsland CR.Peritoneal fluid concentrations of interleuin-8 in women with endometriosis: relationship to stage of disease.Hum Reprod1998,13:1957-1961.
[33] Gabriela F, Meresman,et al. Effect of GnRH analogues on apoptosis and release of interleukin-1β and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Human Reproduction[J].2003,18(9): 1767-1771.
[34] Meresman GF,B ilotasM , BuquetR A,etal. Gonadotorpin-releasing hormone agonist induces apoptosis and rduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertil Steril, 2003, 80: 702 — 707.
[35]Densmore VS,Urbanski HF.(GnRH )-I and GnRH-II on Relative effect of gonadotropin-releasing gonadotropin release.J Clin Endocrinol Hormone Metab. 2003,88(5):2126-2134.
[36]Cheon KW, Lee HS, Parhar IS, et al. Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle.Mol Hum Reprod,2001,7(5):447-452.
[37]刘秋红,黄凤英。Ⅱ型GnRH在子宫内膜异位症患者离体培养的子宫内膜间质细胞中的作用:[硕士学位论文].长沙:中南大学,2008.
[1]Chi Keung Cheng and Peter C.K.Leung.Molecular Biology of Gonadoteopin-releasing hormone GnRH Ⅰ,GnRH Ⅱ,and Their Receptors in Humans.Endocrine Society,2005,26(2):283-306.
[2]Olive DL.Optimizing gonadotropin-releasing hormone agonist therapy in women with endometriosis[J].Treat Endocri2nol,2004,3(2):83-89.
[3]Grundker C,Gunthert AR,Westphalen S & Emons G.Biology of the gonadotropin-releasing hormone(GnRH) system in gynecological cancers.European Journal of Endocrinology 2002,14(6):1-14.
[4]Gunter Emons,Carsten Grunker,et al.Expression of gonadotropin-releasing hormone Ⅱ(GnRH-Ⅱ) receptor in human endometrial and ovarian cancer cells and effects of GnRH-Ⅱ on tumor cell proliferation.Clinical Endocrinology and Metabolism[J],2002,87(3):1427-1430.
[5]Millar RP.GnRH Ⅱ and type Ⅱ GnRH receptors.Trends Endocrinol Metab[J],2003,14(1):35-43.
[6]Khosravi S and Leung PC Differential regulation of gonadotropin-releasing hormone(GnRH)Ⅰ and GnRHⅡ messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells.J Clin Endocrinol Metab 2003,88:663-672.
[7]王蔼明,张志文.家畜内分泌讲座[J].生物学通报,1998,11(33):26-28.
[8]朱新产,张涌.GnRH的调控机制[J].中国兽医学报,1999.
[9]Gabriela F,Meresman,et al.Effect of GnRH analogues on apoptosis and release of interleukin-1β and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis.Human Reproduction[J].2003,18(9):1767-1771.
[10]Gault PM,Morgan K,Pawson AJ,et al.Sheep exhibit novel variations in the organization of the alian type It gonadotropin-releasing hormone receptor gene[J].Endocrinology,2004,145(5):2362-2374.
[11]Enomoto M,Endo D,Kawashima S,et al.Human type Ⅱ GnRH receptor mediates effects of GnRH on cell proliferation.Zool Sci,2004,21(7):763-770.
[12]Perry CM,Brogden RN.Goserelin:a review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in benign gynaecological disorders.Drugs,1996,51:319-346.
[13]Meldrum DR,Chang RJ,Lu J,et al.Medical oophorectomy using a long acting GnRH agonist:a possible new approach to the treatment of endometriosis.J Clin Endocrinol Metab,1982,54:1081-1083.
[14]Meresman GF,B ilotasM,BuquetR A,etal.Gonadotorpin-releasing hormone agonist induces apoptosis and rduces cell proliferation in eutopic endometrial cultures from women with endometriosis.Fertil Steril,2003,80:702-707.
[15]Chatzaki E,Bax CM,Eidne KA,Anderson L,Grudzinskas JG,Gallagher CJ.The expression of gonadotropin-releasing hormonehormone and its receptor in endometrial cancer,and its relevance as an autocrine growth factor.Cancer Res,1996,56:2059-2065
[16]Chegini N,Rong H,Dou Q,Kipersztok S,Williams RS.Gonadotropinreleasing hormone(GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro.Clin Endocrinol Metab J,1996,81:3215-3221
[17]Kobayashi Y,Zhai YL,Iinuma M,Horiuchi A,Nikaido T,Fujii S。Effects of GnRH analogue on human smooth muscle cells cultured from normal myometrial and from uterine leiomyomal tissues.Mol Hum Reprod,1997,3:91-99
[18] Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL. Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue. MolHumReprod , 1998, 4:893-898
[19] Raga F, Casan EM, Krussel JS,WenY, Huang HY, Nezhat C, Polan ML . Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod, 1998 , 59:661-669
[20] Cheon KW, Lee HS, Parhar IS, Kang IS . Expression of the second isoform of gonadotropin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod , 2001 , 7:447-452.
[21] Chieko Morimoto,Tetsu Yano,et al. GnRH II as a possible cytostatic regulator in the development of endometriosis.Human Reproduction[J] . 2005, 20(11):3212-3218.
[22]Xiao ping Luo,Li Ding,Nasser Chegini. Gonadotropin-releasing hormone and TGF-β activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells. Am J Physiol Edocrinol Metab,2004,287: 991-1001.
[23]R M Moretti,M Motta,et al. Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression.Endocrine-Related Cancer,2003, 10:161-167.
[24] Borroni R, Di Blasio AM , Gaffuri B , et al. Expression of GnRH receptor gene in human ectopic endomet rial cells and inhibition of t heir proliferation by leuprolide acetate[J] . Mol Cell Endocrinol, 2000 ,159 (122): 37-43.
[25] Luo X,Xu J,Chegini N.Gonadotropin receasing hormone analogue(GnRHa) alters the expression and activation of smad in human endometrial epithelial and stromal cells[J ].Reprod Biol Ednocrinol:2003,1:215
[26] Sakamoto Y, Harada T, Horie S, et al.Tumor necrosis factor alpha- induced interleukin- 8 ( IL- 8) expression in endometriotic stromal cells probably through nuclear factor - kappa B activation: gonadotropin - releasing hormone agonist treatment reduced IL - 8 expression [J].J Clin Endocrinol Metab,2003, 88( 2) :730-735.
[27] Iwabe T, Harada T, Tsudo T, Tanikawa M, Onohara Y, Terakawa N . Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil Steril, 1998 , 69:924-930
[28] Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M,Terakawa N .Tumor necrosis factor-promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab, 2000, 85:824-829
[29] Vignali M. Molecular action of GnRH analogues on ectopic endometrial cells.Gynecol Obstet Invest. 1998,45:2-5.
[30] Shalev E,Leung P C.Gonadotropin releasing hormone and reproductive medicine [Jpobstet Gynaecol can,2003,25(2):98-113
[31] Grundker C, Schlotawa L, Viereck V, Eicke N, Horst A, Kairies B and Emons G. Antiproliferative effects of the GnRH antagonist cetrorelix and ofGnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor[J]. Eur J Endocrinol,2004, 151,141-149.
[32] Gunter Emons,Carsten Grunker,et al. Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. Clinical Endocrinology and Metabolism[J], 2002,87(3): 1427-1430.
[33] Morgan K, Conklin DC, Pawson AJ, et al. A transcrilc'tional active human type It gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the an tisense orientation on chrmosome 1q, 12 [ J]. Endocrinology, 2003, 144(2): 423-436
[2]郎景和。子宫内膜异位症研究的新里程。中华妇产科杂志,2005,40(1):3-4.
[3]Yin Wang,Hioroya Matsuo,et al.Down-regulation of proliferation and up-regulation of apoptosis by gonadotropin-releasing hormone agnoist in cultured uterine leiomyoma cells.European Journal of Endocrinology,2002,146:447-456.
[4]Klemmt PA,Carver JG,Kennedy SH,et al.Stromal cells from endometriotic lesions and endometrium from women with endometriosis have reduced decidualization capacity.Fertil Steril,2006,85(3):564-572。
[5]冷金花,郎景和,杨桂欣。子宫内膜异位症的诊治进展。中华妇产科杂志,2000,35(1):53-54.
[6]Yokoyama Y,Takahashi,Shinohara.Telomerase activity is found in the epithelial cells but not in the stromal cells in human eddometriosis cell culture[J].Mol Hum Reprod,1998,4(10):985-989.
[7]赵昀,张宏,李亚里等。子宫内膜异位症细胞模型建立及侵袭粘附性研究。解放军医学杂志,2003,28(5):425-427。
[8]Millar RP.GnRH Ⅱ and type Ⅱ GnRH receptors.Trends Endocrinol Metab[J],2003,14(1):35-43.
[9]迟晓丽,周文霞,张永祥,程军平。Ⅱ型GnRH及其受体系统研究进展.军事医学科学院院刊,2006,30(2):169-173。
[10]Khosravi S,Leung PC o Differential regulation of gonadotropin-releasing hormone(GnRH)Ⅰ and GnRHⅡ messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells.Clin Endocrinol Metab[J],2003,88:663-672.
[11]Kyung-chul Chui,Nelly Auersperg,Peter C.K.Leung.Expression and Antiproliferative Effect of A Sceond Form of Gonadotropin-Releasing Hormone in Normal and Neoplastic Ovarian Surface Epithelial Cells.Clin Endocrinol Metab [J],2001,86(10):5075-5078.
[12]Gunter Emons,Carsten Grunker,et al.Expression of gonadotropin-releasing hormone Ⅱ(GnRH-Ⅱ) receptor in human endometrial and ovarian cancer cells and effects of GnRH-Ⅱ on tumor cell proliferation.Clinical Endocrinology and Metabolism[J],2002,87(3):1427-1430.
[13]Chieko Morimoto,Tetsu Yano,et al.GnRH Ⅱ as a possible cytostatic regulator in the development of endometriosis.Human Reproduction[J],2005,20(11):3212-3218.
[14]苑春丽,韩丽英等。人子宫内膜间质细胞的体外培养。吉林大学学报,2004,30(2):267-269
[15]Randolph JF,Peeget H,Ansbacher R,et al.In vitro induction of prolactin and aromatase activity by gonadal steroids exclusively in the stroma of separated proliferative human endometrium.Am J Obstet Gynecol,1990,162(4):1109-1114.
[16]陈峥峥,凌斌.催乳素在子宫内膜异位症发病机制中作用的研究进展。国外医学(计划生育分册),2006,25(5):273-276.
[17]郝敏,石一复,周彩云.异位子宫内膜间质细胞分泌催乳素的体外研究.中华妇产科杂志,2000,35(9):527-529.
[18]司徒镇强,吴军正。西安:世界图书出版公司,2003,67-68。
[19]Salamalekis E,Vasiliadis TX,Kairi P,etal.Perineal endometriosis.Int J Gynecol Obstet,1990,31:75.
[20]杨菠,杨冬梓。GnRHa治疗子宫内膜异位症机制的实验研究。2002.5
[21]Sehalty AV,Nair RM,Redding FW,et al.Isolation of the lute-inizing hormone and follicle-stimulating hormone-releasing hormone from porcine hypothahuni.J Biol Chem,1971,246(23):7230-7236.
[22]Chi Keung Cheng and Peter C.K.Leung.Molecular Biology of Gonadoteopin-releasing hormone GnRH Ⅰ,GnRH Ⅱ,and Their Receptors in Humans.Endocrine Society,2005,26(2):283-306.
[23]Xiao ping Luo,Li Ding,Nasser Chegini.Gonadotropin-releasing hormone and TGF-β activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells.Am J Physiol Edocrinol Metab,2004,287:991-1001.
[24]Luo X,Xu J,Chegini N.Gonadotropin receasing hormone analogue(GnRHa)alters the expression and activation of smad in human endometrial epithelial and stromal cells[J].Reprod Biol Ednocrinol:2003,1:215
[25]Enomoto M,Endo D,Kawashima S,et al.Human type Ⅱ GnRH receptor mediates effects of GnRH on cell proliferation.Zool Sci,2004,21(7):763-770.
[26]Borroni R,Di Blasio AM,Gaffuri B,et al.Expression of GnRH receptor gene in human ectopic endometrial cells and inhibition of their proliferation by leuprolide acetate.Mol Cell Endocrinol,2000,159(1-2):37-43.
[27]Imai A,Takagi A and Tamaya T.Gonadotropin-releasing hormone analog repairs reduced endometrial cell apoptosis in endometriosis in vitro.Am J Obstet Gynecol, 2000,182 (5) : 1142-1146.
[28]Raga F, Casan EM, Krussel JS,WenY, Huang HY, Nezhat C, Polan ML. Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod , 1998,59:661-669.
[29]Carsten Grundker,Lars Scholotawa,et al.Antiproliferative effects of the GnRH antagonist cetrorelix and of GnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor. European Journal of Endocrinology.2004,151:141-149.
[30]Meresman GF,B ilotasM , BuquetR A,etal. Gonadotorpin-releasing hormone agonist induces apoptosis and rduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertil Steril, 2003, 80: 702-707.
[31]Chou CS,Tai CJ,et al. Dose-dependent effects of gondotopin releasing hormone on matrix metalloproproteinase (MMP)-2,and MMP-9 and tissue specific inhibitor of metallo-proteinases-1 nmessenger ribonucleic acid levels in human decidual stromal cells in vitro.Cli Endocrinol Metab [J],2003,88(2): 680-688
[32]Gazvania MR,Chistmas S.Quenby S,Kirwan J,Johnson PM and Kingsland CR.Peritoneal fluid concentrations of interleuin-8 in women with endometriosis: relationship to stage of disease.Hum Reprod1998,13:1957-1961.
[33] Gabriela F, Meresman,et al. Effect of GnRH analogues on apoptosis and release of interleukin-1β and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis. Human Reproduction[J].2003,18(9): 1767-1771.
[34] Meresman GF,B ilotasM , BuquetR A,etal. Gonadotorpin-releasing hormone agonist induces apoptosis and rduces cell proliferation in eutopic endometrial cultures from women with endometriosis. Fertil Steril, 2003, 80: 702 — 707.
[35]Densmore VS,Urbanski HF.(GnRH )-I and GnRH-II on Relative effect of gonadotropin-releasing gonadotropin release.J Clin Endocrinol Hormone Metab. 2003,88(5):2126-2134.
[36]Cheon KW, Lee HS, Parhar IS, et al. Expression of the second isoform of gonadotrophin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle.Mol Hum Reprod,2001,7(5):447-452.
[37]刘秋红,黄凤英。Ⅱ型GnRH在子宫内膜异位症患者离体培养的子宫内膜间质细胞中的作用:[硕士学位论文].长沙:中南大学,2008.
[1]Chi Keung Cheng and Peter C.K.Leung.Molecular Biology of Gonadoteopin-releasing hormone GnRH Ⅰ,GnRH Ⅱ,and Their Receptors in Humans.Endocrine Society,2005,26(2):283-306.
[2]Olive DL.Optimizing gonadotropin-releasing hormone agonist therapy in women with endometriosis[J].Treat Endocri2nol,2004,3(2):83-89.
[3]Grundker C,Gunthert AR,Westphalen S & Emons G.Biology of the gonadotropin-releasing hormone(GnRH) system in gynecological cancers.European Journal of Endocrinology 2002,14(6):1-14.
[4]Gunter Emons,Carsten Grunker,et al.Expression of gonadotropin-releasing hormone Ⅱ(GnRH-Ⅱ) receptor in human endometrial and ovarian cancer cells and effects of GnRH-Ⅱ on tumor cell proliferation.Clinical Endocrinology and Metabolism[J],2002,87(3):1427-1430.
[5]Millar RP.GnRH Ⅱ and type Ⅱ GnRH receptors.Trends Endocrinol Metab[J],2003,14(1):35-43.
[6]Khosravi S and Leung PC Differential regulation of gonadotropin-releasing hormone(GnRH)Ⅰ and GnRHⅡ messenger ribonucleic acid by gonadal steroids in human granulosa luteal cells.J Clin Endocrinol Metab 2003,88:663-672.
[7]王蔼明,张志文.家畜内分泌讲座[J].生物学通报,1998,11(33):26-28.
[8]朱新产,张涌.GnRH的调控机制[J].中国兽医学报,1999.
[9]Gabriela F,Meresman,et al.Effect of GnRH analogues on apoptosis and release of interleukin-1β and vascular endothelial growth factor in endometrial cell cultures from patients with endometriosis.Human Reproduction[J].2003,18(9):1767-1771.
[10]Gault PM,Morgan K,Pawson AJ,et al.Sheep exhibit novel variations in the organization of the alian type It gonadotropin-releasing hormone receptor gene[J].Endocrinology,2004,145(5):2362-2374.
[11]Enomoto M,Endo D,Kawashima S,et al.Human type Ⅱ GnRH receptor mediates effects of GnRH on cell proliferation.Zool Sci,2004,21(7):763-770.
[12]Perry CM,Brogden RN.Goserelin:a review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in benign gynaecological disorders.Drugs,1996,51:319-346.
[13]Meldrum DR,Chang RJ,Lu J,et al.Medical oophorectomy using a long acting GnRH agonist:a possible new approach to the treatment of endometriosis.J Clin Endocrinol Metab,1982,54:1081-1083.
[14]Meresman GF,B ilotasM,BuquetR A,etal.Gonadotorpin-releasing hormone agonist induces apoptosis and rduces cell proliferation in eutopic endometrial cultures from women with endometriosis.Fertil Steril,2003,80:702-707.
[15]Chatzaki E,Bax CM,Eidne KA,Anderson L,Grudzinskas JG,Gallagher CJ.The expression of gonadotropin-releasing hormonehormone and its receptor in endometrial cancer,and its relevance as an autocrine growth factor.Cancer Res,1996,56:2059-2065
[16]Chegini N,Rong H,Dou Q,Kipersztok S,Williams RS.Gonadotropinreleasing hormone(GnRH) and GnRH receptor gene expression in human myometrium and leiomyomata and the direct action of GnRH analogs on myometrial smooth muscle cells and interaction with ovarian steroids in vitro.Clin Endocrinol Metab J,1996,81:3215-3221
[17]Kobayashi Y,Zhai YL,Iinuma M,Horiuchi A,Nikaido T,Fujii S。Effects of GnRH analogue on human smooth muscle cells cultured from normal myometrial and from uterine leiomyomal tissues.Mol Hum Reprod,1997,3:91-99
[18] Dong KW, Marcelin K, Hsu MI, Chiang CM, Hoffman G, Roberts JL. Expression of gonadotropin-releasing hormone (GnRH) gene in human uterine endometrial tissue. MolHumReprod , 1998, 4:893-898
[19] Raga F, Casan EM, Krussel JS,WenY, Huang HY, Nezhat C, Polan ML . Quantitative gonadotropin-releasing hormone gene expression and immunohistochemical localization in human endometrium throughout the menstrual cycle. Biol Reprod, 1998 , 59:661-669
[20] Cheon KW, Lee HS, Parhar IS, Kang IS . Expression of the second isoform of gonadotropin-releasing hormone (GnRH-II) in human endometrium throughout the menstrual cycle. Mol Hum Reprod , 2001 , 7:447-452.
[21] Chieko Morimoto,Tetsu Yano,et al. GnRH II as a possible cytostatic regulator in the development of endometriosis.Human Reproduction[J] . 2005, 20(11):3212-3218.
[22]Xiao ping Luo,Li Ding,Nasser Chegini. Gonadotropin-releasing hormone and TGF-β activate MAP kinase and differentially regulate fibronectin expression in endometrial epithelial and stromal cells. Am J Physiol Edocrinol Metab,2004,287: 991-1001.
[23]R M Moretti,M Motta,et al. Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression.Endocrine-Related Cancer,2003, 10:161-167.
[24] Borroni R, Di Blasio AM , Gaffuri B , et al. Expression of GnRH receptor gene in human ectopic endomet rial cells and inhibition of t heir proliferation by leuprolide acetate[J] . Mol Cell Endocrinol, 2000 ,159 (122): 37-43.
[25] Luo X,Xu J,Chegini N.Gonadotropin receasing hormone analogue(GnRHa) alters the expression and activation of smad in human endometrial epithelial and stromal cells[J ].Reprod Biol Ednocrinol:2003,1:215
[26] Sakamoto Y, Harada T, Horie S, et al.Tumor necrosis factor alpha- induced interleukin- 8 ( IL- 8) expression in endometriotic stromal cells probably through nuclear factor - kappa B activation: gonadotropin - releasing hormone agonist treatment reduced IL - 8 expression [J].J Clin Endocrinol Metab,2003, 88( 2) :730-735.
[27] Iwabe T, Harada T, Tsudo T, Tanikawa M, Onohara Y, Terakawa N . Pathogenetic significance of increased levels of interleukin-8 in the peritoneal fluid of patients with endometriosis. Fertil Steril, 1998 , 69:924-930
[28] Iwabe T, Harada T, Tsudo T, Nagano Y, Yoshida S, Tanikawa M,Terakawa N .Tumor necrosis factor-promotes proliferation of endometriotic stromal cells by inducing interleukin-8 gene and protein expression. J Clin Endocrinol Metab, 2000, 85:824-829
[29] Vignali M. Molecular action of GnRH analogues on ectopic endometrial cells.Gynecol Obstet Invest. 1998,45:2-5.
[30] Shalev E,Leung P C.Gonadotropin releasing hormone and reproductive medicine [Jpobstet Gynaecol can,2003,25(2):98-113
[31] Grundker C, Schlotawa L, Viereck V, Eicke N, Horst A, Kairies B and Emons G. Antiproliferative effects of the GnRH antagonist cetrorelix and ofGnRH-II on human endometrial and ovarian cancer cells are not mediated through the GnRH type I receptor[J]. Eur J Endocrinol,2004, 151,141-149.
[32] Gunter Emons,Carsten Grunker,et al. Expression of gonadotropin-releasing hormone II (GnRH-II) receptor in human endometrial and ovarian cancer cells and effects of GnRH-II on tumor cell proliferation. Clinical Endocrinology and Metabolism[J], 2002,87(3): 1427-1430.
[33] Morgan K, Conklin DC, Pawson AJ, et al. A transcrilc'tional active human type It gonadotropin-releasing hormone receptor gene homolog overlaps two genes in the an tisense orientation on chrmosome 1q, 12 [ J]. Endocrinology, 2003, 144(2): 423-436