【作者】 王焕萍；

【导师】 黄凤英；

【作者基本信息】 中南大学 ， 普通妇科， 2009， 硕士

【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^-10M、10^-8M、10^-6M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^-10M、10^-8M、10^-6M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^-10M、10^-8M、10^-6M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。更多还原

【Abstract】 ObjectiveTo 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.MethodsEutopic 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^-6M GnRHⅡ; 2.treated by 10^-10、10^-8、10^-6M 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.Results1.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^-10M,10^-8M,10^-6M) 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^-10M,10^-8M,10^-6M) 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.更多还原