GnRHⅡ在子宫内膜异位症患者中的表达及其对离体培养内膜间质细胞的作用
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摘要
子宫内膜异位症(endometriosis, EMs),是生育年龄妇女常见病和多发病之一。其发病机制不清、复发率高、治疗棘手而成为研究的难点和热点。因此,研究EMs的发病机制以及寻找行之有效的治疗方法,成为EMs研究中亟待解决的问题。
近年来,促性腺激素释放激素类似物(GnRHa)与腹腔镜手术相结合,在EMs的治疗上得到肯定并得以广泛应用。最近研究发现：Ⅱ型促性腺激素释放激素(GnRHⅡ)在某些生殖系统肿瘤如卵巢癌、乳腺癌和前列腺癌中,能抑制肿瘤细胞生长,且对子宫内膜癌和卵巢癌细胞的抗增殖作用比GnRHⅠ更强。而子宫内膜异位症也是一种激素依赖性疾病,且具有恶性肿瘤的一些生物学特征。
因此,本文试图通过以下三部分来研究GnRHⅡ蛋白在子宫内膜异位症患者中的表达及其对离体培养的异位/在位子宫内膜间质细胞的作用：(一)应用免疫组化SP法检测GnRHⅡ蛋白在子宫内膜异位症患者异位及在位子宫内膜中是否有表达,并以正常子宫内膜作对照,了解其表达是否有差异以及其在正常子宫内膜中是否有周期性变化；(二)对异位及在位子宫内膜组织进行分离,培养其内膜间质细胞并进行鉴定,以不同浓度的GnRHⅡ(10-10M、10-8M、10-6M)进行干预,采用Hoechst染色和流式细胞术检测离体培养的子宫内膜间质细胞的细胞凋亡发生情况,了解GnRHⅡ是否直接对离体培养的子宫内膜间质细胞有促凋亡的作用；(三)以不同浓度的GnRHⅡ(10-10M、10-8M、10-6M)作用于离体培养的子宫内膜间质细胞,并以已知作用的GnRHⅠ类似物(以戈舍瑞林为例)作对比,通过测定间质细胞分泌的血管内皮生长因子(vascular endothelial growth factor, VEGF)的浓度变化、以及比较其对离体培养的子宫内膜间质细胞生长抑制率的影响,来探讨GnRHⅡ对离体培养的子宫内膜间质细胞是否有抗增殖作用。为我们探讨内源性GnRHⅡ蛋白表达是否与内异症发病有关、外源性GnRHⅡ是否可应用于临床治疗子宫内膜异位症及其治疗该病的可能作用机制；提供新的实验和理论依据。关于上述系列研究,目前国内外尚未见报道。
目的：检测GnRHⅡ蛋白在子宫内膜异位症患者异位/在位子宫内膜中的表达情况,并以正常子宫内膜作对照,了解其表达是否有差异；同时分析GnRHⅡ蛋白的表达是否与正常子宫内膜月经周期有关。
材料与方法：选自2007年3月至2008年6月在中南大学湘雅二医院住院、手术及病理切片确诊为内膜异位症、卵巢巧克力囊肿患者30例,取异位/在位子宫内膜组织作试验组；另选同期因非子宫内膜异位症患者的正常子宫内膜组织40例作为对照组(病理切片HE染色内膜正常)。术前半年均无激素应用史及其它疾病史。采用免疫组化SP法检测GnRHⅡ蛋白在异位内膜、在位内膜及正常子宫内膜组织中的表达情况,并分析、比较其表达是否有差异。
结果：
1、GnRHⅡ蛋白在子宫内膜异位症患者异位、在位子宫内膜及正常子宫内膜中均有表达,阳性表达定位于子宫内膜腺体及间质细胞的细胞浆,呈浅黄色、黄色、棕黄色或黄褐色。
2、GnRHⅡ蛋白在内异症组异位内膜、在位内膜及对照组正常内膜的表达依次增强,两两比较差异有显著性(P<0.05)。
3、GnRHⅡ蛋白在正常子宫内膜分泌期表达强于增生期(P<0.05),且以分泌早中期最强,显著强于增生期和分泌晚期,差异有显著性(P<0.01)；而内异症异位组或在位组的分泌期和增生期比较,差异无显著性(P>0.05)。
结论：
1、GnRHⅡ蛋白在子宫内膜异位症患者异位和在位子宫内膜、及正常子宫内膜中均有阳性表达,且正常内膜的表达有周期性变化,提示：GnRHⅡ蛋白可能在人类子宫内膜的病理及生理过程中都起着重要作用。
2、GnRHⅡ蛋白在内异症异位/在位子宫内膜中低表达(P<0.05),提示内源性GnRHⅡ蛋白表达减少可能是内异症发生发展的原因之一。
3、GnRHⅡ蛋白在内异症在位内膜中的表达低于正常子宫内膜(P<0.05),提示内异症的发生可能与在位内膜的异常特性有关。
目的：为探讨外源性的GnRHⅡ蛋白是否对子宫内膜异位症的细胞有直接作用,有必要建立离体的在位及异位子宫内膜间质细胞的体外细胞模型；再用不同浓度GnRHⅡ直接作用于这些间质细胞,检测并分析GnRHⅡ对其细胞凋亡的影响。
方法：取30例内异症异位及在位内膜组织,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。并以不同浓度的GnRHⅡ(0、10-10M、10-8M、10-6M)作用于这些离体培养的子宫内膜间质细胞,采用Hoechst染色和流式细胞术检测离体培养子宫内膜间质细胞的细胞凋亡发生情况。
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、异位内膜间质细胞的培养成活率：红色病灶处为66.67%,褐色病灶处为10.00%。
3、离体培养情况下,对数生长期的子宫内膜间质细胞给予不同浓度的GnRHⅡ(0、10-10 M、10-8 M、10-6M)干预后,采用Hoechst染色对不同处理组细胞进行细胞凋亡形态学的分析。结果显示：不同浓度的GnRHⅡ对离体培养的在位和异位子宫内膜间质细胞可诱导凋亡发生,表现为核碎裂,核溶解,核固缩或凋亡小体。其凋亡率(%)分别为：(5.78±0.53,5.79±0.66),(18.62±2.59,31.30±2.93),(47.41±3.57,59.28±4.25), (66.16±5.46,79.43±6.42),两两比较,差异有显著性(P<0.05),提示：不同浓度的GnRHⅡ对离体培养的异位内膜间质细胞的凋亡率明显高于在位(P<0.05),且呈剂量依赖性(P<0.05)。
4、离体培养情况下,对数生长期的子宫内膜间质细胞给予不同浓度的GnRHⅡ(0、10-10 M、10-8 M、10-6M)干预后,采用流式细胞术对不同处理组细胞进行细胞凋亡率检测,结果显示：不同浓度的GnRHⅡ对离体培养的在位和异位子宫内膜间质细胞可诱导凋亡发生,其凋亡率(%)分别为：(5.87±0.59,5.99±0.78),(19.30±3.28,33.40±3.97), (46.30±5.32,62.41±6.74), (69.70±8.77,83.30±9.42)两两比较,差异有显著性(P<0.05),与HoeChst染色检测的凋亡率变化一致。提示：不同浓度的GnRHⅡ对离体培养的异位内膜间质细胞促凋亡作用明显强于在位内膜间质细胞(P<0.05),且呈剂量依赖性(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研究提供了实验基础。
2.卵巢EMs间质细胞培养成功率之一与所取部位有关,红色病变处成功率较高。
3.外源性的GnRHⅡ可直接促进离体培养的EMs间质细胞的细胞凋亡,呈剂量依赖性,且促凋亡作用异位明显强于在位。为内异症促凋亡方面的新药开发提供实验和理论依据。
目的：通过对子宫内膜异位症(endometriosis, EMs)患者在位及异位内膜间质细胞分离、培养,检测其分泌的血管内皮生长因子(vascular endothelial growth factor, VEGF)的浓度,再用不同浓度的GnRHⅡ进行干预,并以已知作用的GnRHⅠ类似物(以戈舍瑞林为例)作对比,分析其VEGF的浓度变化和GnRHⅡ、GnRHⅠ对其细胞增殖抑制率的影响,为进一步阐明GnRHⅡ的可能作用机制及治疗机制,提供新的实验依据。
方法：
1、在培养液中加入10-10M、10-8M、10-6M的GnRHⅡ,以GnRHⅠ类似物(戈舍瑞林,Goserelin)作对比,同时设对照组(不加GnRH),采用酶联免疫吸附法(ELISA)测定培养液中VEGF浓度,并进行比较。
2、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、EMs患者离体培养的异位子宫内膜间质细胞经48小时培养,能分泌VEGF,分泌量与在位子宫内膜间质细胞的相近,两者比较无统计学差异(P>0.05)。
2、不同浓度的GnRHII对EMs患者离体培养的在位和异位子宫内膜间质细胞VEGF的分泌有明显的抑制作用(P<0.01),呈剂量依赖性(P<0.01),且较GnRH I类似物(戈舍瑞林)的作用更强(P<0.05)。
3、不同浓度的GnRH II对EMs患者离体培养的异位子宫内膜间质细胞VEGF分泌的抑制作用明显强于在位(P<0.01)。
4、用10-100M、10-8M、10-6M的GnRHII对EMs患者离体培养的在位和异位子宫内膜间质细胞进行干预,计算培养24h、48h、72h后的抑制率(%),统计发现：依次升高浓度的GnRHII在相同时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(P<0.05)；相同浓度的GnRHII在相对长时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(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,差异有统计学意义(P<0.05)。
5、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和异位子宫内膜间质细胞干预,计算培养24h、48h、72h后的抑制率(%),统计发现：依次升高浓度的GnRH I类似物在相同时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(P<0.05)；相同浓度的GnRH I类似物在相对长时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(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,差异有统计学意义(P＜0.05)。
6、GnRHII对EMs患者离体培养在位与异位子宫内膜间质细胞增殖的抑制率明显高于GnRH I类似物(戈舍瑞林),差异有统计学意义(P＜0.05)。
结论：
1、EMs患者的异位子宫内膜间质细胞具有分泌VEGF的功能,分泌量与在位子宫内膜的相近,这对EMs的形成和发展可能起重要作用。
2、不同浓度GnRHII对EMs患者离体培养的异位子宫内膜间质细胞VEGF的分泌有明显的抑制作用,呈剂量依赖性,且作用明显强于GnRHI类似物(戈舍瑞林)。
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,尤其是对异位子宫内膜间质细胞,且抑制作用明显强于GnRHI类似物(戈舍瑞林)。
4、在EMs抗血管形成和抗增殖方面的药物治疗中,GnRHII有可能较GnRHI更有效,为寻找EMs新药开发提供新的实验和理论依据。
Endometriosis (short for EMs) is one common and frequently-occurring disease in women of reproductive age. Its pathogenesis is unclear、recurrent rate is high、treatment is thorny, these make EMs become difficult and hot research. Therefore, to study the pathogenesis of EMs and to find effective treatment methods,which becomes an urgent problem. In recent years, the combination of gonadotropin-releasing hormone analogues (GnRHa) and laparoscopic surgery in the treatment of EMs are sure to be useful and effective. Recent study found that:Ⅱ-type gonadotropin-releasing hormone (GnRHⅡ) can inhibit cell growth in some tumors of the reproductive system,such as ovarian cancer、breast cancer and prostate cancer, and had stronger anti-proliferative effects than GnRH I in endometrial cancer and ovarian cancer cells. Endometriosis is a hormone-dependent disease, which has some biological behaviors of malignant tumor. Therefore, our article attempts to study the expression of the GnRH II in patients with endometriosis and its effects on the eutopic and ectopic endometrial stromal cells in vitro culture in the following three parts:(1). Using the method of SP immunohistochemistry to investigate whether there is expression of GnRHⅡprotein in ectopic and eutopic endomentrium from patients with endometriosis, and normal endometrium was used as control, to study whether there are differences in their expression, as well as whether the expression has cyclical changes in the these endometriums. (2) The endometrial stromal cells were isolated、cultured and identified from ectopic and eutopic endometrium tissues In vitro, then different concentrations of GnRH II (0、10-10M、10-8 M、10-6M) were added in the stromal cells in vitro culture, to detect the occurrence of apoptosis in these endometrial stromal cells with the method of Hoechst staining and flow cytometry test, in order to investigate whether GnRHⅡhas a direct inducing apoptosis effect on cultured endometrial stromal cells in vitro; (3) Different concentrations of GnRHⅡ(0、10-10M、10-8M、10-6M) were added in the stromal cells in vitro culture, and compared to GnRHⅠ, to detect the changes of concentrations for VEGF; and to compare these cells proliferation inhibitory effects by analysing cells inhibitory rate in endometrial stromal cell in vitro, so as to investigate whether GnRHⅡhas antiproliferative effects on the endometrial stromal cell in vitro. In short, to investigate the expression of endogenous GnRHⅡprotein whether has relations with the pathogenesis of endometriosis, and exogenous GnRHⅡwhether can be applied to clinical treatment of endometriosis and its possible treatment mechanisms in the patients with EMs, which providing new experimental and theoretical basis. About the above-mentioned serial studies, It has not been reported at home and abroad.
Objective:To study the expression of GnRHⅡprotein in patients with-and without-endometriosis and the relation to the menstrual cycle.
Methods:Thirty ectopic and thirty eutopic biopsy endometrial specimens were obtained from women with untreated endomentriosis and 40 controls were studied, which came from March 2007 to June 2008 in the Second Xiangya Hospital of Central South University. The expression of GnRHⅡprotein were tested from the ectopic、eutopic、and normal endometrium by immuneohistochemistry SP methods, and compared them.
Results:
1. The expression of GnRHⅡprotein positive immune staining is located in cytoplasm of endometrial stromal cells and gland cells in the all specimens.
2 The expression of GnRHⅡprotein was lower in ectopic endometrium than eutopic endometrium of women with endometriosis (P<0.05),and were lower in the eutopic and ectopic endometrium with endometriosis than the nomal endometrium of women without endometriosis (P<0.05).
3. The expression of GnRHⅡprotein was obvious higher in secretory phase than that of in hyperplasia phase in normal endometrial (P<0.05),especially in early and middle secretory phase(P<0.05), but there was no significant difference in eutopic and ectopic endometrium from endometriosis in the menstrual cycle(P>0.05).
Conclusion:
1. The positive expression of GnRHⅡin the ectopic、eutopic、and normal endometrium, and its expression was related to the menstrual cycle in normal not in endometriotic endometrium, suggests:it may play physiological and pathological roles in above endometrium.
2. The lower expression of GnRHⅡin the ectopic、eutopic endometrium (P<0.05) may cause endometriosis.
3. The lower expression of GnRHⅡin the eutopic endometrium suggests endometriosis may be retated to eutopic endometrial abnormal character.
Objective:To culture the eutopic and ectopic endometrial stromal cells from patients with endometriosis and identify them in vitro; Then, To study the effect of GnRH II on these cells apoptosis in cultured endometrial stromal cells in vitro.
Methods:First, thirty ectopic and thirty eutopic biopsy endometrial specimens were obtained from women with untreated endomentriosis, the stromal cells were separated、cultured from the grandular epithelium, then identified in vitro; Second, to detect the cell apoptosis ratio after different concentration (0,10-10 M,10-8 M,10-6 M) GnRH II were used in these stromal cells In vitro by Hoechst staining and flow cytometry test.
Results:
1. Eutopic endometrial stromal cells of survival rate was 83.33%,ectopic endometrial stromal cells of survival rate is 66.67% by the method of trypsin、collagenase、mesh filtration and centeifugation.
2. The ectopic endometrial stromal cells of survival rate(%):the red site is 66.67%, the black site is 10.00%.
3. In vitro culture, the apoptosis cell of endometrial stromal cells can be observed after GnRH II treatment (0,10-10 M,10-8 M,10-6 M), The Hoechst dye showed that eutopic and ectopic endometrial stromal cells can be induced apoptosis:cell shrinkage、nucleus condensation、and sometimes apoptopic body can be seen. The rate of apoptosis(%) is (5.78±0.53,5.79±0.66), (18.62±2.59,31.30±2.93), (47.41±3.57, 59.28±4.25), (66.16±5.46,79.43±6.42), There were statistically significant differences among different concentration experimental groups (P<0.05), the apoptosis rate of ectopic endometrial stromal cells was higher than eutopic endometrial stromal cells (P<0.05),and were dose-dependent.
4. In vitro culture, the apoptosis cell of endometrial stromal cells can be observed after GnRHⅡtreatment (0,10-10 M,10-8 M,10-6 M), the flow cytometry test results showed that eutopic and ectopic endometrial stromal cells can be induced apoptosis. The rate of apoptosis (%) is (5.87±0.59,5.99±0.78), (19.30±3.28,33.40±3.97), (46.30±5.32, 62.41±6.74), (69.70±8.77,83.30±9.42), There were statistically significant differences among different concentration experimental groups (P<0.05), the apoptosis rate of ectopic endometrial stromal cells was higher than eutopic endometrial stromal cells (P<0.05),and were dose-dependent.
Conclusion:
1.Highly purified eutopic and ectopic endometrial stromal cells were seperated and cultured successfully by the method of trypsin、collagenase、mesh filtration and centeifugation, which provides a successful cell model and expremental basis for the pathogenesis and drug therapy of EMs.
2. The one of successful culture of ovarian stromal cells for EMs was related to its site.
3. Exogenous GnRH II can increase stromal cells apoptosis in dose-dependent in vitro for the patients with EMs, and the ectopic was higher than the eutopic, which suggests GnRH II may play an important role in treatment EMs by inducing cell apoptosis.
Objective:
1. To study the effects of GnRHⅡon the secretion of VEGF concentration by eutopic and ectopic endometrial stromal cells cultured in vitro, and contrast with GnRH I, which may provide theoretical basis for exploring GnRH II new treatment for EMs.
2. Different concentrations of GnRH II were added in the cultured stromal cells in vitro, and compared to GnRH I, to detect the cell proliferation inhibitory rates of GnRH II and GnRH I on the endometrial stromal cells in vitro, and compare and analyse them.
Methods:
1. The above eutopic and ectopic stromal cells in vitro were divided into three groups:(1) treated by 10-10M、10-8M、10-6M GnRHⅡ; (2) treated by 10-10 M、10-8 M、10-6M GnRH I; (3) control group, not treated by GnRH. ELISA test was used to measure the concentration of VEGF protein in the medium of above three groups.
2. The above eutopic and ectopic stromal cells in vitro were divided into three groups:(1) treated by 10-10M、10-8M、10-6M GnRHⅡ; (2) treated by 10-10M、10-8M、10-6M GnRH I; (3) control group, not treated by GnRH,and then were continued to culture for 24 hours、48 hours、72hours. MTT method was used to detect survival cell, and calculated to compare the inhibitory rate of stromal cells in vitro.
Results:
1. There is no difference between the VEGF protein concentration by eutopic and ectopic stromal cells secreting in the medium after being cultured for 48 hours in vitro (P>0.05)
2.10-10M、10-8M、10-6M GnRHⅡcan dose-dependent reduce VEGF protein concentration by endometrial stromal cells secreting (P<0.01) and the inhibitory to endometrial stromal cells is stronger than GnRH I (P<0.05).
3.The inhibitory effect of GnRH II on VEGF in ectopic stromal cells is stronger than that of in eutopic stromal cells (P<0.01)
4.The eutopic and ectopic endometrial stromal cell was cultured and treated with gradual high concentrations of GnRH II (10-10M,10-8M,10-6M) for 24 hours、for 48 hours、for 72 hours respectively. The inhibitory rate(%) of cell proliferation in the same endometrial stromal cell with different gradual high concentrations of GnRHⅡat the same time was gradually increased, significant difference was observed(P<0.05); the cell inhibitory rate(%) of the same endometrial stromal cell with same concentrations of GnRH II at different time was gradually increased, significant difference was observed(P<0.05); the cell inhibitory rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell, significant difference was observed (P<0.05).
5.The eutopic and ectopic endometrial stromal cell was cultured and treated with gradual high concentrations of GnRH I (10-10M,10-8M,10-6M) for 24 hours、for 48 hours、for 72 hours respectively. The inhibitory rate(%) of cell proliferation in the same endometrial stromal cell with different gradual high concentrations of GnRH I at the same time was gradually increased, significant difference was observed.(P<0.05),the cell inhibitory rate of the same endometrial stromal cell with same concentrations of GnRH I at 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 inhibitory rate(%) of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell, significant difference was observed (P<0.05).
6. GnRHⅡhad higher inhibitory rate(%) of cell proliferation in endometrial stromal cell in vitro than GnRH I, significant difference was observed (P<0.05).
Conclusions:
1.Ectopic stromal cells cultured in vitro can secrete VEGF, which has no difference with the eutopic stromal cells.This may play an important role in the formation and development of EMs.
2.GnRHⅡcan reduce VEGF protein secreted by endometrial stromal cell cultured in vitro in dose-dependent manner, and the inhibition is stronger than GnRHⅠ.which may provide theoretic basis for exploring new treatment for EMs.
3.GnRHⅡhad more antiproliferative effects on endometrial stromal cell than GnRH I analogues (goserelin) in vitro,especially in ectopic endometrial stromal cells, in dose-and time-dependent manner, which may provide a new theory for researching new drug for EMs.
近年来,促性腺激素释放激素类似物(GnRHa)与腹腔镜手术相结合,在EMs的治疗上得到肯定并得以广泛应用。最近研究发现：Ⅱ型促性腺激素释放激素(GnRHⅡ)在某些生殖系统肿瘤如卵巢癌、乳腺癌和前列腺癌中,能抑制肿瘤细胞生长,且对子宫内膜癌和卵巢癌细胞的抗增殖作用比GnRHⅠ更强。而子宫内膜异位症也是一种激素依赖性疾病,且具有恶性肿瘤的一些生物学特征。
因此,本文试图通过以下三部分来研究GnRHⅡ蛋白在子宫内膜异位症患者中的表达及其对离体培养的异位/在位子宫内膜间质细胞的作用：(一)应用免疫组化SP法检测GnRHⅡ蛋白在子宫内膜异位症患者异位及在位子宫内膜中是否有表达,并以正常子宫内膜作对照,了解其表达是否有差异以及其在正常子宫内膜中是否有周期性变化；(二)对异位及在位子宫内膜组织进行分离,培养其内膜间质细胞并进行鉴定,以不同浓度的GnRHⅡ(10-10M、10-8M、10-6M)进行干预,采用Hoechst染色和流式细胞术检测离体培养的子宫内膜间质细胞的细胞凋亡发生情况,了解GnRHⅡ是否直接对离体培养的子宫内膜间质细胞有促凋亡的作用；(三)以不同浓度的GnRHⅡ(10-10M、10-8M、10-6M)作用于离体培养的子宫内膜间质细胞,并以已知作用的GnRHⅠ类似物(以戈舍瑞林为例)作对比,通过测定间质细胞分泌的血管内皮生长因子(vascular endothelial growth factor, VEGF)的浓度变化、以及比较其对离体培养的子宫内膜间质细胞生长抑制率的影响,来探讨GnRHⅡ对离体培养的子宫内膜间质细胞是否有抗增殖作用。为我们探讨内源性GnRHⅡ蛋白表达是否与内异症发病有关、外源性GnRHⅡ是否可应用于临床治疗子宫内膜异位症及其治疗该病的可能作用机制；提供新的实验和理论依据。关于上述系列研究,目前国内外尚未见报道。
目的：检测GnRHⅡ蛋白在子宫内膜异位症患者异位/在位子宫内膜中的表达情况,并以正常子宫内膜作对照,了解其表达是否有差异；同时分析GnRHⅡ蛋白的表达是否与正常子宫内膜月经周期有关。
材料与方法：选自2007年3月至2008年6月在中南大学湘雅二医院住院、手术及病理切片确诊为内膜异位症、卵巢巧克力囊肿患者30例,取异位/在位子宫内膜组织作试验组；另选同期因非子宫内膜异位症患者的正常子宫内膜组织40例作为对照组(病理切片HE染色内膜正常)。术前半年均无激素应用史及其它疾病史。采用免疫组化SP法检测GnRHⅡ蛋白在异位内膜、在位内膜及正常子宫内膜组织中的表达情况,并分析、比较其表达是否有差异。
结果：
1、GnRHⅡ蛋白在子宫内膜异位症患者异位、在位子宫内膜及正常子宫内膜中均有表达,阳性表达定位于子宫内膜腺体及间质细胞的细胞浆,呈浅黄色、黄色、棕黄色或黄褐色。
2、GnRHⅡ蛋白在内异症组异位内膜、在位内膜及对照组正常内膜的表达依次增强,两两比较差异有显著性(P<0.05)。
3、GnRHⅡ蛋白在正常子宫内膜分泌期表达强于增生期(P<0.05),且以分泌早中期最强,显著强于增生期和分泌晚期,差异有显著性(P<0.01)；而内异症异位组或在位组的分泌期和增生期比较,差异无显著性(P>0.05)。
结论：
1、GnRHⅡ蛋白在子宫内膜异位症患者异位和在位子宫内膜、及正常子宫内膜中均有阳性表达,且正常内膜的表达有周期性变化,提示：GnRHⅡ蛋白可能在人类子宫内膜的病理及生理过程中都起着重要作用。
2、GnRHⅡ蛋白在内异症异位/在位子宫内膜中低表达(P<0.05),提示内源性GnRHⅡ蛋白表达减少可能是内异症发生发展的原因之一。
3、GnRHⅡ蛋白在内异症在位内膜中的表达低于正常子宫内膜(P<0.05),提示内异症的发生可能与在位内膜的异常特性有关。
目的：为探讨外源性的GnRHⅡ蛋白是否对子宫内膜异位症的细胞有直接作用,有必要建立离体的在位及异位子宫内膜间质细胞的体外细胞模型；再用不同浓度GnRHⅡ直接作用于这些间质细胞,检测并分析GnRHⅡ对其细胞凋亡的影响。
方法：取30例内异症异位及在位内膜组织,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。并以不同浓度的GnRHⅡ(0、10-10M、10-8M、10-6M)作用于这些离体培养的子宫内膜间质细胞,采用Hoechst染色和流式细胞术检测离体培养子宫内膜间质细胞的细胞凋亡发生情况。
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、异位内膜间质细胞的培养成活率：红色病灶处为66.67%,褐色病灶处为10.00%。
3、离体培养情况下,对数生长期的子宫内膜间质细胞给予不同浓度的GnRHⅡ(0、10-10 M、10-8 M、10-6M)干预后,采用Hoechst染色对不同处理组细胞进行细胞凋亡形态学的分析。结果显示：不同浓度的GnRHⅡ对离体培养的在位和异位子宫内膜间质细胞可诱导凋亡发生,表现为核碎裂,核溶解,核固缩或凋亡小体。其凋亡率(%)分别为：(5.78±0.53,5.79±0.66),(18.62±2.59,31.30±2.93),(47.41±3.57,59.28±4.25), (66.16±5.46,79.43±6.42),两两比较,差异有显著性(P<0.05),提示：不同浓度的GnRHⅡ对离体培养的异位内膜间质细胞的凋亡率明显高于在位(P<0.05),且呈剂量依赖性(P<0.05)。
4、离体培养情况下,对数生长期的子宫内膜间质细胞给予不同浓度的GnRHⅡ(0、10-10 M、10-8 M、10-6M)干预后,采用流式细胞术对不同处理组细胞进行细胞凋亡率检测,结果显示：不同浓度的GnRHⅡ对离体培养的在位和异位子宫内膜间质细胞可诱导凋亡发生,其凋亡率(%)分别为：(5.87±0.59,5.99±0.78),(19.30±3.28,33.40±3.97), (46.30±5.32,62.41±6.74), (69.70±8.77,83.30±9.42)两两比较,差异有显著性(P<0.05),与HoeChst染色检测的凋亡率变化一致。提示：不同浓度的GnRHⅡ对离体培养的异位内膜间质细胞促凋亡作用明显强于在位内膜间质细胞(P<0.05),且呈剂量依赖性(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研究提供了实验基础。
2.卵巢EMs间质细胞培养成功率之一与所取部位有关,红色病变处成功率较高。
3.外源性的GnRHⅡ可直接促进离体培养的EMs间质细胞的细胞凋亡,呈剂量依赖性,且促凋亡作用异位明显强于在位。为内异症促凋亡方面的新药开发提供实验和理论依据。
目的：通过对子宫内膜异位症(endometriosis, EMs)患者在位及异位内膜间质细胞分离、培养,检测其分泌的血管内皮生长因子(vascular endothelial growth factor, VEGF)的浓度,再用不同浓度的GnRHⅡ进行干预,并以已知作用的GnRHⅠ类似物(以戈舍瑞林为例)作对比,分析其VEGF的浓度变化和GnRHⅡ、GnRHⅠ对其细胞增殖抑制率的影响,为进一步阐明GnRHⅡ的可能作用机制及治疗机制,提供新的实验依据。
方法：
1、在培养液中加入10-10M、10-8M、10-6M的GnRHⅡ,以GnRHⅠ类似物(戈舍瑞林,Goserelin)作对比,同时设对照组(不加GnRH),采用酶联免疫吸附法(ELISA)测定培养液中VEGF浓度,并进行比较。
2、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、EMs患者离体培养的异位子宫内膜间质细胞经48小时培养,能分泌VEGF,分泌量与在位子宫内膜间质细胞的相近,两者比较无统计学差异(P>0.05)。
2、不同浓度的GnRHII对EMs患者离体培养的在位和异位子宫内膜间质细胞VEGF的分泌有明显的抑制作用(P<0.01),呈剂量依赖性(P<0.01),且较GnRH I类似物(戈舍瑞林)的作用更强(P<0.05)。
3、不同浓度的GnRH II对EMs患者离体培养的异位子宫内膜间质细胞VEGF分泌的抑制作用明显强于在位(P<0.01)。
4、用10-100M、10-8M、10-6M的GnRHII对EMs患者离体培养的在位和异位子宫内膜间质细胞进行干预,计算培养24h、48h、72h后的抑制率(%),统计发现：依次升高浓度的GnRHII在相同时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(P<0.05)；相同浓度的GnRHII在相对长时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(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,差异有统计学意义(P<0.05)。
5、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和异位子宫内膜间质细胞干预,计算培养24h、48h、72h后的抑制率(%),统计发现：依次升高浓度的GnRH I类似物在相同时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(P<0.05)；相同浓度的GnRH I类似物在相对长时间、对相同部位间质细胞增殖的抑制率逐渐升高,差异有统计学意义(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,差异有统计学意义(P＜0.05)。
6、GnRHII对EMs患者离体培养在位与异位子宫内膜间质细胞增殖的抑制率明显高于GnRH I类似物(戈舍瑞林),差异有统计学意义(P＜0.05)。
结论：
1、EMs患者的异位子宫内膜间质细胞具有分泌VEGF的功能,分泌量与在位子宫内膜的相近,这对EMs的形成和发展可能起重要作用。
2、不同浓度GnRHII对EMs患者离体培养的异位子宫内膜间质细胞VEGF的分泌有明显的抑制作用,呈剂量依赖性,且作用明显强于GnRHI类似物(戈舍瑞林)。
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,尤其是对异位子宫内膜间质细胞,且抑制作用明显强于GnRHI类似物(戈舍瑞林)。
4、在EMs抗血管形成和抗增殖方面的药物治疗中,GnRHII有可能较GnRHI更有效,为寻找EMs新药开发提供新的实验和理论依据。
Endometriosis (short for EMs) is one common and frequently-occurring disease in women of reproductive age. Its pathogenesis is unclear、recurrent rate is high、treatment is thorny, these make EMs become difficult and hot research. Therefore, to study the pathogenesis of EMs and to find effective treatment methods,which becomes an urgent problem. In recent years, the combination of gonadotropin-releasing hormone analogues (GnRHa) and laparoscopic surgery in the treatment of EMs are sure to be useful and effective. Recent study found that:Ⅱ-type gonadotropin-releasing hormone (GnRHⅡ) can inhibit cell growth in some tumors of the reproductive system,such as ovarian cancer、breast cancer and prostate cancer, and had stronger anti-proliferative effects than GnRH I in endometrial cancer and ovarian cancer cells. Endometriosis is a hormone-dependent disease, which has some biological behaviors of malignant tumor. Therefore, our article attempts to study the expression of the GnRH II in patients with endometriosis and its effects on the eutopic and ectopic endometrial stromal cells in vitro culture in the following three parts:(1). Using the method of SP immunohistochemistry to investigate whether there is expression of GnRHⅡprotein in ectopic and eutopic endomentrium from patients with endometriosis, and normal endometrium was used as control, to study whether there are differences in their expression, as well as whether the expression has cyclical changes in the these endometriums. (2) The endometrial stromal cells were isolated、cultured and identified from ectopic and eutopic endometrium tissues In vitro, then different concentrations of GnRH II (0、10-10M、10-8 M、10-6M) were added in the stromal cells in vitro culture, to detect the occurrence of apoptosis in these endometrial stromal cells with the method of Hoechst staining and flow cytometry test, in order to investigate whether GnRHⅡhas a direct inducing apoptosis effect on cultured endometrial stromal cells in vitro; (3) Different concentrations of GnRHⅡ(0、10-10M、10-8M、10-6M) were added in the stromal cells in vitro culture, and compared to GnRHⅠ, to detect the changes of concentrations for VEGF; and to compare these cells proliferation inhibitory effects by analysing cells inhibitory rate in endometrial stromal cell in vitro, so as to investigate whether GnRHⅡhas antiproliferative effects on the endometrial stromal cell in vitro. In short, to investigate the expression of endogenous GnRHⅡprotein whether has relations with the pathogenesis of endometriosis, and exogenous GnRHⅡwhether can be applied to clinical treatment of endometriosis and its possible treatment mechanisms in the patients with EMs, which providing new experimental and theoretical basis. About the above-mentioned serial studies, It has not been reported at home and abroad.
Objective:To study the expression of GnRHⅡprotein in patients with-and without-endometriosis and the relation to the menstrual cycle.
Methods:Thirty ectopic and thirty eutopic biopsy endometrial specimens were obtained from women with untreated endomentriosis and 40 controls were studied, which came from March 2007 to June 2008 in the Second Xiangya Hospital of Central South University. The expression of GnRHⅡprotein were tested from the ectopic、eutopic、and normal endometrium by immuneohistochemistry SP methods, and compared them.
Results:
1. The expression of GnRHⅡprotein positive immune staining is located in cytoplasm of endometrial stromal cells and gland cells in the all specimens.
2 The expression of GnRHⅡprotein was lower in ectopic endometrium than eutopic endometrium of women with endometriosis (P<0.05),and were lower in the eutopic and ectopic endometrium with endometriosis than the nomal endometrium of women without endometriosis (P<0.05).
3. The expression of GnRHⅡprotein was obvious higher in secretory phase than that of in hyperplasia phase in normal endometrial (P<0.05),especially in early and middle secretory phase(P<0.05), but there was no significant difference in eutopic and ectopic endometrium from endometriosis in the menstrual cycle(P>0.05).
Conclusion:
1. The positive expression of GnRHⅡin the ectopic、eutopic、and normal endometrium, and its expression was related to the menstrual cycle in normal not in endometriotic endometrium, suggests:it may play physiological and pathological roles in above endometrium.
2. The lower expression of GnRHⅡin the ectopic、eutopic endometrium (P<0.05) may cause endometriosis.
3. The lower expression of GnRHⅡin the eutopic endometrium suggests endometriosis may be retated to eutopic endometrial abnormal character.
Objective:To culture the eutopic and ectopic endometrial stromal cells from patients with endometriosis and identify them in vitro; Then, To study the effect of GnRH II on these cells apoptosis in cultured endometrial stromal cells in vitro.
Methods:First, thirty ectopic and thirty eutopic biopsy endometrial specimens were obtained from women with untreated endomentriosis, the stromal cells were separated、cultured from the grandular epithelium, then identified in vitro; Second, to detect the cell apoptosis ratio after different concentration (0,10-10 M,10-8 M,10-6 M) GnRH II were used in these stromal cells In vitro by Hoechst staining and flow cytometry test.
Results:
1. Eutopic endometrial stromal cells of survival rate was 83.33%,ectopic endometrial stromal cells of survival rate is 66.67% by the method of trypsin、collagenase、mesh filtration and centeifugation.
2. The ectopic endometrial stromal cells of survival rate(%):the red site is 66.67%, the black site is 10.00%.
3. In vitro culture, the apoptosis cell of endometrial stromal cells can be observed after GnRH II treatment (0,10-10 M,10-8 M,10-6 M), The Hoechst dye showed that eutopic and ectopic endometrial stromal cells can be induced apoptosis:cell shrinkage、nucleus condensation、and sometimes apoptopic body can be seen. The rate of apoptosis(%) is (5.78±0.53,5.79±0.66), (18.62±2.59,31.30±2.93), (47.41±3.57, 59.28±4.25), (66.16±5.46,79.43±6.42), There were statistically significant differences among different concentration experimental groups (P<0.05), the apoptosis rate of ectopic endometrial stromal cells was higher than eutopic endometrial stromal cells (P<0.05),and were dose-dependent.
4. In vitro culture, the apoptosis cell of endometrial stromal cells can be observed after GnRHⅡtreatment (0,10-10 M,10-8 M,10-6 M), the flow cytometry test results showed that eutopic and ectopic endometrial stromal cells can be induced apoptosis. The rate of apoptosis (%) is (5.87±0.59,5.99±0.78), (19.30±3.28,33.40±3.97), (46.30±5.32, 62.41±6.74), (69.70±8.77,83.30±9.42), There were statistically significant differences among different concentration experimental groups (P<0.05), the apoptosis rate of ectopic endometrial stromal cells was higher than eutopic endometrial stromal cells (P<0.05),and were dose-dependent.
Conclusion:
1.Highly purified eutopic and ectopic endometrial stromal cells were seperated and cultured successfully by the method of trypsin、collagenase、mesh filtration and centeifugation, which provides a successful cell model and expremental basis for the pathogenesis and drug therapy of EMs.
2. The one of successful culture of ovarian stromal cells for EMs was related to its site.
3. Exogenous GnRH II can increase stromal cells apoptosis in dose-dependent in vitro for the patients with EMs, and the ectopic was higher than the eutopic, which suggests GnRH II may play an important role in treatment EMs by inducing cell apoptosis.
Objective:
1. To study the effects of GnRHⅡon the secretion of VEGF concentration by eutopic and ectopic endometrial stromal cells cultured in vitro, and contrast with GnRH I, which may provide theoretical basis for exploring GnRH II new treatment for EMs.
2. Different concentrations of GnRH II were added in the cultured stromal cells in vitro, and compared to GnRH I, to detect the cell proliferation inhibitory rates of GnRH II and GnRH I on the endometrial stromal cells in vitro, and compare and analyse them.
Methods:
1. The above eutopic and ectopic stromal cells in vitro were divided into three groups:(1) treated by 10-10M、10-8M、10-6M GnRHⅡ; (2) treated by 10-10 M、10-8 M、10-6M GnRH I; (3) control group, not treated by GnRH. ELISA test was used to measure the concentration of VEGF protein in the medium of above three groups.
2. The above eutopic and ectopic stromal cells in vitro were divided into three groups:(1) treated by 10-10M、10-8M、10-6M GnRHⅡ; (2) treated by 10-10M、10-8M、10-6M GnRH I; (3) control group, not treated by GnRH,and then were continued to culture for 24 hours、48 hours、72hours. MTT method was used to detect survival cell, and calculated to compare the inhibitory rate of stromal cells in vitro.
Results:
1. There is no difference between the VEGF protein concentration by eutopic and ectopic stromal cells secreting in the medium after being cultured for 48 hours in vitro (P>0.05)
2.10-10M、10-8M、10-6M GnRHⅡcan dose-dependent reduce VEGF protein concentration by endometrial stromal cells secreting (P<0.01) and the inhibitory to endometrial stromal cells is stronger than GnRH I (P<0.05).
3.The inhibitory effect of GnRH II on VEGF in ectopic stromal cells is stronger than that of in eutopic stromal cells (P<0.01)
4.The eutopic and ectopic endometrial stromal cell was cultured and treated with gradual high concentrations of GnRH II (10-10M,10-8M,10-6M) for 24 hours、for 48 hours、for 72 hours respectively. The inhibitory rate(%) of cell proliferation in the same endometrial stromal cell with different gradual high concentrations of GnRHⅡat the same time was gradually increased, significant difference was observed(P<0.05); the cell inhibitory rate(%) of the same endometrial stromal cell with same concentrations of GnRH II at different time was gradually increased, significant difference was observed(P<0.05); the cell inhibitory rate of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell, significant difference was observed (P<0.05).
5.The eutopic and ectopic endometrial stromal cell was cultured and treated with gradual high concentrations of GnRH I (10-10M,10-8M,10-6M) for 24 hours、for 48 hours、for 72 hours respectively. The inhibitory rate(%) of cell proliferation in the same endometrial stromal cell with different gradual high concentrations of GnRH I at the same time was gradually increased, significant difference was observed.(P<0.05),the cell inhibitory rate of the same endometrial stromal cell with same concentrations of GnRH I at 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 inhibitory rate(%) of ectopic endometrial stromal cell was higher than that of eutopic endometrial stromal cell, significant difference was observed (P<0.05).
6. GnRHⅡhad higher inhibitory rate(%) of cell proliferation in endometrial stromal cell in vitro than GnRH I, significant difference was observed (P<0.05).
Conclusions:
1.Ectopic stromal cells cultured in vitro can secrete VEGF, which has no difference with the eutopic stromal cells.This may play an important role in the formation and development of EMs.
2.GnRHⅡcan reduce VEGF protein secreted by endometrial stromal cell cultured in vitro in dose-dependent manner, and the inhibition is stronger than GnRHⅠ.which may provide theoretic basis for exploring new treatment for EMs.
3.GnRHⅡhad more antiproliferative effects on endometrial stromal cell than GnRH I analogues (goserelin) in vitro,especially in ectopic endometrial stromal cells, in dose-and time-dependent manner, which may provide a new theory for researching new drug for EMs.
引文
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