子宫内膜间质细胞体外损伤模型的建立

Establishment of damaged endometrial stromal cells model in vitro

目的探讨子宫内膜间质细胞（ESC）体外损伤模型的建立方法。方法选择2010年6月至12月在南通大学附属医院因子宫肌瘤或宫颈上皮内瘤变行子宫切除患者的子宫内膜组织标本16份，其中增生期8份，分泌期8份。（1）分离、原代培养增生期及分泌期ESC，并鉴定。（2）通过活细胞计数法（CCK．8）检测不同浓度米非司酮作用不同时间及米非司酮撤药后不同时间对ESC增殖抑制率的影响。（3）以米非司酮60μmol／L为实验组，未加米非司酮为对照组，处理ESC48h后撤药，继续培养48h。观察细胞形态变化，流式细胞仪检测增生期、分泌期ESC凋亡率。用荧光定量PCR技术和蛋白印迹法检测ESC中血管内皮生长因子（VEGF）及半胱氨酸天冬氨酸蛋白酶（caspase）3、8、9mRNA和蛋白的相对表达水平。结果（1）16份子宫内膜标本均成功分离并培养ESC。（2）ESC增殖抑制率与米非司酮作用的浓度及时间均呈正相关。撤药后，ESC在一定浓度范围内随撤药时间的延长增殖能力有所恢复，但当米非司酮浓度为100μmol／L时，撤药后ESC增殖能力难以恢复。（3）实验组以米非司酮60μmol／L处理ESC48h后撤药，继续培养48h，可见ESC细胞间距增大，呈长梭形，胞质出现空泡化现象。实验组和对照组增生期ESC凋亡率分别为（52±12）％和（13±5）％，分泌期分别为（534-6）％和（32±3）％，分别比较，差异均有统计学意义（P〈0．05）。增生期实验组和对照组VEGFmRNA相对表达水平为0．52±0．12、1．00±0．17，分泌期实验组与对照组分别为0．19±0．03、0．81±0．07，两组分别比较，差异有统计学意义（P〈0．05）；实验组增生期与分泌期VEGF蛋白相对表达水平均明显低于对照组（P〈0．05）。增生期实验组与对照组caspase-3、8、9mRNA的相对表达水平分别为5．62±0．65、1．00±0．44，5．41±0．53、1．00±0．21，7．22±0．51、1．00±0．32，分泌期实验组与对照组分别为10．22±0．72、1．42±0．14，25．30±1．72、1．14±0．28，9．48±1．89、1．16±0．12．两组分别比较，差异均有统计学意义（P〈0．05）；与对照组比较，实验组在增生期与分泌期caspase-3蛋白表达水平分别上调了2．04和1．60倍，easpase-8上调了4．23倍和1．49倍，easpase-9上调了2．65倍和3．50倍；分别比较，差异有统计学意义（P〈0．05）。结论60μmol／L米非司酮作用于ESC48h后撤药，继续培养48h后可作为ESC的体外损伤模型。

Objective To invDepartment of Obstetrics and Gynecology, Affiliated Hospital of Nantong University, Nantong estigate the method of establishing damaged endometrial stromal cells （ESC） model in vitro. Methods （1） From June to December 2011 ESC from normal endometrim at proliferation phase （ n = 8 ） and secretory phase （ n = 8 ） were isolated, cultured and identified in vitro. （ 2 ） ESC was treated with different concentrations of mifepristone or withdrawal of mifepristone at different time point. The proliferation inhibition percent was measured by cell counting kit-8 （ CCK-8 ）. （ 3 ） 0 μmol/L （control group）and 60 μmol/L（ experimental group） concentration of mifepristone was added into ESC for 48 hours, then withdrew of mifepristone, continued to be cultured for 48 hours. The morphologica~ cha^ges were observed and apoptosis of ESC in different menstrual cycle were detected by flow eytometry. The mRNAand protein level of vascular endothelial growth factor （ VEGF）, caspase-3, 8, and 9 were determined by one-step quantitative real-time PCR （Q-PCR） and western blot. Results （ 1 ） ESC from 16 specimens of endometrium were all isolated and cultured successfully. （2） The proliferation inhibition rate of ESC was correlated with concentration and duration of mifepristone positively. The proliferation of ESC could be recovered at a range of time after withdrawal of mifepristone. However, when the concentration of mifepristone was 100 i^moL/L,the growth of ESC recovered very hardly. （3） The damaged ESC spacing increased, the spindle shape and vacuolization in the cytoplasm were observed in experimental group; the rate of apoptosis of these damaged cells was significantly increased compared with control groups, which were （52 ± 12）% vs. （13 ±5）% at the proliferative phase and （53 ±6）% vs. （32 ±3）% at the secretory phase （all P 〈 0. 05）. The relative mRNA level of VEGF was 0. 52 ± 0. 12 in experimental group and 1.00 ± 0. 17 in control group at proliferation phase （P 〈0. 05）. And the relative mRNA level of VEGF was 0. 19 ±0. 03 in experimental group and 0. 81 ±0. 07 in control group at secretory phase （P 〈 0. 05 ）. The relative level of VEGF protein in the experimental group were both decreased 1.98 and 2. 79 folds at the proliferation phase and the secretory phase when compared with those in control group, respectively （P 〈 0. 05 ）. While the relative levels of caspase-3,8,9 mRNA were 5.62 ±0. 65, 5.41 ±0. 53, 7.22 ±0. 51 in the experimental group and 1.00 ± 0.44, 1.00 ± 0.21,1.00 ± 0. 32 in control group at the proliferative phase. In the mean time, the relative levels of caspase-3,8,9 mRNA were 10.22 ± 0.72, 25.3 ± 1.72, 9.48 ± 1.89 in experimental group and 1.42 ± O. 14, 1.14 ± 0. 28,1.16 ± 0. 12 in control group at the secretory phase, respectively （P 〈 0.05）. Compared with the control group, the levels of caspase protein in the experimental group were increased 2. 04 and 1.60 folds in caspase-3, 4. 23 and 1.49 folds in caspase-8, 2. 65 and 3.5 folds in caspase-9 at the proliferative phase and at the secretory phase, respectively （P 〈0. 05）. Conclusion The damaged model of ESC can be established after 48 hours by the withdrawal of 60 μmol/L mifeoristone in treatment of ESC for 48 hours.