17β-雌二醇和孕酮对永生化山羊子宫内膜细胞及外周血淋巴细胞的影响
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摘要
子宫内膜是一个动态的激素应答的组织,它经历着连续的循环过程,包括增殖、分化和修复。孕酮和雌激素的联合作用使子宫内膜细胞在妊娠期发生形态及生物学特性的变化,这种变化不仅表现在形态的改变上,同时还表现在增殖及分泌活性的改变上。本研究通过对子宫内膜细胞在免疫细胞活化和功能变化中作用的检测,来揭示子宫内膜细胞在子宫局部内分泌-免疫调节中的作用。利用永生化山羊子宫内膜基质细胞(hTERT-ESCs)和子宫内膜上皮细胞(hTERT-EECs)建立体外子宫内膜细胞功能的研究模型,采用MTT法检测细胞生长曲线以评价17β-雌二醇(E_2)和孕酮(P_4)对子宫内膜细胞增殖作用的影响;并在此基础上,应用ELISA方法检测子宫内膜细胞之间及其与外周血淋巴细胞之间相互作用以及E_2和P_4对其分泌功能的影响。
1.对永生化山羊子宫内膜上皮细胞( hTERT-EECs )和子宫内膜基质细胞(hTERT-ESCs)进行生物学特性检测。结果显示细胞生长状态良好,保持正常细胞的特性:具有血清依赖性和接触抑制性,无悬浮生长能力和致瘤能力,染色体数目和形态符合山羊染色体的特征。
2.应用MTT法检测E_2和P_4对hTERT-EECs和hTERT-ESCs增殖活性的影响。结果发现在E_2作用下,hTERT-EECs和hTERT-ESCs生长迅速;而P_4则使细胞增殖缓慢。表明E_2能促进子宫内膜细胞的生长而P_4则对其有抑制作用。
3.应用ELISA方法检测E_2和P_4对hTERT-EECs和hTERT-ESCs分泌活性的影响。结果表明E_2可使hTERT-EECs培养上清中TNF-α和IL-2的含量降低;P_4则使其IFN-γ含量增加而IL-4降低;E_2/P_4共同作用下IL-2含量降低。
在E_2作用下,hTERT-ESCs培养上清液中的IFN-γ减少;E_2/P_4共同作用使TNF-α和IFN-γ增加;而P_4对这些细胞因子没有明显影响。
当hTERT-EECs和hTERT-ESCs共培养时,E_2使hTERT-EECs上清中TNF-α的含量降低;P_4使TNF-α和IL-2的含量减少;E_2/P_4共同作用使TNF-α和IL-2减少而IL-4增加。而此时只有E_2/P_4共同作用使hTERT-ESCs中的IFN-γ含量增加。
4.应用ELISA方法检测E_2和P_4对hTERT-EECs和hTERT-ESCs及山羊外周血淋巴细胞的影响,结果为:hTERT-EECs与PBL共培养时,添加E_2使hTERT-EECs上清中IL-2和TNF-α的含量降低,P_4也使IL-2、IFN-γ和TNF-α减少。同时PBL在无E_2/P_4、E_2单独作用、E_2和P_4共同作用时TNF-α都减少。
当hTERT-ESCs与PBL共培养时,hTERT-ESCs培养液中的上述细胞因子在激素处理或者无激素状态下都没有明显变化;而PBL在E_2作用下IL-2降低,P_4使4种细胞因子都降低,IL-2和IL-4在E_2和P_4共同作用下减少,并且在不加激素共培养时TNF-α含量也降低。
将hTERT-ESCs、hTERT-EECs与PBL共培养,hTERT-ESCs不加激素,其IL-2、IFN-γ和IL-4的含量也上升;并且E_2或P_4都能使IL-2的含量上升,此外,E_2和P_4共同作用使TNF-α减少。同时hTERT-EECs培养液中的TNF-α含量在E_2或P_4单独作用时减少,P_4减少了IFN-γ的分泌,E_2和P_4共同作用使IL-2增加。而激素处理或未处理能都使共培养的PBL分泌的TNF-α减少,而IL-2则在P_4单独作用、E_2和P_4共同作用时减少。
以上研究表明E_2和P_4调节了子宫内膜细胞的增殖和分泌功能,对子宫内膜细胞之间及其与外周血淋巴细胞的相互作用也有影响。本实验将有助于了解妊娠期子宫局部免疫-内分泌的调节和胚胎附植机理,并可为免疫因素引起的繁殖障碍性疾病的防治提供新的研究思路和方法。
Endometrial is a dynamic hormone response organization, what is undergoing continuous cycle, including proliferation, differentiation and repair. The combined effects of progesterone (P_4) and 17β-estrogen (E_2) make the endometrial cells changed in morphology and biological characteristics in the gestation period. This change displays not only in morphology but also in the activation of proliferation and secretion. This study tried to reveal the role of the endometrial cells on the endocrine-immune regulation in local uterus by detecting these cells’effect on activation and function of lymphocyte. We used the immortalized goat endometrial stroma cells (hTERT-ESCs) and endometrial epithelial cells (hTERT-EECs) to establish a model in vitro. MTT assay was used to detect endometrial cells’growth curves and evaluate the effection of E_2 and P_4 on their proliferation. And on the basis of above, the effect of the interaction between hTERT-ESCs and hTERT-EECs, that of endometrial cells and peripheral blood lymphocyte was detected by ELISA method, and the role of E_2 and P_4 on the secretion function of these cells was also detected by ELISA.
1. The biological characteristics of hTERT-ESCs and hTERT-EECs which established and kept in our lab were detected, and the result showed the cells were in good condition maintaining normal cellular characters, such as serum dependence, contact inhibition, chromosome number and morphology, and no suspension growth and tumorigenicity.
2. Detected the effect of E_2 and P_4 on proliferation of the endometrial cells, and found that hTERT-EECs and hTERT-ESCs could growth faster under the treatment of E_2, while P_4 made the cells proliferate slowly what was indicated that E_2 could promote the cells’growth, but P_4 inhibited it.
3. The effect of E_2 and P_4 on secretion of the endometrial cells studied. The result displayed when hTERT-EECs was single-cultured, E_2 made the contents of TNF-αand IL-2 in the culture solution reduced; IFN-γcontent increased and IL-4 lessened with P_4; and the combined action of E_2/P_4 made IL-2 reduce.
When hTERT-ESCs was single-cultured, E_2 made IFN-γreduce; E_2/P_4 made TNF-αand IFN-γincrease; but single P_4 had no effect on these cytokines.
When hTERT-ESCs and hTERT-EECs were coculture, the content of TNF-αin hTERT-EECs culture solution was reduced by E_2 and P_4 made TNF-αand IL-2 reduce; while the coactions of E_2/P_4 made TNF-αand IL-2 reduce but IL-4 increase. For hTERT-ESCs now, only E_2/P_4 combined action made IFN-γincrease.
4. Detected the influence of E_2 and P_4 between the endometrial cells and PBL, the result was that when hTERT-EECs was cultured with PBL, E_2 made IL-2 and TNF-αof hTERT-EECs decrease; P_4 made IL-2, IFN-γand TNF-αreduce. Meanwhile TNF-αof PBL was reduced with or without gonad hormone.
When hTERT-ESCs were cultured with PBL, the cytokines of hTERT-ESCs had no obviously changed regardless of E_2 or P_4. And for PBL, E_2 made IL-2 reduce; P_4 reduced the four cytokines; the combined action of E_2/P_4 made IL-2 and IL-4 reduce; and without hormone the TNF-αalso reduced.
When hTERT-ESCs, hTERT-EECs and PBL were cocultured, IL-2, IFN-γand IL-4 of hTERT-ESCs were increased without E_2 or P_4. And the treatment by E_2 or P_4 all made IL-2 of hTERT-ESCs increased, but TNF-αwas reduced with E_2 and P_4 together. The change in hTERT-EECs was single E_2 or P_4 made TNF-αlessen, single P_4 made IFN-γreduced and IL-2 increased under the coactions of E_2 and P_4. And for PBL, the cocultures with the endometrial cells made TNF-αof PBL reduce whether the hormone appeared or not. While IL-2 also reduced when single P_4 and coactions of E_2 and P_4.
This test indicate that E_2 and P_4 regulate the proliferation and secretion function of endometrial cells, and also effect the interaction of endometrial cells and peripheral blood lymphocyte. It would contribute to understand the endocrine-immunoregulation in the local uterus and the embryos implanted mechanism, and could provide new thought and method for the preventive treatment of reproductive obstacle disease resulted by immune factors.
1.对永生化山羊子宫内膜上皮细胞( hTERT-EECs )和子宫内膜基质细胞(hTERT-ESCs)进行生物学特性检测。结果显示细胞生长状态良好,保持正常细胞的特性:具有血清依赖性和接触抑制性,无悬浮生长能力和致瘤能力,染色体数目和形态符合山羊染色体的特征。
2.应用MTT法检测E_2和P_4对hTERT-EECs和hTERT-ESCs增殖活性的影响。结果发现在E_2作用下,hTERT-EECs和hTERT-ESCs生长迅速;而P_4则使细胞增殖缓慢。表明E_2能促进子宫内膜细胞的生长而P_4则对其有抑制作用。
3.应用ELISA方法检测E_2和P_4对hTERT-EECs和hTERT-ESCs分泌活性的影响。结果表明E_2可使hTERT-EECs培养上清中TNF-α和IL-2的含量降低;P_4则使其IFN-γ含量增加而IL-4降低;E_2/P_4共同作用下IL-2含量降低。
在E_2作用下,hTERT-ESCs培养上清液中的IFN-γ减少;E_2/P_4共同作用使TNF-α和IFN-γ增加;而P_4对这些细胞因子没有明显影响。
当hTERT-EECs和hTERT-ESCs共培养时,E_2使hTERT-EECs上清中TNF-α的含量降低;P_4使TNF-α和IL-2的含量减少;E_2/P_4共同作用使TNF-α和IL-2减少而IL-4增加。而此时只有E_2/P_4共同作用使hTERT-ESCs中的IFN-γ含量增加。
4.应用ELISA方法检测E_2和P_4对hTERT-EECs和hTERT-ESCs及山羊外周血淋巴细胞的影响,结果为:hTERT-EECs与PBL共培养时,添加E_2使hTERT-EECs上清中IL-2和TNF-α的含量降低,P_4也使IL-2、IFN-γ和TNF-α减少。同时PBL在无E_2/P_4、E_2单独作用、E_2和P_4共同作用时TNF-α都减少。
当hTERT-ESCs与PBL共培养时,hTERT-ESCs培养液中的上述细胞因子在激素处理或者无激素状态下都没有明显变化;而PBL在E_2作用下IL-2降低,P_4使4种细胞因子都降低,IL-2和IL-4在E_2和P_4共同作用下减少,并且在不加激素共培养时TNF-α含量也降低。
将hTERT-ESCs、hTERT-EECs与PBL共培养,hTERT-ESCs不加激素,其IL-2、IFN-γ和IL-4的含量也上升;并且E_2或P_4都能使IL-2的含量上升,此外,E_2和P_4共同作用使TNF-α减少。同时hTERT-EECs培养液中的TNF-α含量在E_2或P_4单独作用时减少,P_4减少了IFN-γ的分泌,E_2和P_4共同作用使IL-2增加。而激素处理或未处理能都使共培养的PBL分泌的TNF-α减少,而IL-2则在P_4单独作用、E_2和P_4共同作用时减少。
以上研究表明E_2和P_4调节了子宫内膜细胞的增殖和分泌功能,对子宫内膜细胞之间及其与外周血淋巴细胞的相互作用也有影响。本实验将有助于了解妊娠期子宫局部免疫-内分泌的调节和胚胎附植机理,并可为免疫因素引起的繁殖障碍性疾病的防治提供新的研究思路和方法。
Endometrial is a dynamic hormone response organization, what is undergoing continuous cycle, including proliferation, differentiation and repair. The combined effects of progesterone (P_4) and 17β-estrogen (E_2) make the endometrial cells changed in morphology and biological characteristics in the gestation period. This change displays not only in morphology but also in the activation of proliferation and secretion. This study tried to reveal the role of the endometrial cells on the endocrine-immune regulation in local uterus by detecting these cells’effect on activation and function of lymphocyte. We used the immortalized goat endometrial stroma cells (hTERT-ESCs) and endometrial epithelial cells (hTERT-EECs) to establish a model in vitro. MTT assay was used to detect endometrial cells’growth curves and evaluate the effection of E_2 and P_4 on their proliferation. And on the basis of above, the effect of the interaction between hTERT-ESCs and hTERT-EECs, that of endometrial cells and peripheral blood lymphocyte was detected by ELISA method, and the role of E_2 and P_4 on the secretion function of these cells was also detected by ELISA.
1. The biological characteristics of hTERT-ESCs and hTERT-EECs which established and kept in our lab were detected, and the result showed the cells were in good condition maintaining normal cellular characters, such as serum dependence, contact inhibition, chromosome number and morphology, and no suspension growth and tumorigenicity.
2. Detected the effect of E_2 and P_4 on proliferation of the endometrial cells, and found that hTERT-EECs and hTERT-ESCs could growth faster under the treatment of E_2, while P_4 made the cells proliferate slowly what was indicated that E_2 could promote the cells’growth, but P_4 inhibited it.
3. The effect of E_2 and P_4 on secretion of the endometrial cells studied. The result displayed when hTERT-EECs was single-cultured, E_2 made the contents of TNF-αand IL-2 in the culture solution reduced; IFN-γcontent increased and IL-4 lessened with P_4; and the combined action of E_2/P_4 made IL-2 reduce.
When hTERT-ESCs was single-cultured, E_2 made IFN-γreduce; E_2/P_4 made TNF-αand IFN-γincrease; but single P_4 had no effect on these cytokines.
When hTERT-ESCs and hTERT-EECs were coculture, the content of TNF-αin hTERT-EECs culture solution was reduced by E_2 and P_4 made TNF-αand IL-2 reduce; while the coactions of E_2/P_4 made TNF-αand IL-2 reduce but IL-4 increase. For hTERT-ESCs now, only E_2/P_4 combined action made IFN-γincrease.
4. Detected the influence of E_2 and P_4 between the endometrial cells and PBL, the result was that when hTERT-EECs was cultured with PBL, E_2 made IL-2 and TNF-αof hTERT-EECs decrease; P_4 made IL-2, IFN-γand TNF-αreduce. Meanwhile TNF-αof PBL was reduced with or without gonad hormone.
When hTERT-ESCs were cultured with PBL, the cytokines of hTERT-ESCs had no obviously changed regardless of E_2 or P_4. And for PBL, E_2 made IL-2 reduce; P_4 reduced the four cytokines; the combined action of E_2/P_4 made IL-2 and IL-4 reduce; and without hormone the TNF-αalso reduced.
When hTERT-ESCs, hTERT-EECs and PBL were cocultured, IL-2, IFN-γand IL-4 of hTERT-ESCs were increased without E_2 or P_4. And the treatment by E_2 or P_4 all made IL-2 of hTERT-ESCs increased, but TNF-αwas reduced with E_2 and P_4 together. The change in hTERT-EECs was single E_2 or P_4 made TNF-αlessen, single P_4 made IFN-γreduced and IL-2 increased under the coactions of E_2 and P_4. And for PBL, the cocultures with the endometrial cells made TNF-αof PBL reduce whether the hormone appeared or not. While IL-2 also reduced when single P_4 and coactions of E_2 and P_4.
This test indicate that E_2 and P_4 regulate the proliferation and secretion function of endometrial cells, and also effect the interaction of endometrial cells and peripheral blood lymphocyte. It would contribute to understand the endocrine-immunoregulation in the local uterus and the embryos implanted mechanism, and could provide new thought and method for the preventive treatment of reproductive obstacle disease resulted by immune factors.
引文
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