Lowered expression of CCN5 in endometriotic tissues promotes proliferation, migration and invasion of endometrial stromal cells / 南方医科大学学报

OBJECTIVE@#To explore the expression of CCN5 in endometriotic tissues and its impact on proliferation, migration and invasion of human endometrial stromal cells (HESCs).@*METHODS@#We collected ovarian endometriosis samples from 20 women receiving laparoscopic surgery and eutopic endometrium samples from 15 women undergoing IVF-ET for comparison of CCN5 expression. Cultured HESCs were transfected with a recombinant adenovirus Ad-CCN5 for CCN5 overexpression or with a CCN5-specific siRNA for knocking down CCN5 expression, and the changes of cell proliferation, migration and invasion were evaluated using CCK-8 assay, wound healing assay and Transwell chamber assay. RT-qPCR and Western blotting were used to examine the expression levels of epithelial-mesenchymal transition (EMT) markers including E-cadherin, N-cadherin, Snail-1 and vimentin in HESCs with CCN5 overexpression or knockdown.@*RESULTS@#CCN5 expression was significantly decreased in ovarian endometriosis tissues as compared with eutopic endometrium samples (P < 0.01). CCN5 overexpression obviously inhibited the proliferation, migration and invasion of HESCs, significantly increased the expression of E-cadherin and decreased the expressions of N-cadherin, Snail-1 and vimentin (P < 0.01). CCN5 knockdown significantly enhanced the proliferation, migration and invasion of HESCs and produced opposite effects on the expressions of E-cadherin, N-cadherin, Snail-1 and vimentin (P < 0.01).@*CONCLUSION@#CCN5 can regulate the proliferation, migration and invasion of HESCs and thus plays an important role in EMT of HESCs, suggesting the potential of CCN5 as a therapeutic target for endometriosis.

Progress on roles and mechanisms of CCAAT/enhancer binding protein β in endometrium decidualization / 上海交通大学学报（医学版）

CCAAT/enhancer binding protein β (C/EBPβ) is a critical member of C/EBP family.C/EBPβ,based on the leucine zipper located in C terminal of the protein,can regulate transcriptional activities of downstream target genes involved in diverse cellular processes,such as proliferation and differentiation.Recently published studies have identified that C/EBPβ is essential in the decidualization of endometrial stromal cells.This review summarizes the roles and mechanisms of C/EBPβ during the courses of decidualization,which is aimed at providing novel insights for dysfunctional decidualization.

Effects of H2 O2 on endometrial stromal cells in the decidual mouse / 医学研究生学报

Objective Among the factors affecting embryo implantation, oxidative stress is one that receives much medical attention. The purpose of the study was to explore the effect of H2 O2 on the endometrial stromal cells ( ESCs) in the decidual mouse model of oxidative stress of ESCs. Methods Primary ESCs cultured by enzymatic digestion with a mesh filter were divided into a control, an experimental, and a model group. Decidualization was induced in the mice of the experimental and model groups by inter-vention with 10 nM E2 and 1μmol/L P4 for 72 h in vitro. Then, the animals of the model group were treated with different concentra-tions of H2 O2 for 4 hours. The primary ESCs were identified by immunohistochemistry, the expression of dPRP mRNA determined byRT-PCR, the proliferation of the decidual ESCs treated with H2 O2 analyzed by CCK-8, and the level of ROS detected by flow cytometry. Results Primary mouse ESCs were successfully isolated, cultured, and identified, which were shaped like spindles and polygons, radial-ly aligned under the microscope. Immunofluorescence analysis showed positive expression of vimentin and negative expression of cytokeratin. The purity of the primary mouse ESCs was ( 96. 3 ± 0. 49 )%. Theexpression of dPRP mRNA was significantly higher in the ESCs treated with E2 and P4 than in the control (0.0002±0.0000 vs 1.0010±0.0011, P<0.01). H2O2 at ≥150μmol/L suppressed the proliferation of the decidual ESCs by 6.9% (P<0.05), and in a concen-tration-dependent manner, reaching the maximum inhibition rate of 70.6% at the concentration of 300μmol/L. The level of intracellular ROS was markedly increased in the ESCs treated with H2O2 at 50μmol/L (27.77±4.20) and 100μmol/L (43.57±6.58), with statisti-cally significant difference from that in the control group (17.47±0.61) (P=0.001). Conclusion H2O2 at 100μmol/L can signifi-cantly elevate the intracellar ROS level without affecting the proliferation of decidual primary ESCs, and therefore can be used for estab-lishing the model of oxidative stress of ESCs in decidual mice.

Effect of GnRH Ⅱ and GnRH Ⅰ on secretion of VEGF by eutopic and ectopic endometrial stromal cells of endometriosis patients / 中南大学学报(医学版)

Objective To determine the effect of GnRH Ⅰ and GnRH Ⅱ on the secretion of VEGF by eutopic and ectopic endometrial stromal cells cultured in vitro, and to provide theoretical basis for exploring new treatments for endometriosis (EMs).Methods Eutopic and ectopic endometrium stromal cells cultured in vitro were treated with different concentrations of GnRH Ⅱ and a GnRH I(goserelin), and a control group was not treated by GnRH Ⅱ and GnRH Ⅰ. Enzyme linked immunosorbent assay (ELISA) was used to measure the content of vascular endothelial growth factor (VEGF) protein in the medium of the above 2 groups.Results (1)There was no difference in the VEGF protein secreted by eutopic and ectopic stromal cells in the medium after being cultured in vitro for 48 h (P＞0.05).(2)10-10, 10-8, and 10-6mol/L GnRH Ⅱ dose-dependently reduced VEGF protein secreted by endometrial stromal cells (P＜0.05),and the inhibition effect was stronger than that of GnRH Ⅰ (P＜0.05).(3)The inhibition effect of GnRH Ⅱ on VEGF in ectopic stromal cells was stronger than that of eutopic stromal cells (P＜0.05).Conclusion (1)Ectopic stromal cells cultured in vitro can secrete VEGF,which has no difference from the eutopic stromal cells, and which may play an important role in the formation and development of EMs.(2)GnRH Ⅱ can dose-dependently reduce VEGF protein secreted by ectopic and eutopic endometrial stromal cells cultured in vitro,and the inhibition effect is stronger than that of GnRH Ⅰ, providing theoretical basis for exploring new treatments for EMs.

Effects of simvastatin on the proliferation and apoptosis of human endometrial stromal cells from women with endometriosis / 대한산부인과학회지

OBJECTIVE: To evaluate the effects of simvastatin on the proliferation and apoptosis of human endometrial cells from women with endometriosis. METHODS: Endometrial tissues were obtained from four women with endometriosis. The endometrial stromal cells isolated from tissue were cultured with 0, 2 and 10 micrometer simvastatin treatments for 48 hours. The proliferation of endometrial stromal cells was inhibited with 2 and 10 micrometer simvastatin treatments compared to control. The effect of simvastatin on the sub-G1 phase of cell cycle was determined by flow cytometry. The expression of apoptosis related molecule (Bcl-2, Bax and caspase-3) was examined in control and simvastatin treatments using western blot. RESULTS: The sub-G1 phase was higher in 10 micrometer simvastatin than in control and 2 micrometer simvastatin (P<0.05). This result showed that simvastatin could induce apoptosis of stromal cells. The expression of Bcl-2 was increased in simvastatin treatments slightly (P<0.05) and the expression of Bax was not different between control and experimental groups. The activation of caspase-3 was significantly higher in 10 micrometer simvastatin treated group than control and 2 micrometer simvastatin treated groups (P<0.05). CONCLUSION: Simvastatin induces apoptosis of endometrial stromal cells and inhibits their proliferation. It was considered that simvastatin could potentially be a therapeutic agent for the treatment of endometriosis.

Effect of Transforming Growth Factor (TGF)-beta and Peroxisome Proliferator-Activated Receptor (PPAR)-gamma in Endometrial Decidualization / 대한산부인과학회지

OBJECTIVE: To ascertain the expression of transforming growth factor (TGF)-beta receptors in normal human endometrium during the menstrual cycle, and the action of TGF-beta and peroxisome proliferator-activated receptor (PPAR)-gamma during endometrial decidualization using cultured human endometrial stromal cells. METHODS: Human endometrial tissues were examined immunohistochemically for the expression of TGF-beta receptors and Smad. Western blotting, confocal microscopy and viable cell counting were performed on cultured human endometrial stromal cells which were treated with TGF-beta (10 ng/mL) and PPAR-gamma agonists (Rosiglitazone(R) 50 nM). Thereafter we compared the effect of TGF-beta and PPAR-gamma on the Smad phosphorylation, prolactin expression, and cellular proliferation in vitro human endometrial decidualization. RESULTS: The results revealed significantly increased expression of both TGF-beta receptor-I and -II proteins in the secretory stromal cells compared to the epithelial cells of human endometrium. The degree of expression and translocation into the nucleus of the phosphorylated Smad2/3 was also increased in the secretory endometrium compared to the proliferative endometrium. In the stromal cell culture, the decidualization process associated with TGF-beta and pSmad is inhibited by the PPAR-gamma agonist. In contrast to the PPAR-gamma agonist, TGF-beta inhibits cellular proliferation. CONCLUSION: TGF-beta/Smad signaling pathway is essential for endometrial decidualization and closely related to cellular differentiation. PPAR-gamma plays a conflicting role by directly acting on the Smad protein and blocking the TGF-beta/Smad signaling pathway.

Alteration of Gene Expressions in Human Endometrial Stromal Cells by Exogeneous FSH Treatments / 대한불임학회지

OBJECTIVE: To evaluate the effects of recombinant FSH (rFSH) and urinary FSH (uFSH) on the gene expressions of human endometrial stromal cells in vitro. METHODS: Endometrial tissue was obtained from a pre-menopausal women undergoing hysterectomy. Primary endometrial stromal cells were isolated and in vitro cultured with FBS-free DMEM/F-12 containing 0, 10, 100, and 1,000 mIU/ml of rFSH and uFSH for 48 hours, respectively. Total RNA was extracted from the cultured cells and subjected to real time RT-PCR for the quantitative analysis of progesterone receptor (PR), estrogen receptor alpha/beta (ER-alpha/beta), cyclooxygenase 2 (Cox-2), leukemia inhibitory factor (LIF), homeobox A10-1 and -2 (HoxA10-1/-2). RESULTS: Both hormone treatments slightly increased (< 3 folds) the expressions of PR, ER-beta and HoxA10-1/-2 gene. However, ER-alpha expression was increased up to five folds by treatments of both FSH for 48 hours. The LIF expression by the 10 mIU/ml of uFSH for 12 hours was significantly higher than that of rFSH (p<0.01). After 24 hours treatment of two kinds of hormones, the expression patterns of LIF were similar. The 100 and 1,000 mIU/ml of rFSH induced significantly higher amount of Cox-2 expression than those of uFSH, respectively (p<0.05). CONCLUSION: This study represents no adversely effect of exogeneous gonadotropins, rFSH and uFSH, on the expression of implantation related genes. We suggest that rFSH is applicable for the assisted reproductive technology without any concern on the endometrial receptivity.

The Effect of IL-6 and IL-8 on the MMP-9 Secretion of Endometrial Stromal Cells in Women with Endometriosis / 中国医师杂志

Objective To study the effect of IL-6 and IL-8 on the MMP-9 secretion of endometrial stromal cells in women with endometriosis to explore the function of IL-6 and IL-8 in the adherence and planting process of ectopic endometrium. Methods The endometrial stromal cells were cultured in DMEM/F12 medium containing different concentrations of IL-6 or IL-8 for 24 hours, then the media were collected to measure the levels of MMP-9 by ELISA. Results The levels of MMP-9 in control stromal cells cultured with high concentration of IL-6 were significantly higher than those without IL-6 (P

Cultivation of the Isolated Bovine Endometrial Stromal Cells and the Effect of Interleukin - 2 on Its Proliferation / 대한면역학회지

At the time in vitro fertilization and embryo transfer, patients with unsuitable endometrium recovered by hormone. However, the overtreatment of hormone causes indispositionly the uterus internal secretion and finally induces endometriosis. Therefore, this study was done to inverstigate the effects of interleukin-2, which was known to differentiator and proliferator of T cells, on proliferation of the endometrial stromal cells in vitro. We have exammined the effects of interleukin-2, on the proliferation of bovine endometrial stromal cells in vitro, assessed by ['H]-thymidine incorporation and MTT assay methods. Results indicate that we isolated endometrial stromal cells from bovine uterus and established in vitro culture system. And interleukin-2 showed distint stimulatory effect on proliferation of the established stromal cells. These stimulative effects were not affected by estrogen and progesterone indirectly. In conclusion, these data imply that interleukin-2 may proliferate bovine endometrial stromal, cells, and it provides clue for understanding of direct actions of cytokines on the endometriat cells.