• Clinical and Experimental Reproductive Medicine
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• v.30 no.4
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• pp.317-324
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• 2003

목 적: 본 실험의 목적은 자궁내막세포를 분리 및 배양법 확립과 함께 불멸화 시키는 것이다. 방 법: 자궁내막에서 상피세포(epithelial cells)와 기질세포(stromal cells)의 분리는 Satyawaroop 등(1979)의 방법에 기초를 두었다. 자궁내막에서 상피세포와 기질세포의 순수 분리도를 확인하고, 불멸화된 기질세포에서 SV40 large T antigen을 확인하기 위하여 면역형광 염색(immunocytochemistry)과 Western blot 기법을 이용하였다. 정상 기질세포의 경우 subconfluence (60%) 상태에서 transfection을 진행하였다. 순수 분리된 plasmid DNA와 Qiagen 사의 superfect를 이용하여 transfection을 실시하였다. 결 과: 본 연구에서 우리는 두 가지 형태의 자궁내막 세포의 분리 및 배양에 성공하였다. 상피세포는 다면체의 형태를 띠며, 선(grandular)조직의 조각으로부터 나선형으로 자란다.기질 세포는 길쭉한 형태를 띠며, 상피세포에 비해 오래 살고, 빠르게 증식하여 나란한 형태로 배열된 세포 다발(cell bundle)을 형성한다. 이렇게 분리된 세포들은 95%의 균질성을 보였으며, 면역형광염색과 western blot을 통해 확인 하였다. 한편 SV40(Simian Virus 40) large T 항원을 암호화 하고 있는 염기 서열을 포함한 플라스미드 벡터로 안정적인 트랜스펙션을 시킴으로써 불멸화 된 자궁내막의 기질 세포주를 확립하였다. 불멸화 된 세포는 그 세포가 유래한 정상의 세포와 동일한 표현형을 가지고 있었다. 결 론: 본 연구에서, 우리는 자궁내막에서 상피세포(epithelial cells)과 기질세포(stromal cells)를 분리하여 배양법을 확립하였다. 동시에 SV40 large T antigen을 이용하여 불멸화된 세포주를 확립하였다. 이렇게 확립된 세포주는 자궁의 생리작용 연구 및 자궁내막증(Endometriosis)과 자궁암(Endometrial cancer) 등과 같은 여러 자궁관련 질병 연구에 많은 도움이 될 것으로 사료된다.

• Journal of Life Science
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• v.22 no.8
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• pp.1034-1040
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• 2012

Synovium is the soft tissue that lines the non-cartilaginous surfaces within joints. It has been reported that synovial cells are activated during the pathogenesis of rheumatoid arthritis. In this study, we quantitate and compare the cellular composition of synovia derived from individuals with non-inflammatory osteoarthritis (OA) and those with inflammatory rheumatoid arthritis (RA). Synovia from OA (n=8) and RA (n=5) patients were used for hematoxylin and eosin (H&E) staining. A light microscopic examination has shown that RA synovia were morphologically thickened and hypertrophied as compared to OA synovia. We also performed an immunohistochemistry (IHC) analysis to classify cell types in the synovia using CD68, CD90, or PGP9.5 markers. As a result, we obtained quantitative data regarding the cell populations, which are macrophages in the lining layer and FLSs in the subintimal layer of the synovium. Further Photoshop analyses of the H&E images could allow the counting of the number and layer of the cells in the synovium. The number and layers of the macrophage cells were increased in the lining layer of the RA synovia as compared to the OA synovia. FLS cells also were increased in the subintimal layer of RA synovia. Therefore, quantification of the H&E stained images via Photoshop is a possible analysis protocol for synovium study. This quantitation also supports the idea that the increases in cell number and cell activation are important processes for RA pathogenesis.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.183-190
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• 2000

연구목적: 포유류의 착상은 배아가 모체의 자궁벽에 매몰되는 현상으로 부착과 침투 과정을 거쳐 진행되며, 이 과정은 스테로이드 호르몬, 성장인자, 세포점착분자, 그리고 cytokine 등의 상호작용으로 이루어진다. 이 시기에 Interleukin-1 (IL-1)과 leukemia inhibitory factor (LIF) 등이 발현되는 것으로 알려져 있다. 본 실험에서는 이들의 발현이 착상과정에 어떠한 역할을 하는지 그 상관관계를 알아보고자 하였다. 재료 및 방법: 착상 전후의 배아와 자궁내막세포에서 LIF 유전자의 발현양상과 $IL-1{\beta}$와 IL-1 receptor antagonist (IL-1ra)를 처리한 LIF 유전자의 발현양상을 역전사중합효소연쇄반응 (RT-PCR)을 통해 비교하였다. 결과: 배아에서의 LIF 유전자 발현은 in vivo와 in vitro 모두에서 상실기와 포배기에 발현되었고, 자궁내막에서는 임신 1일과 4일째에 발현되었는데, 상실기보다는 포배기에, 그리고 임신 1일보다는 착상시기인 4일째의 자궁내막세포에서 발현양이 많은 것으로 나타났다. 자궁내막세포를 배양한 경우 LIF 유전자는 in vivo에서의 발현양상과 동일하게 임신 1일과 4일에 발현되었으며, 배양액에 $IL-1{\beta}$(500pgml)를 처리하였을 경우 LIF 유전자가 초기 임신 (1~5일) 중 발현되는 것으로 나타났다. 2-세포기 배아의 배양시에 $IL-1{\beta}$를 처리한 경우 8-세포기부터 LIF 유전자가 발현되었으며, 또한 IL-1ea(60 ng/ml)를 배양액에 첨가하였을 경우에는 임신1일째 자궁내막에서는 LIF 유전자가 발현되지 않은 반면, 임신 4일째의 자궁내막세포와 상실기, 포배기 배아 모두에서 LIF 유전자 발현이 감소하는 경향을 보였다. 결론: 이러한 결과는 착상 전후 배아와 자궁내막세포에서 IL-1에 의해 LIF 유전자 발현이 조절되며, 그 결과 착상에 영향을 줄 수 있다는 것을 의미한다. 또한 배아와 자궁내막세포에서 IL-1이 LIF 유전자 발현에 영향을 주는 것으로 보아 착상을 위해 IL-1과 LIF의 상호작용이 중요한 요인이라는 것을 확인할 수 있었다.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.2
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• pp.159-168
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• 2010

Objective: The purpose of this study was to evaluate the correlation between the expression pattern of vascular endothelial growth factor (VEGF) in endometrium and the pathogenesis of endometriosis by investigating VEGF expression patterns and their difference between eutopic endometrium of patients with endometriosis and that of normal controls without endometriosis. Methods: Endometrial sections were obtained from 64 hysterectomy specimens from women under age of 40, who had undergone hysterectomies and had histological evidence of endometriosis, with stage 3 and 4 according to the revised American Society for Reproductive Medicine classification. As for controls, 37 sections were gained from women diagnosed as having cervical intraepithelial neoplasia (CIN) of the uterine cervix and without evidence of pelvic endometriosis or adenomyosis during their operation. The VEGF content was evaluated immunohistochemically in the eutopic endometrium from 64 patients with endometriosis and 37 normal controls. Histological semiquantitative score (H-score) was calculated and compared between study group and control group throughout the menstrual cycle. Results: There was no significant difference in the H-score of VEGF in the eutopic endometrium between patients with endometriosis and controls without endometriosis when compared according to the same phase of the cycle, although the H-score of the study group was significantly higher in the secretory phase than the proliferative phase. Conclusion: The VEGF expression in the eutopic endometrium of women with endometriosis was not different from that of women without endometriosis. This study suggests VEGF expression in eutopic endometrium is unlikely associated with the pathogenesis of endometriosis.

• Journal of Embryo Transfer
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• v.22 no.3
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• pp.149-154
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• 2007

Endometrial cells play important roles in implantation and during pregnancy. This study was carried out to identify whether two-pore domain $K^+\;(K_{2P})$ channels are expressed in endometrial cells of Korean cattle. $K_{2P}$ channels set the resting membrane potential of many kinds of neuronal cells in the central and peripheral nervous systems. RT-PCR data showed that TASK-1, TASK-3, TREK-1, TREK-2, and TRAAK were expressed in bovine endometrial cells, and the mRNA expression levels were similar between endometrial cells with or without endometritis. The protein expression was confirmed by using commercially available polyclonal antibodies (TASK-3, TREK-1, TREK-2, and TRAAK). TASK-3 and TREK-1 were expressed in all area of endometrial cells including nuclei, while TREK-2 and TRAAK were expressed in all area of cells except nuclei. These results demonstrate for the first time the presence of $K_{2P}$ channel in endometrial cells of Korean cattle.

• Journal of Life Science
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• v.33 no.1
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• pp.102-113
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• 2023

This review discusses the cellular and molecular mechanisms by which the endometrial estrogen and progesterone receptors regulate local estrogen production, expression of the specific estrogen receptors, progesterone resistance, inflammatory responses and the differentiation and survival of endometriotic cells in endometrial inflammation. The epigenetic aberrations of endometrial stromal cells play an important role in the pathogenesis and progression of endometriosis. In particular, differential methylation of the estrogen receptor genes changes in the stromal cells the dominancy of estrogen receptor from ERα into ERβ, and results in the abnormal estrogen responses including inflammation, progesterone resistance and the disturbance of retinoid synthesis. These stromal cells also stimulate local estrogen production in response to PGE2 and the SF-1 mediated induction of steroidogenic enzyme expression, and the increased estradiol then feeds back into the ERβ to repeat the vicious inflammatory cycle through the activation of COX-2. In addition, high levels of ERβ expression may also change the chromatin structure of endometrial mesenchymal stem cells, and together with the repeated menstrual cycles can induce formation of the endometriotic tissue. The cascade of these serial events then leads to cell adhesion, angiogenesis and survival of the differentiation-disregulated stromal cells through the action of inflammatory factors such as ERβ-mediated estrogen, TNF-α and TGF-β1. Therefore, understanding of the dynamic hormonal changes during the menstrual cycle and the corresponding signal transduction mechanisms of the related nuclear receptors in endometrium would provide new insights for treating inflammatory diseases such as the endometriosis.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.1
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• pp.49-60
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• 2008

Objective: This study was carried out to investigate the effects of 3-dimensional co-culture of human endometrial cells decidualized with progesterone and TGF-${\beta}1$ on the development of 2-cell mouse embryos. Methods: Stromal and epithelial cells isolated from human endometrial tissue were immunostained for cytokeratin and vimentin. Expression of TGF-${\beta}1$, its receptor-1, -2, integrin-${\beta}3$ and prolactin in mono or co-culture according to three different hormone conditions was investigated by RT-PCR. Differential staining was used to investigate the number of ICM and trophectoderm of hatched mouse blastocysts in different three conditions. Results: The immunohistochemical study was positive for cytokeratin or vimentin and confirmed that epithelial and stromal cells were isolated from endometrial tissue successfully. In co-culture, TGF-${\beta}1$, its receptor-1, integrin-${\beta}3$ and prolactin except TGF-${\beta}1$-r2 were expressed in progesterone dominant condition. The hatching and attaching rate were higher in the co-culture with decidualized cells (p<0.05). However, we observed that lots of the incomplete hatched blactocysts attached on non-decidualized cells. The ICM number of hatched mouse blastocysts was higher in co-culture with decidualized and non decidualized cells than media only culture (p<0.05). The trophectoderm number of hatched blastocyst was higher in the co-culture with decidualized cells than non-decidualized cells or media only culture (p<0.05). Conclusion: The administration of progesterone, estrogen and TGF-$\beta$ could induce decidualization of stromal and epithelial cells isolated from human endometrial tissue using 3-dimensional co-culture, and the decidualization of human endometrial cells could increase the hatching and attaching rate of 2-cell mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.2
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• pp.99-113
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• 2010

Objective: It has been recently reported that very small stem cells with pluripotency are detected in murine and human. The purposes of this study are to confirm whether very small putative stem cells (VSPSCs), which have the proper characteristics of stem cells as well as the expression of stem cell markers, are detected in human endometrium. Methods: The endometrial cells of 5 women, which were obtained by endometrial biopsy, were cultured for 2 weeks and were confirmed for the expressions of alkaline phosphatase, OCT-4 and CXCR4 by immunochemistry. Subsequently VSPSCs were separated by percoll density gradient method and were cultured. Also VSPSCs and their derived cells were confirmed for the expressions of OCT-4 and CXCR4. Results: The colonies, which is composed with VSPSCs less than 3 ${\mu}m$ and the 5~15 ${\mu}m$ sized hyperchromatic round cells, were detected in the endometrium of all of women and showed the strong expressions of alkaline phosphatase, OCT-4 and CXCR4. In culture after the separation of VSPSCs by percoll, these cells showed the morphological and functional characteristics of stem cells; self-renewal, colony formation, embryoid body-like formation and differential plasticity. VSPSCs formed gradually the 5~15 ${\mu}m$ sized hyperchromatic round cells and the 10~20 ${\mu}m$ sized sphere-shaped cells by cell-to-cell aggregation or cell fusion. Then these cells differentiated the various cells including fibroblast-like cells, nerve-like cells and endothelium-like cells. VSPSCs and their derived cells often showed the strong expressions of OCT-4 and CXCR4. Conclusion: VSPSCs less than 3 ${\mu}m$ and their derived cells are detected in human endometrium and these cells have the proper characteristics of stem cells and the expressions of stem cell markers as alkaline phosphatase, OCT-4 and CXCR4.

• Journal of Chest Surgery
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• v.40 no.4 s.273
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• pp.256-263
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• 2007

Background: Intimal hyperpiasia is characterized by a proliferation of vascular smooth muscle cells in the intimal layer Epigallocatechin-3-gallate (EGCG) is known to suppress smooth muscle cell proliferation. We propose that EGCG may have a protective effect against the development of intimal hyperplasia through the suppression of smooth muscle cell proliferation. Material and Method: Human umbilical vein endothelial cells (HUVEC) and rat aortic smooth muscle cells (RASMC) were cultured with different concentrations of EGCG, and proliferation and migration speed were measured. In 20 dogs, the autologous jugular veins were interposed into the carotid arteries. For the study group (n=10), the graft was stored for 30 minutes in EGCG solution and 300mM EGCG was applied to the perivascular space after grafting. After 6 weeks, the intimal and medial thickness was measured. Result: The proliferation of RASMC and HUVEC was suppressed with EGCG. The migration of RASMC was suppressed with EGCG, but that of HUVEC was not affected. In the in vivo study, the intimal thickness was thinner in EGCG group than in the control group (p<0.05), but the medial thickness did not show any difference. The intimal/medial thickness ratio was lower in the EGCG group (p<0.05). Conclusion: EGCG suppresses intimal hyperplasia after vascular grafting, and this may be mediated by prevention of migration and proliferation of vascular smooth muscle cells. The use of EGCG may offer new therapeutic modality to prevent intimal hyperplasia.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.3
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• pp.207-216
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• 2010

Objective: This study was performed to clarify the role of HomeoboxA (HOXA) and its related signaling molecules in the decidualization of primary cultured endometrial cells. Methods: Human endometrial tissues were obtained by curettage of hysterectomy specimens from patients with conditions other than endometrial diseases. Tissues were minced and digested with Trypsin-EDTA for 20 min, $37^{\circ}C$. Cells were cultured with DMEM/F12 medium in $37^{\circ}C$, 5% $CO_2$ incubator for 24 hrs. Cells were treated with HOXA10 siRNA and added transforming growth factor (TGF)-${\beta}1$ (10 ng/mL) for 48 hrs to induces decidualization in vitro. Reverse transcription polymerase chain reaction analysis was accomplished to observe the expression of HOXA10, prolactin, cyclooxygenase (COX)-2, peroxisome proliferatoractivated receptor (PPAR)-$\gamma$, and wingless-type MMTV integration site family (Wnt). Results: HOXA10 expression was increased (1.8 fold vs. non-treated control) in TGF-${\beta}1$ treated cells. Decidualization marker, prolactin, was significantly increased in TGF-${\beta}1$ treated cells compared with HOXA10 siRNA treated cells. Endometrial cell differentiation marker, COX-2 was down-regulated by HOXA10 siRNA even if cells were treated with TGF-${\beta}1$. Wnt4 was down-regulated by treated with HOXA10 siRNA, this expression patters was not changed by TGF-${\beta}1$. Expression of PPAR-$\gamma$ was down regulated by TGF-${\beta}1$ in regardless of HOXA10 siRNA treatment. Conclusion: TGF-${\beta}1$ which is induced by progesterone in endometrial epithelial cells may induces stromal cell decidualization via HOXA10 and Wnt signaling cascade.