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Development of in vitro model for implantation using the endometrial spheroid

使用子宫内膜球体开发体外植入模型

基本信息

批准号：
18580282
负责人：
YAMAUCHI Nobuhiko
金额：
$ 2.45万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580282/
关键词：
spheroid embryo rat three-dimensional culture implantation MMP 子宮内膜間質細胞

项目摘要

The endometrium is one of the most complex tissues; it undergoes dynamic changes in response to implantation and pregnancy processes. An in vitro model may provide a tool for clarifying the complex implantation process. However, there is no suitable in vitro model for the investigation of endomotrial functions. The spheroid has been utilized in cell biology research because it appears to mimic morphology and physiology of cells in living tissues and organs, which is unlike conventional monolayer culture. Multicellular spheroids composed of normal adult cells may provide a more useful model for the study of endometrium. The purpose of the present study is to develop the spheroid composed of rat endometrial stromal cells(RES) as an in vitro model for analysis of the endometrial functions.The RES were prepared from rat endometrium at day 5 of pregnancy. Spheroids were generated using salmon aterocollagen(SAC). The cells were plated on 12 well SAC gels(Imoto Suisan Co.) and cultured in the … More culture medium(DMEM/F12) containing 10% foetal bovine serum. After the cells reached confluence at day 7 in culture, SAC gels were digested by collagenase to promote the detachment of RES cell sheets. Then the floating cell sheets were transferred to agarose-coated plates and cultured to form spheroids. The cell-sheet shrank and became an aggregated cell mass in a few days; it finally formed a round-shaped spheroid. Diameters of the spheroids were about 500μm at day 5 after detachment from SAC gels.TUNEL examination of cell viability in the spheroid suggested no cell was apoptotic until 15 days after cell sheet detachment, TUNEL-positive cells appeared at 20 days. Additionally, no positive staining of PCNA was observed in the spheroids in contrast to the strong staining in the proliferating monolayer cultured cells. The results of gelatin zymography showed that both MMP-2 and -9 were produced in monolayer culture. However, after the detachment of the cell sheet, the production of both MMPs decreased immediately and could not be detected until 15 days after detachment.In vitro decidualization of the spheroids were induced by arachidonic acid(AA) treatment. RT-PCR analysis showed that typical marker genes for the decidualization, desmin and d/tPRP, were expressed in the spheroids after 2 days of AA treatment, but not in the control groups without treatment.The results of the present study indicate that rat endometrial stromal cells are capable of being regenerated as a spheroid using SAC gels in vitro. Importantly, the present spheroid displays an endometrium-mimic feature in both the structural and functional similarities. The present method is simple and convenient, and therefore provides a new insight into the study of endometrial functions and implantation. Less

子宫内膜是最复杂的组织之一;它经历动态变化，以响应着床和妊娠过程。体外模型可以提供用于阐明复杂的植入过程的工具。然而，目前还没有合适的体外模型用于研究肌内膜功能。球状体已被用于细胞生物学研究，因为它似乎模仿活组织和器官中细胞的形态和生理学，这与传统的单层培养不同。由正常成体细胞组成的多细胞球体可能为子宫内膜的研究提供一个更有用的模型。本研究的目的是建立大鼠子宫内膜基质细胞（RES）的体外模型，用于子宫内膜功能的研究。使用鲑鱼缺角胶原（SAC）产生球体。将细胞接种在12孔SAC凝胶（Imoto Suisan Co.）并在 ...更多信息含10%胎牛血清的培养基（DMEM/F12）。在培养的第7天细胞达到汇合后，用胶原酶消化SAC凝胶以促进RES细胞片的脱离。然后将漂浮的细胞片转移到琼脂糖包被的平板上并培养以形成球状体。细胞片层在几天内收缩并变成聚集的细胞团，最终形成圆形球体。从SAC凝胶上脱落后第5天，细胞球体直径约为500μm，TUNEL法检测球体内的细胞活力，直到脱落后第15天才发现有细胞凋亡，第20天出现TUNEL阳性细胞。此外，在球状体中未观察到PCNA阳性染色，而在增殖的单层培养细胞中观察到强染色。明胶酶谱分析结果表明，MMP-2和MMP-9均在单层培养中产生。然而，在细胞片脱离后，这两种MMPs的产生立即下降，直到脱离后15天才被检测到。RT-PCR分析表明，典型的蜕膜化，desmin和d/tPRP的标记基因，表达的球体后，AA处理2天，而不是在对照组无treatment. Results的本研究表明，大鼠子宫内膜间质细胞是能够再生为一个球体使用SAC凝胶在体外。重要的是，目前的球体显示子宫内膜模拟功能的结构和功能的相似性。该方法简便易行，为研究子宫内膜功能和着床提供了新的思路。少

项目成果

期刊论文数量（0）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Effects of tumor necrosis factor-alpha on cell proliferation,prostaglandins and matrix-metalloproteinases production in rat endometrial stromal cells cultured in vitro

肿瘤坏死因子-α对体外培养的大鼠子宫内膜基质细胞增殖、前列腺素和基质金属蛋白酶产生的影响

DOI：
发表时间：
2007
期刊：
J Exp Zool Part A Ecol Genet Physiol 307
影响因子：
0
作者：
Gamo T;et. al.
通讯作者：
et. al.

Effects of tumor necrosis factor-α on cell proliferation, prostaglandins and matrix-metalloproteinases production in rat endometrial stromal cells cultured in vitro

肿瘤坏死因子-α对体外培养的大鼠子宫内膜基质细胞增殖、前列腺素和基质金属蛋白酶产生的影响

DOI：
发表时间：
2007
期刊：
Reproduction, Fertility and Development (掲載決定)
影响因子：
0
作者：
T.;Gamo;N.;Yamauchi;K.;Nishimura;R.;Watanabe;K.;Matsumoto;P-J.;He;T.;Soh;M-A.;Hattori;T gamo et al.
通讯作者：
T gamo et al.

生体外におけるラット子宮内膜スフェロイドの脱落膜化

大鼠子宫内膜球体的体外蜕膜化