Coculture of preimplantation embryo with an immortalized human oviductal epithelial cells

植入前胚胎与永生化人输卵管上皮细胞的共培养

• 批准号: 06671654
• 负责人: HARADA Tasuku
• 金额: $ 1.09万
• 依托单位: Tottori University, Faculty of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06671654/
• 关键词: Oviductal epithelial cells; immortalize; basis fibroblast growth factor; FGF receptor 1; 6FGF

We have tried establish an immortalized cell line of human tubal epithelium. It is not possible to obtain immortalized cells after repeated experiments. We also have performed experiments using preimplantation embryos. We determined the expression of fibroblast growth factor (FGF) -2 and the FGF receptor 1 (FGFR-1) mRNA in the mouse embryos and in epithelial cells of the uterine endometrium using reverse transcription-polymerase chain reaction (RT-PCR). No FGF-2 mRNA was detected in mouse embryos but FGFR-1 mRNA was expressed at the blastocyst stage. These results suggest importance of FGFR-1 in preimplantation development of embryo. To examine the mechanism of FGFR-1 expression, the arrangement of exons and introns encoding 5'-side of murine FGFR-1 gene was mapped. A large intron with a size of 14kb was identified between exon 1 and 2. All subtypes of FGFR-1 was obserbed to be generated through alternative aplicing. Furthermore, we characterized a transcription element of this gene. The basal promoter element was mapped to the 5'-flanking region from -89 to -43. The DNase I protection assay and gel shift analysis revealed that a nuclear protein could bind to the nucleotide sequence from -62 to -42. In conclusion, FGF-2 produced by the female reproductive tract may exert paracrine effects on preimplantation embryos. FGFR-1 gene has an unique genomic structure. The 5'-flanking region from -62 to -42 plays a pivotal role in FGFR-1 gene expression.

我们尝试建立人输卵管上皮永生化细胞系。反复实验后不可能获得永生化细胞。我们还使用植入前胚胎进行了实验。我们确定成纤维细胞生长因子（FGF）-2和FGF受体1（FGFR-1）mRNA的表达在小鼠胚胎和子宫内膜上皮细胞，使用逆转录-聚合酶链反应（RT-PCR）。在小鼠胚胎中未检测到FGF-2 mRNA，但在囊胚期表达FGFR-1 mRNA。这些结果表明FGFR-1在胚胎着床前发育中的重要性。为了研究FGFR-1表达的机制，对编码小鼠FGFR-1基因5 '端的外显子和内含子的排列进行了定位。在外显子1和2之间发现了一个大小为14 kb的大内含子。FGFR-1的所有亚型均通过交替应用产生。此外，我们还鉴定了该基因的一个转录元件。将基础启动子元件定位于-89至-43的5 '侧翼区。DNA酶I保护试验和凝胶位移分析表明，核蛋白可以结合到-62至-42的核苷酸序列。总之，女性生殖道产生的FGF-2可能对植入前胚胎产生旁分泌作用。FGFR-1基因具有独特的基因组结构。从-62至-42的5 '侧翼区在FGFR-1基因表达中起关键作用。

项目成果

Saito H: "Mapping of a transcviption clement cvitical for expression of the fibroblast growth facfor receptor 1 gene" Biochem Biophys Res Commun. 198. 1020-1026 (1994)

Saito H：“对成纤维细胞生长因子受体 1 基因表达至关重要的转录因子的作图”Biochem Biophys Res Commun。

Tasuku Harada: "Murine fibroblast growth factor receptor 1 gene generates multiple messenger RNAs containing two open reading frames via alteernative splicing" Biochemical and Biophysical Research Communications. 205(2). 1057-1063 (1994)

Tasuku Harada：“鼠成纤维细胞生长因子受体 1 基因通过选择性剪接产生包含两个开放阅读框的多个信使 RNA”《生物化学和生物物理研究通讯》。

Harada T: "Murine fibroblast growth factor receptor 2 genc generates mucltigle messenger RNAs containing two open reading framesvia alternative splicing" Biochem Biophys Res Commun. 205. 1057-1063 (1994)

Harada T：“鼠成纤维细胞生长因子受体 2 基因通过选择性剪接产生含有两个开放阅读框的多信使 RNA”Biochem Biophys Res Commun。

Harada T: "Hurine fibroblast growth factor receptor 1 gene generates multiple messenger RNAs containing two open reading frames via alternative splicing" Biochem Biophys Res Commun. 205. 1057-1063 (1994)

Harada T：“人成纤维细胞生长因子受体 1 基因通过选择性剪接产生包含两个开放阅读框的多个信使 RNA”Biochem Biophys Res Commun。

Hiroshi Saito: "Mapping of a transcription element critical for expression of the fibroblast growth factor receptor 1 gene" Biochemical and Biophysical Research Communications. 198(3). 1020-1026 (1994)

Hiroshi Saito：“对成纤维细胞生长因子受体 1 基因表达至关重要的转录元件的图谱”生物化学和生物物理研究通讯。