Isolation and identification of cDNAs related with mouse tooth germs and its localization on human odontogenic tumor.

小鼠牙胚相关cDNA的分离鉴定及其在人牙源性肿瘤中的定位。

• 批准号: 08457482
• 负责人: OKABE Haruo
• 金额: $ 4.67万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457482/
• 关键词: TOOTH GERM; IN SITU HYBRIDIZATION; RT-PCR; T-T DIMER

We isolated cDNAs from excised tooth germs and giniva of mouse embryo (E13-E17) and compared expression of cDNAs of each tissue by using RT-PCR and differential display. The eluted PCR products of tooth germ-spcecific cDNAs from acrylamide gels were thymine-thymine (T-T) dimerized by ultra violet irradiation. The specificity of the cDNAs were confirmed by in situ hybridization applied to tooth germs of mouse embryo (E14-17) by using the each T-T dimerized cDNA probe. By this method, we isolated mouse amelogenin cDNA and the coding region had 83% identity with human amelogenin.Moreover, we performed in situ hybridization for the same amelogenin cDNA probe on human odontogenic tumor : Ameloblastoma (plexiform type, follicular type and mixed type), Calcifying odon togenic tumor, and Cementoma. The amelogenin was detected on ameloblastoma both plexiform and follicular type but not calcifying odontogenic tumor and cementoma, because of previous decalcification by hydrochloric acid.These results reveals that the in situ hybridization using T-T dimerized cDNA probe were available for screening and isolation of novel tissue specific mRNA.

我们从小鼠胚胎（E13-E17）切除的牙胚和牙龈中分离cdna，并采用RT-PCR和差异显示的方法比较各组织的cdna表达。从丙烯酰胺凝胶中洗脱的牙齿细菌特异性cdna PCR产物在紫外线照射下被胸腺嘧啶-胸腺嘧啶（T-T）二聚体化。利用T-T二聚体探针对小鼠胚胎（E14-17）牙胚进行原位杂交，证实了cDNA的特异性。用该方法分离得到小鼠淀粉原蛋白cDNA，编码区与人淀粉原蛋白同源性达83%。此外，我们对人类牙源性肿瘤：成釉细胞瘤（丛状型、滤泡型和混合型）、钙化牙源性肿瘤和骨水泥瘤进行了相同的淀粉原蛋白cDNA探针的原位杂交。在丛状和滤泡型成釉细胞瘤中检测到淀粉原蛋白，但在钙化性牙源性肿瘤和骨水泥瘤中未检测到淀粉原蛋白，因为之前曾用盐酸脱钙。这些结果表明，利用T-T二聚体cDNA探针原位杂交可用于筛选和分离新的组织特异性mRNA。