Telemere length and telomerase activity in cultured amniotic fluid cell

培养羊水细胞的端粒长度和端粒酶活性

• 批准号: 17591141
• 负责人: NINOMIYA Shinsuke
• 金额: $ 0.96万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591141/
• 关键词: amniotic fluid cell; chromosome; telomerase; テロメア長; テロメラーゼ活性

Amniotic fluid cells are cultured in the condition of 37°C and 5% CO_2 using AmnioMax initially. Amniotic fluid cells were passed by 1st, 2nd, 5th respectively. In the passage 0.25% trypsin and MEM+10% FBS were used. The number of cells was treated 1X10^4.The pellet was reserved to measure telomerase activity at-80°C. We prepared 6 amniotic fluid cell samples, 3 chorionic villi samples and 1 fetus skin fibroblast.We used HeLa cells which have strong activity to perform the quantitative analysis about telomerase activity and made a measurement curve. We prepared 2 X10^4 cell count for HeLa cells and diluted cells by 5 steps respectively. We analyzed telomerase activity by using TeloChaser. The cell extract containing telomerase was prepared and telomere repeat sequence was added to substrate primer. After purification PCR was performed using reverse primer added. PCR products were analyzed by electrophoresis staining with ethidium bromide and a measurement curve was completed.The telomerase activity about amniotic fluid cells was analyzed using the same as above mentioned method. 6 amniotic fluid cells samples with normal chromosome karyotype had no telomerase activity.

用AmnioMax在37℃、5%CO_2的条件下培养羊水细胞。羊水细胞分别传代第1、2、5代。传代中使用0.25%胰酶和MEM+10%胎牛血清。取细胞数处理1×10~4，在-80℃条件下留取颗粒进行端粒酶活性测定，制备6例羊水细胞、3例绒毛组织和1例胎儿皮肤成纤维细胞，用活性较强的HeLa细胞进行端粒酶活性定量分析，并绘制测量曲线。用5步法分别制备HeLa细胞和稀释液细胞的2×10~4细胞计数。用TeloChaser软件分析端粒酶活性。制备含有端粒酶的细胞提取液，在底物引物中加入端粒重复序列。纯化后，加入反向引物进行聚合酶链式反应。聚合酶链式反应产物用溴化乙锭进行电泳法染色，绘制测量曲线，用上述方法分析羊水细胞端粒酶活性。6例染色体核型正常的羊水细胞无端粒酶活性。