| 饶亚岚,陈肖华,张军权,魏巍,董波,毛秉智.SSH方法分离大剂量照射后小鼠骨髓差异表达EST序列[J].中华放射医学与防护杂志,2003,23(3):163-166 |
| SSH方法分离大剂量照射后小鼠骨髓差异表达EST序列 |
| Expressed sequence tags isolated from mouse bone marrow cells irradiated with high-dose gamma rays by suppression subtractive hybridization (SSH) |
| 投稿时间：2002-09-27 |
| DOI： |
| 中文关键词: 小鼠骨髓 电离辐射 抑制消减杂交 差异表达序列标签 |
| 英文关键词:Mouse bone marrow Ionizing radiation Suppression subtractive hybridization Expressed sequence tags |
| 基金项目:全军“十五”医药卫生科研基金资助项目(01L018) |
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| 中文摘要: |
| 目的 为探讨骨髓辐射损伤的分子机理,研究了整体照射条件下,辐射前后骨髓基因表达的变化。方法 小鼠7Gyγ射线照射后4h提取骨髓总RNA为实验方(tester),对照组动物骨髓总RNA为驱动方(driver);实验方、驱动方总RNA由SMART cDNA合成方法合成双链cDNA,进而由抑制消减杂交(SSH)方法获得消减杂交产物,消减杂交产物克隆到T载体建立消减文库;对部分克隆进行杂交筛选及序列比对。结果 消减杂交文库挑取800个克隆进行PCR扩增,阳性率约为86%;部分克隆经两轮杂交筛选获得14个差异片段;7个差异片段与鼠EST库已有序列高度相似,其余7个差异片段可能是新的EST序列。结论 大剂量照射后小鼠骨髓cDNA消减文库的建立以及部分差异表达EST的验证为进一步研究辐射相关差异表达基因奠定基础。 |
| 英文摘要: |
| Objective To explore the molecular mechanism of bone marrow injury caused by irradiation, gene expression of bone marrow cells in mice after whole body irradiation was studied. Methods Total RNA extracted from bone marrow cells in mice at 4 h after whole body irradiation with 7 Gy γ-rays was taken as the tester and the total RNA from control mice as the driver cDNA subtraction was performed using the protocols described in the Clontech SMART PCR cDNA Sythesis Kit and PCR-select cDNA Subtraction Kit.The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library.Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank. Results A total of 800 clones selected from the subtracted cDNA library were PCR-amplified and about 86% had inserts.Fourteen differential cDNA fragments were acquired after two round hybridization screening, 7 of them showed high similarity to the expressed sequence tags (ESTs) in the mouse EST database and the other 7 cDNA fragments were possibly new ESTs. Conclusion The succesessfully constructed subtracted cDNA library of bone marrow cells in mice after whole body high-dose irradiation and the validation of some differential ESTs establish the basis for further research of radiation-related genes. |
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