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New classification of leukemic cells by using of biotecnology (Differential expression of selected genes in human leukemia leukocytes)

利用生物技术对白血病细胞进行新分类（人白血病白细胞中选定基因的差异表达）

基本信息

批准号：
60570293
负责人：
SHIOSAKA Takahiko
金额：
$ 0.9万
依托单位：
Ehime University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1986
项目状态：
已结题

项目摘要

Human leukemias are considered monoclonal disease viewed as impairing the hematopoietic cell differentiation process. It is reasonable to except that such impairment would be reflect in quantiative changes in mRNA. By using recombinant DNA technology to prepare a cloned library of expressed gene sequences discrete RNA species present in differential amounts in normal and malignant cells can now be examined. The relative abundance of a clonal sequence of cellular RNA can be estimated by nucleic acid hybridization. Selected and analysis of clones containing copies of cDNA transcripts of mRNA was accomplised as shown below.1.Preparation of cDNA library from total mRNA of CLL cells. And screening of library with [ <^(32)P> ]-cDNA from CLL and placental cells. Selection of 21 clones that react positively with [ <^(32)P> ]-cDNA from CLL but do not react with [ <^(32)P> ]-cDNA from placental cells.2.The use of these DNA segments to measure the population of mRNA for neoplastic cell specific expressed genes in various leukemic cells by Dot blot hybridization method.3.Determination of DNA sequence of cDNA insert in each clone.Total cellular RNAs from a variety of hematopoietic neoplastic cells and normal lymphocytes were analyzed for homology with 21 recombinant DNA segments complementary to the specific mRNA of cells from a patiant with CLL.Twenty of the clones (except for clone 8-6G) were not significantly represented in the mRNA from normal lymphocytes. The relative concentrations of three clones (6-1E,7-3G and 9-5C) in the RNAs from a variety of normal leukemic leukocytes and human hematopoietic cell lines were measured by Northern blot hybridization. This showed that 7-3G,6-1G and 9-5C RNA were highly abundant in RNA from hematopoietic neoplastic leukocytes. Also, clone 7-2D was highly represented in B cell type hematopoietic neoplastic leukocytes,6-1G was highly represented in Raji cell while clone 6-1E was highly represented in Molt 3 cell.

人类白血病被认为是一种损害造血细胞分化过程的单克隆性疾病。这是合理，但这种损害将反映在定量变化的信使核糖核酸。通过使用重组DNA技术准备表达基因序列的克隆文库，现在可以检测正常细胞和恶性肿瘤细胞中存在的不同数量的离散RNA物种。细胞RNA克隆序列的相对丰度可以通过核酸杂交来估计。如下所示：1.从CLL细胞的总mRNA中制备cDNA文库。从CLL和胎盘细胞中筛选含[&lt；^(32)P&gt；]-cDNA文库。从CLL中筛选出与[&lt；^(32)P&gt；]-cDNA呈阳性反应但不与[&lt；^(32)P&gt；]反应的21个克隆。2.利用这些DNA片段通过斑点杂交法测定不同白血病细胞中肿瘤细胞特异性表达基因的mRNA群体。3.每个克隆插入的DNA序列的测定。对来自各种造血肿瘤细胞和正常淋巴细胞的细胞总RNA进行同源性分析，其中21个重组DNA片段与一名CLL患者细胞的特异性mRNA互补，其中20个克隆(克隆8-6G除外)在正常淋巴细胞的mRNA中没有显著表达。用Northern印迹杂交法测定了3个克隆(6-1E、7-3G和9-5C)在多种正常白血病细胞和人造血细胞系的RNA中的相对浓度。这表明7-3G、6-1G和9-5C RNA在造血肿瘤白细胞的RNA中高度丰富。克隆7-2D在B细胞型造血肿瘤细胞中高表达，6-1G在Raji细胞中高表达，6-1E在Molt3细胞中高表达。