人巨核白血病细胞诱导分化后差异表达基因的mRNA差别显示和克隆

Cloning of differentially expressed genes in a human leukemic megakaryocytic cell line HIMeg after induced differentiation using mRNA differential display

目的：克隆巨核白血病细胞诱导分化后表达变化的ｍＲＮＡ，从中寻找对诱导分化起关键作用的基因，最终揭示巨核白血病细胞诱导分化的分子机制。方法：人巨核白血病细胞ＨＩＭｅｇ经１３－顺式维甲酸处理４ｄ后，收集细胞总ＲＮＡ，进行ｍＲＮＡ差别显示、ｃＤＮＡ片段克隆和序列分析。结果：获得５个差示ｃＤＮＡ片段，经ＲＮＡ点杂交分析后发现其中３个为假阳性，１个因没有杂交信号而无法确定，只有１个ｃＤＮＡ片段得到证实，其对应的ｍＲＮＡ在诱导分化后表达下降。该ｃＤＮＡ片段的核苷酸序列已在ＧｅｎＢａｎｋ中登录，登录号为ＡＦ０２６５２６。结论：克隆到一个未知基因的ｃＤＮＡ片段，其全长ｃＤＮＡ序列。

Objective: In a human leukemic megakaryocytic cell line HIMeg, differentially expressed genes regulated by induced differentiation will be cloned, and from which the key genes in the process of induced differentiation will be identified. Methods: The human megakaryocytic leukemia cell line HIMeg was treated with 13 cis retinoic acid for 4 d, then total cellular RNA was extracted and reverse transcribed to first strand cDNA. The first strand cDNA was subjected to PCR based mRNA differential display, cloning and sequence analysis. Results: From 5 cDNA fragment candidates obtained, RNA dot blot analysis demonstrated non regulation in 3 fragments, undetectable signals in one fragment, and altered gene expression in one cDNA fragment which was designated MDI 1 (mRNA downregulated after induced differentiation).The nucleotide sequence data reported here will appear in the GenBank under the accession number AF026526. Conclusion: A cDNA fragment encoded by a unknown gene was cloned. Further studies are required to determine its full length sequence, gene structure and biological function(s).