cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE_1表达差异cDNA序列的初步研究

PRIMARY STUDY OF DIFFERENTIALLY EXPRESSED cDNA SEQUENCES IN CELL LINE HNE 1 OF HUMAN NASOPHARYNGEAL CARCINOMA BY cDNA REPRESENTATIONAL DIFFERENCE ANALYSIS

目的寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因。方法应用ｃＤＮＡ代表性差异分析法（ＲＤＡ），分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，并用链终止法测序。结果在第４轮杂交及扩增反应后，获得４条差异性条带。Ｓｏｕｔｈｅｒｎ杂交及Ｎｏｒｔｈｅｒｎ杂交证明，这些差异性片段来自作为“检测”扩增子的正常人鼻咽上皮，在鼻咽癌细胞株ＨＮＥ１中不表达或表达降低。序列分析这些差异性片段的克隆，发现一些序列是与已知基因高度同源的基因，包括一些看家基因；另有一些基因则为新基因序列。结论鼻咽癌的发生是一个多基因参与的过程，所获得的差异性片段中，与之同源的一些已知基因具有抑瘤功能。

Objective To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma(NPC), including the candidates of tumor suppressor genes. Methods cDNA representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE 1. The sources of differentially expressed products were proved by Southern blot and Northern blot. The fragments were cloned with pGEM T easy kit and sequenced by the chain termination reaction.Results Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human primary cultures of nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE 1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down regulated in the NPC HNE 1 cells. Of these obtained clones, some are the fragments of the human known genes including house keeping genes, the others are novel genes.Conclusion NPC involves alteration of multiple genes. Some of known genes matched with the differentially expressed sequences have an effective suppressive ability on the carcinoma.