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目的 探讨白血病细胞多药耐药发生的分子机理,寻找新的多药耐药相关基因。方法 以HL- 6 0细胞为“驱动方”,以多药耐药细胞系HL- 6 0 / VCR为“实验方”,建立人白血病多药耐药细胞系HL-6 0 / VCR抑制消减杂交文库,利用基因芯片技术从中筛选出一条在野生型HL - 6 0细胞中基本不表达,而在HL - 6 0 / VCR耐药细胞中呈低丰度差异表达的新表达序列标记序列。用表达序列标记拼接的同源基因克隆法得到人类新基因HV12 6序列,再利用生物信息学数据库和软件对其进行功能的预测和分析。最后针对推导序列开放阅读框设计引物,逆转录-聚合酶链反应检测推导HV12 6基因序列在临床白血病患者化疗前后白血病细胞中的表达。结果 HV12 6的c DNA序列长1991bp,编码36 5个氨基酸,其编码蛋白序列局部与新近发现的在细胞凋亡与炎症反应中起重要作用的蛋白基序“PYRIN”存在约4 3%的相似性。逆转录-聚合酶链反应结果提示该基因与白血病细胞受化疗药物的作用和耐药存在联系。结论 HV12 6可能是与白血病细胞耐药相关的一条新基因,有必要对该基因进行进一步的实验室克隆与功能研究。
Objective To investigate the molecular mechanism of multidrug resistance in leukemia cells and to search for new multidrug resistance related genes. Methods HL-60 cells were used as driver and HL-60 / VCR multi-drug resistant cell line was used as experimental group to establish a human leukemia multidrug-resistant cell line HL-60 / VCR suppression subtractive hybridization Library, a new expression sequence tagged in HL - 60 / VCR multidrug resistant cell line was screened out by using gene chip technique. Homologous gene cloning with expression sequence tagging was used to obtain the human HV12 6 gene sequence, which was predicted and analyzed by using bioinformatics database and software. Finally, primers were designed according to the deduced open reading frame sequence. The expression of HV12 6 gene in leukemic cells of patients with clinical leukemia before and after chemotherapy was determined by reverse transcription polymerase chain reaction (RT-PCR). Results The DNA sequence of HV12 6 was 199 bp in length and encoded 36 5 amino acids. The coding sequence of HV12 6 was about 43% similar to the newly discovered protein motif “PYRIN”, which was found to play an important role in apoptosis and inflammation. Sex. The result of RT-PCR indicated that the gene and leukemia cells are affected by chemotherapeutic drugs and drug resistance. Conclusion HV12 6 may be a new gene related to drug resistance in leukemia cells. It is necessary to further investigate the cloning and function of this gene in laboratory.