抑制性消减杂交技术检测反式二羟环氧苯并芘转化细胞与正常细胞基因表达差异

Gene expression differences between the anti-BPDE-transformed and normal cells by suppression subtractive hybridization

目的 筛选 7,8 二羟 9,10 环氧苯并芘 (BPDE)转化细胞与正常细胞的基因表达差异。方法 以BPDE转化支气管上皮细胞 (16HBE)细胞为检测子 (tester) ,正常 16HBE细胞为驱动子 (driver) ,应用抑制性消减杂交方法构建cDNA消减文库 ,经 2次消减杂交和 2次PCR后 ,将巢式PCR产物插入TA载体克隆 ,随机挑选克隆进行鉴定和测序 ,并用斑点杂交法验证其同源性 ,将所得序列进行GenBank的BLAST分析。结果 发现了 7个在BPDE转化细胞中高表达的基因 ,为翻译延长因子 1α1、线粒体基因、溶解物载体家族 2 5、EphA2、酪氨酸 3 加单氧酶 色氨酸 - 5 -加单氧酶活化蛋白、乳酸脱氢酶A、TRIM2 8等 ,尚有一条在GenBank的已知基因中找不到对应序列 ,在EST序列中找到全长对应序列。结论 发现的高表达基因可能在BPDE的致癌机制中起作用 ,另外 ,可能有未知基因与BPDE的恶性转化作用有关。

Objective To screen the gene expression differences between the BPDE-transformed and normal cells. Methods Suppression subtractive hybridization was performed to profile differentially expressed genes between BPDE and normal 16HBE cells. The BPDE-transformed cells was used as tester,and the normal cells as driver. The nested PCR products were inserted into TA vector after two substractive hybridization and two PCR. The clons were picked up randomly and analyzed with PCR. Dot blot was used to tested the same source with the tester. The overexpressed cDNA fragments in BPDE cells were sequenced and compared with known genes in Genbank. Results Seven genes were identified being overexpressed in BPDE-transformed cells: translation elongation factor 1 alpha 1, mitochondrion gene, solute carrier family 25, EphA2, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,lactate dehydrogenase A (LDHA), tripartite motif-containing 28 (TRIM28), ect. One fragment could not be found in the knowm genes, it could be found in the EST database. Conclusion These over-expressed genes may be related to the carcinogenesis of BPDE, in addition , one unknown gene may be responsible for the malignant transforming effect of BPDE.