【摘要】 目的　用荧光差异显示PCR(fluoroDD PCR)方法寻找低剂量的甲醛(FA)诱导人胚肺成纤维细胞(MRC 5 )损伤耐受差异表达的基因。方法　用MTT方法得到FA对MRC 5毒性的剂量-反应关系。根据剂量-反应关系选择对MRC 5几乎没有损伤或有增殖的剂量作为低剂量,对细胞有明显损伤的剂量为高剂量,然后用低剂量、高剂量、损伤耐受模式(低剂量预刺激后继而用高剂量刺激)三种方式处理细胞,通过荧光差异显示PCR技术寻找不同处理组与对照组比较差异表达的基因。对其中的11个差异条带进行二次PCR、克隆、测序,在GeneBank上通过Blast鉴定基因。结果　根据FA对MRC 5毒性的剂量-反应关系,选择10 0 μmol L为低剂量、10mmol L为高剂量。然后按方法中所述的方式处理细胞后,用荧光差异显示PCR的方法发现有6 1个差异表达的基因。对其中的11个差异条带进行鉴定,发现有2个已知基因,分别与nuclearfactorofactivatedT cells 5 (NFAT5 )和tetratricopeptiderepeatdomain 3(TPRD 3)高度同源,其余9个为新基因。结论　FA在低剂量可以促进MRC 5的增殖,通过荧光差异显示PCR获得6 1个FA诱导MRC 5损伤耐受差异表达的基因,通过对其中11个差异条带的鉴定,为进一步研究FA诱导MRC 5损伤耐受的机制提供线索。更多还原

【Abstract】 Objective To observe the differentially expressed genes of the human embryo lung fibroblast (MRC-5) induced by formaldehyde (FA) of low dose using fluoro DD-PCR. Methods The dose-effect relation of FA toxicity to MRC-5 was acquired, No observed damage effect or proliferation concentration was used as low dose, and obvious damage concentration was used as high dose. MRC-5 was treated with low and high dose, and treated with high dose after pretreated with low dose for some time. Then Fluoro differential display polymerase chain reaction (Fluoro DD-PCR) was used to search differentially expressed genes of the differently treated groups of FA. 61 differential display straps were acquired and 11 of them were reamplified, cloned, sequenced and blasted. Results According to the dose-effect relation of FA toxicity to MRC-5, 100μmol/L was choosed as low dose and 10 mmol/L was choosed as high dose. Samples of differently treated groups were amplified by means of fluro-DD-PCR, 61 differentially expressed straps were acquired. 11 differential display straps have been cloned,sequenced and blasted. Two of them were known genes:one was highly homologous to nuclear factor of activated T-cells 5(NFAT5)and the other was highly homologous to tetratricopeptide repeat domain 3(TPRD-3). Nine of them were new genes. Conclusion It seemed that FA of low dose could promote MRC-5 proliferation and find 61 differentially expressed genes of differently treated groups, and the result of clone could provide scientific base for mechanism of FA toxicity research.更多还原