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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。
OBJECTIVE: To study the expression of differentially expressed genes in human lung fibroblasts (MRC 5) induced by formaldehyde (FA) induced by low dose of fluoroDD PCR. Methods The dose-response relationship of FA to MRC 5 toxicity was obtained by MTT method. According to the dose-response relationship, MRC 5 was selected as a low dose with little or no proliferative dose. The cells with significant damage to the MRC 5 were treated with high dose and then treated with low dose, high dose and injury tolerance mode (after low dose pre-stimulation Followed by high-dose stimulation) three ways to deal with cells by fluorescence difference display PCR technology to find different treatment groups compared with the control group differentially expressed genes. The 11 differential bands were subjected to secondary PCR, cloning and sequencing, and the genes were identified by Blast on GeneBank. Results According to the dose-response relationship of FA to MRC 5 toxicity, 10 μmol L was chosen as the low dose and 10 mmol L was given as the high dose. After the cells were treated in the manner described in the method, 61 differentially expressed genes were found by fluorescent differential display PCR. Of the 11 differential bands identified, two known genes were found that were highly homologous to nuclear factor of activated T cells 5 (NFAT5) and tetratricopeptide repeat domain 3 (TPRD 3), respectively, and the remaining nine were novel genes. Conclusion FA can promote the proliferation of MRC 5 at low dose. Sixty FA-induced differentially expressed genes in MRC 5 lesions were obtained by fluorescence differential display PCR. Through the identification of 11 differential bands, FA was selected to further study the effect of FA-induced MRC 5 mechanisms of damage tolerance provide clues.