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目的寻找低剂量三氯乙烯(TCE)诱导人正常肝细胞(L02)损伤耐受差异表达的基因。方法参照本实验室用MTT法所得的TCE对L02毒性剂量-效应关系,选择5μmolL的三氯乙烯为低剂量,40μmolL为高剂量,按以下方式对细胞进行染毒:空白对照、低剂量、高剂量、损伤耐受组(先用低剂量刺激24小时,再用高剂量刺激6小时),然后用荧光差异显示PCR(FluoroDDPCR)寻找不同方式刺激差异表达的基因,并对差异表达基因进行克隆、鉴定。结果按方法所示的方式处理细胞,处理完毕后提取各组细胞总RNA,进行荧光差异显示PCR,找到51个差异条带。对其中的11个差异条带进行克隆鉴定同源性比较,发现其中9个已知基因,2个新基因。结论用荧光差异显示PCR方法寻找TCE诱导L02损伤耐受过程差异表达的基因,通过鉴定基因,为进一步研究TCE诱导L02的损伤耐受的机理提供科学依据。
Objective To find a low-dose trichlorethylene (TCE) -induced differential expression of human lung injury tolerance genes. Methods With reference to the dose-effect relationship of TCE obtained by MTT method in our laboratory with L02 toxicity, we selected 5 μmol L of trichlorethylene as the low dose and 40 μmol L as the high dose. The cells were exposed to the following methods: blank control, low dose and high dose Dose and injury tolerance group (stimulated by low dose for 24 hours and then by high dose for 6 hours). Then FluoroDDPCR was used to find different ways to stimulate differentially expressed genes and to clone differentially expressed genes. Identification. Results The cells were treated as shown in the method. After treatment, the total RNA of each group was extracted. Fluorescence differential PCR was carried out to find 51 differential bands. One of the 11 differential bands was cloned to identify homology, and nine of them were found and two of them were new genes. Conclusion The fluorescence differential display PCR method is used to find the genes that TCE induces the differentially expressed genes in the process of L02 injury tolerance. By identifying the genes, it provides a scientific basis for further study on the mechanism of T02-induced L02 injury tolerance.