The Establishment And Application Of Biological Detection Method For Environmental Estrogens

Environmental estrogens (EEs) have estrogenic activity in low level and when it’s enrichedin organism through the food chain it can produce adverse effects. Water pollution in China havebeen very serious, and the estrogen pollution in waters also has drawn widely attention. However,there is no a high sensitive detection method to detect the potential comprehensive biologicalactivities. Environmental estrogen receptor α (ERα) can either activate or suppress transcriptionof ER-responsive gene, hence, environmental estrogens produce estrogenic effects. A highsensitive detection system has been established by using of ERα, Estrogen Receptor responseElement4(ERE4) and luciferase reporter gene assays based on stably transfected humancervical cancer (HeLa) cell line. The study includes below aspects:I. According to the human ERα gene complete open reading frame (ORF) sequencedesigned specific amplification primers, we acquired approximately2kb segment DNAsequences by RT-PCR technology from human placenta tissue. Then cloned the gene topEASY-T1vector, sequencing results showed that obtained ERα gene successfully, whichincludes1788nucleotides, coding595amino acid residue. Cloning the gene to eukaryoticexpression vector constructed recombinant plasmid pEGFP-ERα. Following characterization byPCR, enzyme digestion and sequencing evaluation methods, the pEGFP-ERα recombinantplasmid was built correctly. Then transfected the recombinant plasmid into HeLa cells, under theG418selection-medium screening, the HeLa cells obtained stably expressed ERα gene. Thedesignated HeLa/ERα identified through fluorescence quantitative PCR found the ERα mRNAtranscription level is3.45times of the negative control group, P value analysis is lower than0.05,which indicated the HeLa monoclonal cell line has established successfully.II. The artificial synthesis of a four times series estrogen receptor response elements (ERE4)gene was inserted into pGL3-basic eukaryotic vector MCS site, to construct a recombinantplasmid pGL3-ERE4-luc which including luciferase reporter gene downstream. The correctlyestablished recombinant plasmid transfected into the designated HeLa/ERα cells then,maintained the cells in Hygromycin B (HygB) antibiotics selection-medium for approximately2months, Then chemiluminescence detection showed the fluorescence absorption up to229,000by10-3mmol/L17-βestradiol (E2) inducing ERα expressed, indicating that HeLa/ERα cellsintake the ERE4gene permanently. Named the cell line contained both pEGFP-ERα and pGL3-ERE4-luc recombinant plasmids HeLa/ERα/ERE4.III. By using the HeLa/ERα/ERE4monoclonal cell line detection system to assess theestrogen level of Qiantang river in Hangzhou section water and Xiasha University District zone,Hangzhou, Zhejiang province, of both areas the population density is high, and have masssewage water. Obtained the same period water samples altogether16samples which distributesin10points,2positive samples detected among them contained0.2-0.3ng/L whole estrogenicactivity (equivalent to E2).IV. In order to explore the EEs pollution impacting on insect’s growth and development, theestrogen polluted water is used to feed the Jingsong×Haoyue Bombyx mori silkworm to feedthem during eggs to5ages. Then detected the estrogenic concentration of5ages silkwormhemolymph. High content estrogen (final concentration is4×10-3pmol/g, with10pmol/L E2solution prepared feeds) feeding silkworms’ size are smaller and grew slower than the controlgroup. Overall perform a not synchronous growth phenomenon. And many matured Bombyxmori can’t callous, furthermore, calloued silkworm appeared dead cage symptom. In addition,estrogen final content is4×10-3pmol/g feeded Bombyx mori’s hemolymph estrogenconcentration dropped instead.This research used ERE4regulation function and luciferase reporter gene established a highsensitivity and high-throughput of the integrated biological detection method through the twostage amplification methods.