發(fā)布時間：2018-09-11 08:18

【摘要】：遺傳毒性試驗可以考察受試物是否具有潛在致癌性或者致突變性，是藥物毒性評價和環(huán)境致癌物研究的重要組成部分。建立一個具有良好特異性和敏感性并且操作簡便的體外試驗體系，可以在藥物早期研發(fā)過程中提供重要的參考依據(jù)，降低研發(fā)風險。此外，在體外實驗體系中采用合理的代謝活化條件，可以更加科學(xué)地評價化合物的遺傳毒性。 我們應(yīng)用人淋巴瘤母細胞（TK6）建立了優(yōu)化的遺傳毒性組合試驗體系。在同一處理條件下的不同時間點對受試物分別進行彗星試驗，體外微核試驗和TK基因突變試驗。該體系的優(yōu)點為TK6細胞為人源細胞，與常用的小鼠細胞相比，，p53基因表達正常；此外，體系具有良好特異性與敏感性，操作簡便；該檢測方法覆蓋了DNA損傷、染色體異常和基因突變?nèi)齻€遺傳終點。在本研究中我們運用該試驗體系考察了中藥單體——漢防己甲素和雷公藤甲素的遺傳毒性。彗星試驗結(jié)果顯示漢防己甲素可能導(dǎo)致DNA的斷裂，體外微核試驗表明雷公藤甲素可能導(dǎo)致染色體損傷，提示兩者都具有潛在的遺傳毒性。在致癌物代謝活化中，細胞色素P450酶CYP1A1和CYP2E1是常見的兩種代謝酶。本研究應(yīng)用可穩(wěn)定表達CYP1A1的AHH-1細胞株和同時穩(wěn)定表達CYP1A1和CYP2E1的h2E1V2細胞株，建立了體外TK基因突變實驗體系，考察了環(huán)境致癌物丙烯酰胺通過CYP1A1和CYP2E1代謝后的遺傳毒性。試驗結(jié)果表明，丙烯酰胺對h2E1V2細胞具有致突變作用，表明CYP2E1在其代謝活化中發(fā)揮重要作用。 綜上所述，本研究建立了兩個優(yōu)化哺乳動物細胞的遺傳毒性試驗組合體系，具有良好特異性、敏感性，并且操作簡便。對兩種中藥單體受試物和一種環(huán)境致癌物進行了毒性評價和初步機制探討，驗證了實驗體系并得到了毒性評價結(jié)果。
[Abstract]:Genotoxicity test is an important part of drug toxicity evaluation and environmental carcinogen research, which can be used to investigate the potential carcinogenicity or mutagenicity of the tested material. The establishment of an in vitro test system with good specificity and sensitivity and simple operation can provide an important reference for early drug development and reduce R & D risk. In addition, the genetic toxicity of the compounds can be evaluated more scientifically by using reasonable metabolic activation conditions in vitro. We have established an optimized genotoxicity combination test system using human lymphoma mother cells (TK6). Comet assay in vitro micronucleus test and TK gene mutation test were carried out at different time points under the same treatment conditions. The advantages of this system are that TK6 cells are derived from human cells, and the expression of p53 gene is normal compared with common mouse cells. In addition, the system has good specificity and sensitivity, and is easy to operate. The detection method covers DNA damage. Chromosomal abnormalities and gene mutations are three genetic endpoints. The genotoxicity of Tetrandrine and Tripterygium wilfordii were investigated by using this experimental system. Comet assay showed that Tetrandrine might lead to DNA breakage. Micronucleus test in vitro indicated that triptolide might cause chromosome damage, suggesting that both of them have potential genotoxicity. Cytochrome P450 enzyme CYP1A1 and CYP2E1 are two common metabolic enzymes in the metabolic activation of carcinogens. In this study, AHH-1 cell lines expressing CYP1A1 stably and h2E1V2 cells expressing CYP1A1 and CYP2E1 stably at the same time were used to establish TK gene mutation assay system in vitro. The genotoxicity of environmental carcinogen acrylamide via CYP1A1 and CYP2E1 metabolism was investigated. The results showed that acrylamide had mutagenicity on h2E1V2 cells, suggesting that CYP2E1 played an important role in the activation of CYP2E1 metabolism. In conclusion, two optimal genetic toxicity test systems for mammalian cells were established, which were specific, sensitive and easy to operate. The toxicity evaluation and preliminary mechanism of two Chinese medicine monomers and one environmental carcinogen were carried out. The experimental system was verified and the results of toxicity evaluation were obtained.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

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