【摘要】：目的檢測高分子避孕復(fù)合材料和金屬銅在體外對哺乳動物細(xì)胞致遺傳毒性的作用，評價(jià)該材料的安全性。 方法采用小鼠淋巴瘤實(shí)驗(yàn)（Mouse Lymphoma Assay, MLA）和彗星實(shí)驗(yàn)即單細(xì)胞凝膠電泳實(shí)驗(yàn)（SCGE）檢測各材料組對tk基因致突變能力和導(dǎo)致單個細(xì)胞DNA損傷程度。各材料RMPI-1640浸提液經(jīng)火焰原子吸收分光光度法測定銅離子濃度，預(yù)實(shí)驗(yàn)后選擇金屬銅組50%、25%、12.5%浸提液濃度，含銅復(fù)合材料組100%、50%、25%浸提液濃度，無銅復(fù)合材料組100%、50%、25%浸提液濃度分別測定致突變能力和DNA損傷程度，溶劑對照作為陰性對照，小鼠淋巴瘤實(shí)驗(yàn)中在有無S9mix時(shí)分別選擇環(huán)磷酰胺和甲磺酸甲酯作為陽性對照。小鼠淋巴瘤實(shí)驗(yàn)通過計(jì)數(shù)處理當(dāng)天平板、表達(dá)后平板和TFT拮抗平板的集落數(shù)測定PE0、PE2和MF。彗星實(shí)驗(yàn)以Tail DNA%和Olive尾距評價(jià)細(xì)胞DNA損傷程度。 結(jié)果在含有銅離子的浸提液中，細(xì)胞均出現(xiàn)了不同程度的突變，突變頻率較陰性對照組有明顯升高，其中金屬銅50%、25%和含銅復(fù)合材料組100%組可以致遺傳毒性，含銅復(fù)合材料50%組為可疑致遺傳毒性。彗星實(shí)驗(yàn)結(jié)果表明，各含銅復(fù)合材料組和金屬銅組均可導(dǎo)致細(xì)胞DNA損傷，Tail DNA%和OTM與陰性對照組相比有顯著性差異，損傷程度與浸提液中銅離子濃度有一定關(guān)系。無銅復(fù)合材料在各濃度下突變頻率、Tail DNA%和OTM均無明顯增高 結(jié)論含銅復(fù)合材料、金屬銅組由于銅離子的存在體外可以致DNA損傷和細(xì)胞突變，具有一定的體外遺傳毒性。損傷程度與銅離子濃度存在劑量反應(yīng)關(guān)系。 目的研究高分子避孕復(fù)合材料和金屬銅哺乳動物體內(nèi)致遺傳毒性的能力。 方法KM小鼠（體重：25-30g）作為實(shí)驗(yàn)動物，各材料組生理鹽水浸提，火焰原子吸收分光光度法測定銅離子濃度，選擇金屬銅組50%、25%、12.5%浸提液濃度,含銅復(fù)合材料組100%、50%、25%浸提液濃度，無銅復(fù)合材料組100%、50%、25%浸提液濃度，陽性對照選擇環(huán)磷酰胺，濃度為40mg/kg體重，陰性對照為生理鹽水。各組浸提液按照1.5ml/100g體重腹腔注射染毒動物，24小時(shí)后頸椎脫臼處死動物，胸骨骨髓涂片Giemsa染色計(jì)數(shù)含微核的嗜多染紅細(xì)胞數(shù)量評定各材料體內(nèi)遺傳毒性。 結(jié)果染毒24小時(shí)胸骨骨髓微核計(jì)數(shù)結(jié)果顯示，無銅材料組各染毒濃度下微核數(shù)量無明顯增加。含銅復(fù)合材料組100%、50%和25%組微核數(shù)目（每1000PCE）分別為16.0、14.6和13.4，金屬銅組50%、25%和12.5%分別為11.1、14.1和12.1。與陰性對照組相比均有顯著性差異（P0.05），但各組微核數(shù)量與染毒濃度并未表現(xiàn)出明顯的劑量反應(yīng)關(guān)系。 結(jié)論含銅復(fù)合材料和金屬銅組微核數(shù)目與陰性對照組相比均有顯著性差異，但并未表現(xiàn)出明顯的劑量反應(yīng)關(guān)系，按照OECD474指導(dǎo)標(biāo)準(zhǔn)，尚不能確定此兩組材料具有體內(nèi)遺傳毒性。無銅材料組無體內(nèi)遺傳毒性。 目的檢測體外含銅復(fù)合材料浸提液處理細(xì)胞后,去除處理物細(xì)胞DNA損傷修復(fù)情況。 方法用含銅復(fù)合材料50%和25%浸提液濃度處理小鼠淋巴瘤細(xì)胞3小時(shí)，溶劑作為陰性對照。處理后取部分細(xì)胞采用彗星實(shí)驗(yàn)檢測細(xì)胞DNA損傷程度，其余細(xì)胞在去除處理物后繼續(xù)培養(yǎng)3小時(shí)，同樣采用彗星實(shí)驗(yàn)測定細(xì)胞DNA損傷程度。彗星實(shí)驗(yàn)結(jié)果經(jīng)CASP軟件分析后，Tail DNA%、OTM作為DNA損傷觀測指標(biāo)。含銅復(fù)合材料25%浸提液處理后以及繼續(xù)培養(yǎng)3小時(shí)后檢測細(xì)胞內(nèi)ROS熒光強(qiáng)度，Image ProPlus分析其平均熒光強(qiáng)度。 結(jié)果細(xì)胞經(jīng)兩個不同含銅復(fù)合材料浸提液濃度處理3小時(shí)后，，細(xì)胞DNA受損，繼續(xù)培養(yǎng)3小時(shí)后，彗星實(shí)驗(yàn)結(jié)果表明，細(xì)胞受損程度較處理后有所增加，Tail DNA%和OTM與處理后相比有顯著性差異(P0.05)，但繼續(xù)培養(yǎng)3小時(shí)后細(xì)胞間DNA損傷程度差異增大，表明受損少的細(xì)胞得到一定的修復(fù)。細(xì)胞內(nèi)ROS含量在處理后和繼續(xù)培養(yǎng)3小時(shí)平均熒光強(qiáng)度與陰性對照組相比有明顯升高，分別為79.84、86.29和49.74。 結(jié)論細(xì)胞經(jīng)處理后胞內(nèi)ROS含量較處理前明顯增高并持續(xù)存在，繼續(xù)培養(yǎng)后由于細(xì)胞內(nèi)ROS的升高可以使細(xì)胞DNA繼續(xù)受損。損傷修復(fù)不明顯。 目的機(jī)體對高濃度銅離子染毒造成DNA損傷修復(fù)能力檢測，以及與血漿SOD含量的關(guān)系。 方法用金屬銅100%浸提液連續(xù)7天按1.5ml/100g體重腹腔注射染毒小鼠，分別在染毒前以及每次染毒后24小時(shí)斷尾取血，全血細(xì)胞進(jìn)行彗星實(shí)驗(yàn)檢測血細(xì)胞DNA損傷程度，血漿用WST法測定超氧化物歧化酶活力。彗星實(shí)驗(yàn)結(jié)果經(jīng)CASP軟件分析后，Tail DNA%、OTM作為DNA損傷觀測指標(biāo)。 結(jié)果金屬銅100%浸提液濃度染毒連續(xù)染毒小鼠7天彗星實(shí)驗(yàn)結(jié)果顯示，雌性小鼠于染毒后第2、3、4天DNA損傷程度最嚴(yán)重（Tail DNA%為分別為55.41%、51.05%和51.99%），雄性小鼠于染毒后第4天DNA損傷最嚴(yán)重（57.20%）。雌性第小鼠第5天DNA損傷有明顯降低（18.91%），雄性小鼠第6天明顯降低（30.89%）。第7天雌雄小鼠血細(xì)胞Tail DNA%分別為11.25%和10.81%。WST法測定血漿內(nèi)SOD含量表明，雄性小鼠體內(nèi)SOD含量于第4天和第7天顯著升高，雌性小鼠于第3天達(dá)到高峰。 結(jié)論連續(xù)染毒處理小鼠后，金屬銅100%浸提液可以導(dǎo)致小鼠體內(nèi)血細(xì)胞DNA明顯受損。同時(shí)，體內(nèi)的損傷修復(fù)機(jī)制可以使受損細(xì)胞修復(fù)并抑制銅離子進(jìn)一步對細(xì)胞的損傷，SOD在損傷修復(fù)中發(fā)揮一定的作用。
[Abstract]:Objective To investigate the genotoxic effect of polymer contraceptive composites and metal copper on mammalian cells in vitro and evaluate the safety of the materials.
Methods the Mouse Lymphoma Assay (MLA) and comet assay (SCGE) were used to detect the mutagenic ability of TK gene and the degree of DNA damage in each cell group. The concentrations of 50%, 25%, 12.5% extract, 100%, 50%, 25% extract, 100%, 50%, 25% extract of copper-containing composite, 100%, 50%, 25% extract of copper-free composite, and 25% extract of copper-free composite were used to determine the mutagenicity and DNA damage degree respectively. Solvent control was used as negative control. Cyclophosphamide and methyl methylsulfonate were used as the choices of mice lymphoma experiment in the presence or absence of S9 mix. The colony number of PE0, PE2 and MF. Comet assay was used to evaluate the degree of cell DNA damage by Tail DNA% and Olive tail distance.
Results In the extract containing copper ions, the mutation frequency of the cells was significantly higher than that of the negative control group. 50%, 25% and 100% of the copper-containing composite materials could cause genetic toxicity, and 50% of the copper-containing composite materials could be suspected to cause genetic toxicity. There was a significant difference between Tail DNA% and OTM and negative control group, and the degree of damage was related to the concentration of copper ion in the extract.
Conclusion Copper-containing composites can cause DNA damage and cell mutation in vitro due to the presence of copper ions. The degree of damage is dose-dependent on the concentration of copper ions.
Objective to study the genetic toxicity of polymer contraceptive composite materials and metallic copper lactating animals.
Methods KM mice (body weight: 25-30g) were used as experimental animals. The concentration of copper ion was determined by flame atomic absorption spectrophotometry. The concentration of copper ion was 50%, 25%, 12.5% in copper group, 100%, 50%, 25% in copper composite group, 100%, 50%, 25% in non-copper composite group, 50%, 25% in copper composite group, and positive control group. Cyclophosphamide, 40 mg/kg body weight, negative control was normal saline. The animals in each group were injected intraperitoneally at the weight of 1.5 ml/100 g. The animals were sacrificed after cervical dislocation 24 hours. The number of micronucleated polychromatic erythrocytes in the sternal bone marrow smears was counted by Giemsa staining.
Results The number of micronuclei in sternal bone marrow of non-copper group did not increase significantly at different concentrations after 24 hours exposure. The number of micronuclei in 100%, 50% and 25% copper-containing composite group (16.0, 14.6 and 13.4 per 1000 PCE), 50%, 25% and 12.5% copper group (11.1, 14.1 and 12.1 respectively) were significantly higher than those in negative control group. Differences (P0.05), but the number of micronucleus in each group did not show a dose-response relationship with the concentration.
Conclusion The number of micronuclei in copper-containing composites and copper-containing metals group was significantly different from that in negative control group, but there was no significant dose-response relationship. According to OECD474 standard, it was not determined that the two materials had genotoxicity in vivo.
Objective To detect the DNA damage and repair of cells treated with copper-containing composite extract in vitro.
Methods Mouse lymphoma cells were treated with 50% and 25% copper-containing composite extract for 3 hours, and the solvent was used as negative control. Some of the treated cells were tested for DNA damage by comet assay. The remaining cells were cultured for 3 hours after removal of the treated materials. The degree of DNA damage was also determined by comet assay. Results After analysis by CASP software, Tail DNA and OTM were used as the indexes of DNA damage observation. The ROS fluorescence intensity was measured after 25% extract of copper-containing composite material was treated and cultured for 3 hours. The average fluorescence intensity was analyzed by Image ProPlus.
Results After treated with two different concentrations of copper-containing composite extracts for 3 hours, the DNA damage of the cells was observed. After continued culture for 3 hours, the comet assay showed that the damage degree of the cells was increased. There was significant difference between Tail DNA% and OTM (P 0.05), but the damage degree of DNA between the cells was different after 3 hours of continuous culture. Compared with the negative control group, the average fluorescence intensity of ROS content in the treated and cultured cells after 3 hours was significantly higher, 79.84, 86.29 and 49.74 respectively.
Conclusion The content of ROS in the treated cells was significantly higher than that before treatment and persisted. After continuous culture, the DNA of the cells could continue to be damaged due to the increase of ROS.
Objective To study the DNA damage repair ability of human body exposed to high concentration of copper ion and its relationship with plasma SOD content.
Methods The mice were injected intraperitoneally with 100% copper extract for 7 days at a weight of 1.5ml/100g. Blood samples were taken at the tail of the mice before and 24 hours after each exposure. The whole blood cells were tested for DNA damage by comet assay and the activity of superoxide dismutase (SOD) by WST in plasma. L DNA% and OTM were used as indicators of DNA damage observation.
Results The results of comet assay in mice exposed to 100% copper extract for 7 days showed that the DNA damage of female mice was the most serious (55.41%, 51.05% and 51.99% respectively) on the 2nd, 3rd and 4th day after exposure. The DNA damage of male mice was the most serious (57.20%) on the 4th day after exposure. On the 7th day, Tail DNA content in blood cells of male and female mice was 11.25% and 10.81%, respectively. The content of SOD in plasma of male mice increased significantly on the 4th and 7th days, and reached the peak on the 3rd day.
Conclusion After continuous exposure to copper, 100% extract of copper can induce DNA damage of blood cells in mice. At the same time, the mechanism of damage repair in vivo can repair damaged cells and inhibit the further damage of copper ions to cells. SOD plays a certain role in the repair of injury.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R114

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相關(guān)期刊論文 前1條

1 何黎黎;楊立開;鄧?yán)?龔濤;孫迅;張志榮;;S9代謝活化酶系統(tǒng)制備及蛋白含量的測定[J];西南民族大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年02期

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