【作者】 陈静；

【导师】 王根林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2012， 硕士

【摘要】 本试验使用不同浓度雌二醇、槲皮素单独作用及共同作用于奶牛乳腺上皮细胞,对比三种处理下细胞抗氧化性及相关基因的表达水平,并通过建立H202诱导的氧化应激模型,进一步研究槲皮素在氧化应激下对细胞的保护功能。本试验分为三个部分：一、雌激素对奶牛乳腺上皮细胞抗氧化性及HSP70mRNA表达量的影响试验分溶剂对照组(CK)和I-V浓度分别为0.5、1、5、10、100nmol/L雌二醇(E2)处理组,培养奶牛乳腺上皮细胞4h、8h和12h后,MTT法检测细胞存活率情况,比色法检测细胞培养液中乳酸脱氢酶(LDH),比色法和荧光定量RT-PCR法分别检测培养12h各组细胞中超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量及HSP70mRNA表达量,旨在筛选体外培养奶牛乳腺上皮细胞雌激素较适添加浓度。结果显示：(1)与对照组相比,Ⅰ-Ⅲ组细胞存活率升高(P<0.05)；Ⅳ～Ⅴ组细胞存活率降低且培养液中LDH活性显著升高(P<0.01)。(2)Ⅰ-Ⅳ组细胞中HSP70mRNA表达量、SOD活性高于对照组(P<0.05)；V组乳腺细胞的HSP70mRNA表达量、SOD活性低于对照组(P<0.05)。结论：低浓度E2能够上调奶牛乳腺上皮细胞的存活率,诱导乳腺上皮细胞中HSP70mRNA表达,进而通过调节SOD活性发挥抗氧化作用,长期处于高浓度E2环境下,下调乳腺细胞存活率,降低抗氧化性并引起细胞损伤,5nmol/L E2可作为体外培养奶牛乳腺上皮细胞较适添加浓度。二、槲皮素对奶牛乳腺上皮细胞抗氧化性及HSP70、Bcl-2/Bax mRNA表达量的影响试验分溶剂对照组(CK)和Ⅰ～Ⅴ浓度分别为12.5、25、50、100、200μmol/L槲皮素(Que)处理组,培养奶牛乳腺上皮细胞4h、8h和12h后,检测细胞存活率情况；检测培养12h后丙二醛(MDA)含量、超氧化物歧化酶(SOD)、乳酸脱氢酶(LDH)活性和Hsp70、Bcl-2/Bax mRNA的表达量的变化。结果表明：与CK组相比,Ⅰ～Ⅲ组呈浓度和时间依赖性上调细胞存活率,上调SOD活性和胞内Bcl-2/Bax表达量,下调MDA含量及胞外LDH活性,V组各检测值则呈完全相反的变化(P<0.05)。结论：12.5～50μmol/L槲皮素能够上调奶牛乳腺上皮细胞存活率,提高抗氧化和抗凋亡的能力,发挥保护作用,100～200μmol/L槲皮素结果相反,发挥抗雌激素作用。三、槲皮素对奶牛乳腺上皮细胞双雌激素样作用及抗氧化应激作用的研究试验分为溶剂对照组(CK)、5nmol/L E2对照组、5nmol/L E2分别与12.5、25、50、100、200umol/L Que共同作用试验组(I-V),在培养奶牛乳腺上皮细胞4h、8h、12h后检测细胞存活率情况；检测培养12h后MDA含量、SOD、LDH活性及HSP70mRNA的表达量。H2O2损伤组：含200pmol/L H2O2的培养液作用乳腺上皮细胞4h后,换常规培养液培养12h；Que前保护组：含50μmol/L Que的培养液作用于乳腺上皮细胞12h后,换含200μmol/L H2O2的培养液培养4h; Que后保护组：含200μmol/L H2O2的培养液作用乳腺上皮细胞4h后换含50μmol/L Que的培养液培养12h,对各组细胞MDA、SOD、LDH进行检测。旨在进一步研究Que对氧化应激细胞的保护作用以及其在不同E2水平对健康奶牛乳腺上皮细胞存活率、抗氧化性及HSP70mRNA表达的影响,并筛选出在不同E2水平下槲皮素的双雌激素样作用浓度。与试验二结果对比,Que对氧化应激损伤的细胞通过提高SOD活性,降低脂质化反应发挥有效的保护作用,并且Que对氧化应激的预防作用强于修复作用,Que单独使用和与E2共同作用时抗雌激素浓度分别是100～200μmol/L和50～100μmol/L,并且共同作用与单独作用比,Que上调细胞存活率及提高抗氧化性的作用更显著。更多还原

【Abstract】 This experiment compared antioxidation and gene expression levels with different does of estradiol, quercetin and both of these two, respectively, on bovine mammary epithelial cells. The further study forced on the protective function of quercetin in cells under H2O2-induced oxidative stress.This thesis includes three sections.1. Effects of estrogen on antioxidation and HSP70expression in bovine mammary epithelial cellsExperimental groups were divided into solvent control group (CK) and Ⅰ-Ⅴ experimental groups, which were treated with estradiol (E2) at0.5,1,5,10and100nmol/L respectively. After cultured for4h,8h,12h the viability of mammary epithelial cells was assessed by MTT assay and the activities of lactate dehydrogenase (LDH) in culture medium was assayed by a colorimetric method. After cultured forl2h the activities of superoxide dismutase (SOD) and malonaldehyde(MDA) in the cells also detected by a colorimetric method. HSP70mRNA expression were determined by real-time fluorescent quantitative PCR. The purpose was to obtain the does of estradiol adapting to bovine mammary epithelial cells in vitro. The result showed that:1. Compared with the control group, Ⅰ-Ⅲ E2-treated groups showed increased cell viabilities (P<0.05), Ⅳ-ⅤE2-treated groups showed exhibited cell viabilities(P<0.05) and whose LDH activity was significantly higher (P<0.01).2. Compared with the control group, HSP70mRNA level and SOD activity in Ⅰ-Ⅲ E2-treated groups were higher (P<0.05). HSP70mRNA level and SOD activity in V E2-treated groups were lower (P<0.05). Conclusion:Low concentration of E2could promote the proliferation of mammary epithelial cells, induced expression of HSP70mRNA, which might play antioxidant role by increasing intracellular SOD activity. High concentration of E2inhibited proliferation and antioxidation of mammary epithelial cells, caused cell damage with long-term stimulation.5nmol/L E2can be used as the optimal concentration to keep normal gowth of bovine mammary epithelial cells cultured in vitro. 2. Effects of Quercetin on antioxidation and HSP70,BcⅠ-2/Bax expression in bovine mammary epithelial cellsExperimental groups were divided into solvent control group (CK) and I-V experimental groups with Que at12.5,25,50,100and200μmol/L respectively. Then cells were cultured in4h,8h and12h for the test of proliferation. The12h cells and culture medium were used to determin the content of malonaldehyde(MDA) and the acivities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD), as well as HSP70and Bcl-2/Bax mRNA expression. The Result showed:the cell proliferation was promoted in dose and time dependent way in I-IIIQue-treated groups, besides up-regulation of SOD activity and Bcl-2/Bax RNA expression and down-regulation of MDA and LDH. However, the opposite results were shown in V Que-treated group (P<0.05). Conclusion:The treatment of Que at12.5-50μmol/L could promote the proliferation and improve the antioxidant and antiapoptosis function for the protection. The result in100-200umoi/L Que-treat group was opposite, which play anti-estrogenic function.3. Study on the estrogenicity and antioxidative stress function of quercetin on bovine mammary epithelial cellsExperimental groups were divided into solvent control group (CK) and Ⅰ～Ⅴ experimental groups with5nmol/L E2and Que at12.5,25,50,100and200μmol/L respectively. Then cells were cultured in4h,8h and12h for the test of proliferation. The12h cells and culture medium were used to determine the content of MDA and the acivities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD), as well as HSP70mRNA expression.H2O2damage group:the cells were incultrued with200μmol/L H2O2for4h followed by subsequent incubation with DMEM for12h. Que pre-protecion group:prior to incubation with200μmol/L H2O2for4h, then the cells were cultured with50μmol/L Que for12h. Que post-protection group:after incubation with200μmol/L H2O2for4h, then the cells were cultured with50μmol/L Que for12h. Then cells for the test of content of MDA and the acivities of Lactate dehydrogenase (LDH) and superoxide dismutase (SOD). The purpose was to further study protective roles of Que under oxidative stress and effects of Que on cells proliferation, anti-oxidation and HSP70mRNA expression in mammary epithelial cells. Then the antiestrogen does of the estogennicity of Quercetin was selected in two different situations. Comparison with the results of experiment two:Que protects mammary epithelial cells against oxidative stress-induced injury through improving SOD activity and decreasing lipid reaction and the preventive effect was stronger than repairing effect. When only Que was used or used together with E2, the antiestrogen concentration range was50～100μmol/L and25～50umol/L respectively, and the effect on promoting proliferation and increasing antioxidation was more significant when both E2and Que were used.更多还原