【作者】 吴清安；

【导师】 王家富； 李祥安； 商战平；

【作者基本信息】 泰山医学院 ， 病理学与病理生理学， 2010， 硕士

【摘要】 目的通过观察清道夫受体(Scavenger receptor class B type I, SR-BI)基因敲除小鼠脾脏组织中含铁血黄素的沉积的变化;比较氮基三醋酸铁(ferric nitrilotriacetate, Fe-NTA)诱导的细胞毒性对HepG2(-)细胞SR-BI蛋白表达水平的影响；研究SR-BI蛋白的表达对Fe-NTA、血红蛋白(hemoglobin)或高铁血红素(hemin)等高价铁(Fe3+)诱导的细胞毒性的影响以及探讨Fe-NTA诱导细胞毒性的发生机制,为SR-BI可以抑制Fe-NTA、Fe-NTA-H2O2、hemoglobin-H2O2和hemin-H2O2等高价铁(Fe3+)所诱导的细胞毒性提供一定的理论和实验依据,为SR-BI功能研究提供新的关注点。方法1.SR-BI杂合子(SR-BI+/-)小鼠交配繁殖,筛选SR-BI正常和SR-BI基因敲除小鼠,8-12周龄小鼠麻醉处死,分离摘取脾脏组织,常规组织切片、HE染色和普鲁士蓝染色,显微镜下观察比较两组小鼠脾脏组织结构、脾脏红髓中含铁血黄素(haemosiderin)沉积量的变化。2.采用蛋白质印迹(Western blot)技术检测不同浓度Fe-NTA培养的人肝癌HepG2(-)细胞中SR-BI蛋白表达的变化；利用细胞转染技术将human-SR-BI-cDNA转染中国仓鼠卵巢(CHO)细胞,筛选建立CHO-SR-BI和CHO-Vector细胞株。选取2-3代的培养细胞,分别单独给予浓度都为0、0.2、0.4、0.8、1.0mM的Fe-NTA、H2O2,浓度为0、0.8、1.0、2.0mM的NTA,显微镜下观察细胞生长状态的变化,检测细胞培养基中乳酸脱氢酶(lactate dehydrogenase, LDH)的含量,探讨SR-BI蛋白的表达对Fe-NTA诱导的细胞毒性的影响以及NTA、H2O2对细胞生长的影响。3.选取2-3代培养的CHO-SR-BI细胞和CHO-Vector细胞,分别给予0.2mM Fe-NTA培养24小时和0.05mM Fe-NTA培养48小时,显微镜下观察细胞生长状态变化,提取细胞DNA,利用琼脂糖凝胶电泳DNA鉴定技术,检测有无DNA梯状条带的形成,探讨Fe-NTA诱导的细胞毒性的发生机制；分别给予浓度为0、0.2、0.4、0.8、1.0mM的Fe-NTA-H2O2,浓度为0、6.25、12.5、25.0uM血红蛋白-H2O2、高铁血红素-H2O2,显微镜下观察细胞生长状态的变化,检测CHO-SR-BI细胞和CHO-Vector细胞的培养基中LDH的含量,比较SR-BI蛋白的表达对上述各种含高价铁(Fe3+)混合物所诱导的细胞毒性的影响。结果1.HE染色和普鲁士蓝染色：与SR-BI正常小鼠相比,SR-BI基因敲除小鼠脾脏红髓、白髓分布不规律,红白髓界限不清,白髓大小不一,尤其是红髓区域内出现大量含铁血黄素的沉积(p<0.05)2.在本实验条件下,实验结果表明Fe-NTA可以诱导细胞出现非凋亡性的损伤和死亡；蛋白印迹技术检测结果显示Fe-NTA诱导的细胞毒性对HepG2(-)细胞中SR-BI蛋白表达水平没有明显影响(p>0.05)。3.H2O2对于Fe-NTA诱导的细胞毒性有明显的增强作用(p<0.05)；给予不同浓度的Fe-NTA、Fe-NTA-H2O2、血红蛋白-H2O2及高铁血红素-H2O2培养后,CHO-SR-BI细胞培养基中LDH释放百分比例显著低于CHO-Vector细胞组,两组的差异具有统计学意义(p<0.05),说明细胞中SR-BI蛋白的表达可以明显抑制Fe-NTA、Fe-NTA-H2O2、血红蛋白-H2O2、高铁血红素-H2O2等含高价铁(Fe3+)的混合物所诱导的细胞毒性。。结论1.SR-BI基因敲除小鼠脾脏红髓、白髓分布不规律,红白髓界限不清,白髓大小不一,尤其是红髓区域内出现大量含铁血黄素的沉积。2. Fe-NTA诱导浓度依赖性的细胞毒性,并导致细胞出现非凋亡性的损伤和死亡,而Fe-NTA对HepG2(-)细胞中SR-BI蛋白的表达并没有明显影响。3.H2O2明显增强Fe-NTA诱导的细胞毒性；SR-BI不仅明显抑制Fe-NTA诱导的细胞毒性,而且显著抑制Fe-NTA-H2O2、血红蛋白-H2O2、高铁血红素-H2O2等含高价铁(Fe3+)的混合物所诱导的细胞毒性。更多还原

【Abstract】 OBJECTIVETo evaluate the histological changes and iron deposition in the Red Pulp of spleens of SR-BI-deficient mice, effects of Fe-NTA on SR-BI expression in HepG2(-) cells and effects of SR-BI on suppression of cytotoxicity-induced by trivalent iron, such as Fe-NTA, hemoglobin or hemin. Furthermore, explore the underlying mechanism of the cell damage and death induced by Fe-NTA, so to provide a theoretical and experimental evidence of SR-BI suppresses cytotoxicity-induced by trivalent iron and a new focus on the function of SR-BI research.METHODS1. Heterozygous SR-BI+/- mice on a mixed C57BL/6 x129-background were obtained from the Jackson Laboratory. SR-BI-/- and SR-BI+/+ littermates were generated by mating SR-BI+/- mice.8-12 week-old mice were sacrificed by anesthesia. Spleens were isolated and removed for the conventional tissue sections and HE and Perls Prussian Blue staining. Evaluate the histological changes and hemosiderin deposition in the Red Pulp of spleens of SR-BI+/+ and SR-BI-/- mice under microscope.2. Using Western blot to detect the expression levels of SR-BI in HepG2 (-) cells that treated by various concentrations of Fe-NTA; transfect the human SR-BI-cDNA into CHO cells to generate CHO-Vector cells and CHO-SR-BI cells, then 2- to 3-generations used in experiments were treated with different concentrations of Fe-NTA (0,0.2,0.4,0.8 and 1.0mM), NTA (0,0.8,1.0 and 2.0mM) and H2O2 (0,0.2,0.4 0.8 and 1.0mM). Assess the cells ability by microscope, detect the LDH levels in the cultured cells medium and the effects of SR-BI on the cytotoxicity induced by Fe-NTA.3.2- to 3-generations of CHO-SR-BI cells and CHO-Vector cells were treated with0.2 mM Fe-NTA for 24 hours and 0.05mM Fe-NTA for 48 hours. Assess the cells ability using the microscope, extract cells DNA and identificate DNA ladder of the cell apoptosis by using DNA agarose gel electrophoresis to explore the underlying mechanism of cytotoxicity induced by Fe-NTA.2- to 3-generations of cultured CHO-SR-BI cells and CHO-Vector cells were treated with different concentrations of Fe-NTA-H2O2 complexes (0,0.2,0.4,0.8 and 1.0mM), hemoglobin-H202 complexes (0,6.25,12.5 and 25.0uM) and hemin-H2O2 complexes (0,6.25,12.5 and 25.0uM). Assess the cells ability using the microscope, detect the LDH levels in the two cultured cells medium and the effects of the expression of SR-BI on cytotoxicities induced by complexes used in our experiments.RESULTS1. HE and Perls Prussian Blue staining:Compared to SR-BI+/+ littermates, the histological structure of SR-BI-/- mice was changed, the distribution of red pulp and the various size of white pulp is irregular, and the amount of hemosiderin and the content of iron in the red pulp region of spleens of SR-BI-/- mice were significantly increased.2. In our experimental conditions, the results indicated that Fe-NTA could result in the non-apoptotic cell damage and death. Western blot detection and assay:There was no obviously up-regulation expression of SR-BI of the human HepG2 (-) cells under the various concentrations of Fe-NTA (p>0.05)3. Cytotoxicity-induced by Fe-NTA was markly enhanced by H2O2 (p<0.05); compared to control groups (CHO-Vector cells), the LDH release percents were obviously lower in the experiment groups (CHO-SR-BI) in the different concentrations of Fe-NTA, Fe-NTA and H2O2 complexes, hemoglobin and H2O2 complexes or hemin and H2O2 complexes, there is significant difference of the percent between control groups and experiment groups (p<0.05).CONCLUSION1. Compared to SR-BI+/+ littermates, the histological structure of SR-BI-/- mice was changed, the distribution of red pulp and the various size of white pulp is irregular, and the amount of hemosiderin and the content of iron in the red pulp region of spleens of SR-BI-/-mice were significantly increased.2. Cytotoxicity induced by Fe-NTA is in a dose-dependent way to result in the non-apoptotic cell damage and death in our experimental conditions. There were no significant changes of SR-BI expression levels in HepG2 (-) cells treated by various concentrations of Fe-NTA.3. Cytotoxicity induced by Fe-NTA was markly enhanced by H2O2 SR-BI suppresses cytotoxicity-induced by trivalent iron, such as Fe-NTA, Fe-NTA-H2O2 complexes, hemoglobin-H2O2 complexes or hemin-H2O2 complexes.更多还原