【作者】 蒋军军；

【导师】 哈小琴； 伏小平；

【作者基本信息】 甘肃农业大学 ， 动物预防医学， 2010， 硕士

【摘要】 研究背景铅是环境中普遍存在的污染物,在人体内可以长期蓄积,而产生全身性多系统的损伤,尤其对儿童的危害更为严重[1]。研究表明铅具有很强的神经亲和性,可在神经组织中蓄积,引起神经系统功能长期不可逆的损害[2]。肾脏作为铅分布和排泄的主要器官,也是铅中毒的靶器官之一[3]。近些年发现,铅的毒性作用没有安全阈值,即体内有铅便有毒[4]。虽然许多国家采取了一些降低环境铅污染的措施,但慢性铅中毒依然是现代城市居民所面临的主要健康问题之一[5]。血-脑屏障(Blood brain barrier ,BBB)是中枢神经系统的重要结构,其形态学基础包括血液与神经元之间的一系列解剖结构:脑内毛细血管的内皮细胞及内皮细胞之间的紧密连接、基膜以及神经胶质细胞突起(胶质膜)。BBB保证了中枢神经系统所需内环境的高度稳定,是中枢神经系统正常进行各项机能活动的基础。肾单位(renal unit)主要由肾小球和肾小管组成,是肾脏结构与功能的基本单位,肾小球由毛细血管丛组成,期间有系膜组织支持,系膜细胞发挥着重要作用;肾小管是单层上皮性小管,有重吸收原尿中某些成分和排泄废物等的作用。可见,BBB或肾单位结构和/或功能的变化均是引起许多疾病病理生理变化的核心。目前,有关铅致脑、肾损伤的研究多以动物模型和流行病学调查为主[ 6 ]。在细胞上的研究也主要以海马、大脑皮层神经元细胞和人肾小管上皮细胞(HK-2)为研究对象,在星形胶质细胞(C8)、脑毛细血管内皮细胞和人肾小球系膜细胞(HMC)上的系统研究相对较少。本研究是在我们前期动物实验基础上,以C8、人股动脉内皮细胞(HFAEC)和HMC、HK-2为研究对象,观察醋酸铅对体外培养C8、HFAEC和HMC、HK-2的影响,以探讨醋酸铅致脑损伤及肾损伤的细胞及分子机制。第一部分醋酸铅对星形胶质细胞和人股动脉内皮细胞的影响目的通过观察醋酸铅对C8和HFAEC细胞形态学、生化指标、凋亡相关蛋白表达及凋亡细胞数量的影响,探讨醋酸铅对C8和HFAEC的损伤机制,为铅致BBB的损伤提供理论基础。方法1.以不同浓度(5、10、20、40μmol/L )醋酸铅作用(染毒)对数生长期C8和HFAEC 6、12、24、48h,同时以不加醋酸铅组为对照组,MTT法检测醋酸铅对细胞生长抑制情况。2.选用5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,同样以不加醋酸铅组为对照组,采用Giemsa、HE染色及透射电子显微镜观察细胞形态学变化。3. 5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,分光光度计检测染毒组和对照组C8和HFAEC培养上清中乳酸脱氢酶(LDH)和丙二醛(MDA)含量,间接了解细胞被损伤程度。4. 5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,DNA Ladder法观察染毒组和对照组C8和HFAEC基因组DNA损伤。5. 5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,免疫组织化学法检测染毒组和对照组C8和HFAEC中c-jun、P53、Bax、Bcl-2、Caspase-3蛋白的表达。6. 5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,RT-PCR法检测染毒组和对照组C8和HFAEC Caspase-3 mRNA的转录。7. 5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,Western blotting法检测染毒组和对照组C8和HFAEC Caspase-3蛋白的表达情况。8. 5、10、20μmol/L醋酸铅染毒C8和HFAEC 24h后,PI-Hoechst33342双重荧光染色、流式细胞仪检测染毒组和对照组C8和HFAEC的凋亡率。结果1.不同浓度醋酸铅(5、10、20、40μmol/L )染毒C8、HFAEC不同时间(6、12、24、48h)后,各染毒组细胞生长抑制率较其对照组均显著升高(P<0.01),并存在浓度与时间依赖关系。2.醋酸铅(5、10、20μmol/L)染毒C8、HFAEC 24h后,倒置相差显微镜观察发现细胞密度较对照组降低、胞体变小,且星形胶质细胞突触缩短变细,细胞间连接减少;Giemsa、HE染色法观察发现染毒组较对照组细胞胞浆浓缩,大量细胞核浓染、固缩,核仁裂解,部分细胞核成肾型、马蹄型等,电镜观察同样发现染毒组细胞核质固缩、边移,核质间隙增大,并随醋酸铅浓度加大有凋亡小体形成。3.分光光度计检测培养上清LDH、MDA含量发现5、10、20μmol/L醋酸铅染毒组C8、HFAEC培养上清中LDH及MDA含量较其对照组均显著升高(P<0.01)。4. DNA ladder法检测C8、HFAEC结果发现5、10、20μmol/L醋酸铅染毒组各细胞较其对照组细胞基因组出现较为明显的DNA损伤现象。5.免疫组织化学法检测结果显示5、10、20μmol/L醋酸铅染毒组C8、HFAEC细胞中c-jun、P53、Bax、Caspase-3蛋白表达较其对照组细胞表达量明显上升,而Bcl-2蛋白的表达量醋酸铅染毒组细胞较对照组细胞降低。6. RT-PCR法检测结果显示5、10、20μmol/L醋酸铅染毒组C8、HFAEC的Caspase-3 mRNA转录较其对照组细胞转录水平明显升高。7. Western blotting法检测也显示5、10、20μmol/L醋酸铅染毒组C8、HFAEC Caspase-3蛋白表达较其对照组细胞表达水平明显升高,且与醋酸铅剂量呈正相关。8. PI-Hoechst33342双重染色和流式细胞仪检测结果显示,5、10、20μmol/L醋酸铅染毒组C8、HFAEC凋亡率较其对照组细胞凋亡率均显著升高(P<0.01),且存在剂量关系。结论醋酸铅可通过生成脂质过氧化物致C8和HFAEC基因组DNA损伤,进一步影响凋亡相关因子表达量的改变,使Bax/Bcl-2比值升高,激活Caspase-3凋亡基因的高表达,最终促使C8和HFAEC凋亡。并可能通过促使C8和HFAEC的凋亡破坏血脑屏障的结构和功能。第二部分醋酸铅对人肾小球系膜细胞和人肾小管上皮细胞的影响目的通过观察醋酸铅对HMC和HK-2细胞形态学、生化指标、凋亡相关蛋白表达量及凋亡细胞数量的影响,探讨醋酸铅对HMC和HK-2的损伤机制,为铅致肾脏的损伤提供理论基础。方法同第一部分结果1.不同浓度醋酸铅(5、10、20、40μmol/L )染毒HMC和HK-2不同时间(6、12、24、48h)后,各染毒组细胞生长抑制率较其对照组均显著升高(P<0.01),并存在浓度与时间依赖关系。2. 5、10、20μmol/L醋酸铅染毒HMC和HK-2 24h后,倒置相差显微镜观察发现细胞密度较对照组降低、胞体变小变圆;Giemsa、HE染色观察发现染毒组较对照组细胞胞浆浓缩,大量细胞核浓染、固缩,核仁裂解,部分细胞核成肾型、马蹄型等,同样电镜观察发现染毒组细胞核质固缩、边移,核质间隙增大,且随醋酸铅浓度加大凋亡小体形成并增多。3.分光光度计检测培养上清LDH、MDA含量结果显示,5、10、20μmol/L醋酸铅染毒组HMC和HK-2培养上清中LDH及MDA含量较其对照组均显著升高(P<0.01)。4. DNA ladder检测HMC和HK-2基因结果显示, 5、10、20μmol/L醋酸铅染毒组HMC和HK-2基因较其对照组细胞基因组出现较为明显的DNA损伤现象。5.免疫组织化学法检测HMC和HK-2凋亡相关蛋白表达,结果显示5、10、20μmol/L醋酸铅染毒组细胞中c-jun、P53、Bax、Caspase-3表达较其对照组细胞表达量明显升高,而Bcl-2蛋白的表达量在醋酸铅染毒组较其对照组表达量却明显降低。6. RT-PCR法检测发现5、10、20μmol/L醋酸铅染毒HMC和HK-2细胞24h后Caspase-3 mRNA转录较其对照组细胞明显升高。7. Western blotting法检测也显示5、10、20μmol/L醋酸铅染毒HMC和HK-2细胞24h后Caspase-3蛋白表达量较其对照组也明显升高。8. PI-Hoechst33342双重染色和流式细胞仪检测结果显示,5、10、20μmol/L醋酸铅染毒组HMC和HK-2凋亡率较其对照组细胞凋亡率均显著升高(P<0.01),且与醋酸铅剂量呈正相关。结论醋酸铅可通过生成脂质过氧化物致HMC和HK-2基因组DNA损伤,进一步影响凋亡相关因子表达量的改变,使Bax/Bcl-2比值升高,激活Caspase-3凋亡基因的高表达,最终促使HMC和HK-2凋亡。并可能通过促使HMC和HK-2的凋亡破坏肾脏的结构与功能。更多还原

【Abstract】 BACKGROUNDLead,which is one of the most popular contaminations in the environment, is able to cumulate for a long time and damage various of tissue systems of human body, especially on the children. Recent study showed that Lead acquires neurotropism and cumulates in the nervous system, then further induces long and inconvertible damage. The kidney, as the main organ of distribution and excretion of Lead, is one of the target organs of Lead damage. Some researches demonstrated that there is no safe threshold in the Lead toxicity, that’s to say, the toxicity exist when lead invades the human body. Although many countries’governments have reduced the Lead contaminations in the environment through many measures, chronic harm of Lead on the body is still one of the most essential healthy problems which people have to face nowadays.Blood brain barrier (BBB), which consists of the endothelial cells of brain blood capillaries, tight junction between endothelial cells, basement membrane and neuroglial cells(gelatinous membrane), is not only an important part of the central nervous system but also ensures internal environment of the nervous system to keep highly stable. Therefore, BBB supplies the base for various normal activities of central nervous system (CNS). The nephron is the structural and functional unit of the kidney, comprising renal corpuscle and an epithelial renal tubule. The renal corpuscle consists of blood capillaries and mesenteria which develop a significant function, while the renal tubule functions as reabsorption and excretion. The structural and functional changes of BBB or renal unit may lead to pathophysiological changes of many diseases.Nowadays many researches about Lead toxicity on the brain and kidney mainly focus on the animals models, epidemiological investigations, and some cells like hippocampal neurons and human kidney epithelial cell line(HK-2), however, studies on the relationship between Lead and mouse astrocytes(C8), cerebral capillary vessel endothelial cell and human glomerular mesangial cells are rare. Based on previous animal experience, we studied the effects of Lead toxicity on C8, human femoral artery endothelial cell (HFAEC), Human mesangial cells (HMC) and HK-2, and tried to explore the cellular and molecular mechanisms of Lead effecting on the brain and kidney. PartⅠEffects of Lead toxicity on astrocytes(As) and human femoral arterial endothelial cell(HFAEC)Objection To explore Lead acetate inducing apoptosis of mice astrocytes(C8) and human femoral arterial endothelial cell (HFAEC), and provide theoretical basis for the damage of Lead to blood-brain barrier and nephron.Methods1. C8 and HFAEC in logarithmic growth phase were treated to different concen- -tration Lead acetate(5,10,20,40μmol/L)for different time (6,12,24,48h). Mean- -while, the cell treated without Lead acetate was served as the control group, and the prorolifcations of the cells were detected by MTT.2. C8 and HFAEC were treated with 5,10,20μmol/L Lead acetate for 24h respect- -ively .the cell treated without Lead acetate was served as the control group, Cell morphology changes were observed by Giemsa and HE stain and cell ultrastruc- -tures were observed by transmission electron microscopy.3. LDH activity and MDA content of cellular supernatant of Lead acetate-treated groups (5, 10, 20μmol/L) for 24h and blank group of C8 and HFAEC were detected by spectrophotometer in order to investigate cell damage level.4. DNA damage of Lead acetate-treated grous (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by DNA ladder.5. The expression of c-jun、P53, Bax, Bcl-2 and Caspase-3 of Lead acetate-treated groups (5,10,20μmol/L) and control group of C8 and HFAEC were detected by cell immunohistochemistry.6. Caspase-3 mRNA level of Lead acetate-treated groups (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by RT-PCR .7. Caspase-3 protein level of Lead acetate-treated groups (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by Western blotting. 8. Cell apoptosis rates of Lead acetate-treated groups (5, 10, 20μmol/L) and control group of C8 and HFAEC were detected by PI-Hoechst33342 stain and flow cytometry.Results1. With different concentration of Lead acetate(5,10,20,40μmol/L)and different time (6,12,24,48h), the cell growth activity in treated groups reduced significantly compared to the control group (P<0.01), presenting a dose and time-dependent manner.2. C8 and HFAEC treated with the Lead acetate (5,10,20μmol/L)for 24h showed that cell density was lower, and synapses became shorter and thinner, and intercellular junction reduced compared to control group. Giemsa and HE stain showed that cell nucleolus and cytoplasm dense stained, pyknosis,nucleolus cracking, and part of nucleolus showed kindey and horseshoe type. Electron microscopy showed nuclear pyknosis, side shift, nucleus and cytoplasm space increased on exposed groups of Lead acetate, and with the increasing concentration of Lead acetate, the apoptotic bodies appeared.3. LDH and MDA content in supernatant of treated groups (5, 10, 20μmol/L) increased significantly compared to control group (P<0.01).4. The result of DNA ladder showed that, diffused bands appeared in treated groups (5, 10, 20μmol/L) compared to control group by agarose gel electrophoresis.5. Results of immunohistochemistry showed that the expression of c-jun, P53, Bax and Caspase-3 increased, meanwhile Bcl-2 decreased in treated groups (5, 10, 20μmol/L) compared to control group.6. The result of RT-PCR showed that Caspase-3 mRNA level increased in 5, 10, 20μmol/L toxicant groups compared to control group,and the expression of Capase-3 was positively correlated with Lead dose.)7. Western blotting suggested that Caspase-3 expression of C8 and HFAEC in the treated group was higher than that in the control group,and the expression of Capase-3 was positively correlated with Lead dose.8. PI-Hoechst33342 double staining and flow cytometry demonstrated that cell apoptosis rates of 5, 10, 20μmol/L toxicant groups increased significantly compared to control group(P<0.01)。 Conclusion Lead acetate was able to induce the formation of lipid peroxide and result in the damage of genome DNA of C8 and HFAEC, and then influence the expression of apoptosis-related factors, increase the ratio of Bax/Bcl-2, activate the high expression of Caspase-3, finally lead to the apoptosis of C8 and HFAEC and destroy the structure and function of brain blood barrier. PartⅡEffects of Lead toxicity on human mesangial cells and human renal tubular epithelial cellsObjetion To find the effects of Lead acetate induced apoptosis of human mesangial cells(HMC) and human renal tubular epithelial cells(HK-2), and provide theoretical basis for the damage of Lead to nephron. Methods The same to the PartⅠResults1. With different concentration of Lead acetate(5,10,20,40μmol/L)and different time (6,12,24,48h), the cell growth activity in toxicant groups reduced significantly compared to the control group (P<0.01), presenting a dose and time-dependent manner.2. Under inverted phase contrast microscope observing,the Lead acetate(5,10,20μmol/L)treated HMC and HK-2 for 24h showed that cell density was lower, synapses became shorter and thinner and intercellular junction reduced compared to control group. Giemsa and HE stain showed that cell nucleolus and cytoplasm dense stained, pyknosis, nucleolus cracking, and part of nucleolus showed kindey and horseshoe type. Electron microscopy showed nuclear pyknosis, side shift, nucleus and cytoplasm space increased on exposed groups of Lead acetate, and with the increasing concentration of Lead acetate, the apoptotic bodies appeared.3. LDH and MDA content in supernatant of treated groups (5, 10, 20μmol/L) increased significantly compared to control group (P<0.01).4. The result of DNA Ladder showed that, diffused bands appeared in treated groups(5,10,20μmol/L) compared to control group by agarose gel electrophoresis.5. Results of immunohistochemistry showed that the expression of c-jun, P53, Bax and Caspase-3 increased, meanwhile Bcl-2 decreased in treated groups(5,10,20μmol/L) compared to control group.6. The result of RT-PCR showed that Caspase-3 mRNA level increased in treated groups (5,10,20μmol/L) compared to control group,and the transcription of Capase-3 was positively correlated with Lead dose.7. Western blotting suggested that Caspase-3 expression of HMC and HK-2 in the treated group was higher than that in the control group,and the expression of Capase-3 was positively correlated with Lead dose.8. PI-Hoechst33342 double staining and flow cytometry demonstrated that cell apoptosis rates of treated groups (5, 10, 20μmol/L) increased significantly compared to control group(P<0.01).Conclusion Lead acetate was able to induce the formation of lipid peroxide and result in the damage of genome DNA of HMC and HK-2, and then influence the expression of apoptosis-related factors, increase the ratio of Bax/Bcl-2, activate the high expression of Caspase-3, finally lead to the apoptosis of HMC and HK-2 and damage the structure and function of the kidney.更多还原