慢性微量铬（Ⅵ）暴露对小鼠肝细胞氧化应激的影响

发布时间：2018-04-26 15:13

本文选题：金属对金属髋关节置换 + 铬(Ⅵ)　；参考：《南方医科大学》2012年硕士论文

【摘要】：背景自1988年第二代金属对金属(Metal-on-Metal,MOM)关节由B.GWeber应用于临床以来,每年有超过275000例Metasul关节在全球范围内被植入人体,被认为对于年轻且活跃的病人较为适用。金属对金属髋关节假体按照是否保留股骨颈可分为金属对金属全髋关节假体及表面髋关节假体。因其具有良好的刚度和强度、优越的抗磨损性和耐腐蚀性,被认为具有良好的应用前景。然而,金属假体植入人体后不断承受弯曲、撞击、摩擦等机械外力作用,将导致关节假体表面惰性氧化层的破坏,导致金属离子不断释放。现在普遍认为MOM假体植入术后会引起人体金属离子浓度的升高。Sauve等经过30年的随访研究发现,使用MOM关节假体的患者其血清中钴、铬金属离子浓度较对照组升高3-5倍。Maezawa,K等对44例行金属对金属全髋关节置换的患者进行了为期7年的随访研究发现,患者术后3年血中金属离子浓度逐渐增加,至后4年会有25%的患者金属离子浓度有所下降,但仍会有16.3%的患者金属离子浓度逐渐升高。我国学者曾对25例行表面髋关节置换的患者进行了2年随访,结果显示患者术后6月内金属离子浓度逐渐上升,以后逐渐下降趋于平稳,但仍高于正常值。多中心临床研究已证实：与金属对聚乙烯假体相比,MOM会产生较少的磨损颗粒,但假体周围、血液及组织中的金属离子浓度较高。随着现代冶金技术及生物材料的发展,钴铬合金(其中铬含量约占50-70%)因有较好的理学及机械学特性,已成为当代金属对金属髋关节假体的主要材料。随着假体的植入,铬会不断的缓慢释放从而对人体产生影响。目前,大多数研究集中于金属离子对假体周围的骨组织、软组织以及免疫细胞等的影响：①假体周围不断释放的金属离子对于周围软软组织的影响。如引起炎性假瘤等。②假体无菌性松动。假体松动是THR后中、晚期最主要的并发症之一,大部分学者认为假体松动主要是因为假体周围骨溶解引起。③金属离子能够诱导单核细胞及巨噬细胞释放多种细胞因子,从而引起免疫反应。肝脏是人体最大的解毒腺体,具有及其复杂多样的生物化学功能。体内包括铬在内的金属离子均需在肝细胞内进行生物学转化。当前,对于来源于假体的不断释放的铬离子对远隔靶器官如肝脏的生物学影响研究尚不多。铬是人体必需的微量元素之一,其在体内的含量随着年龄的增大而逐渐减少。生理剂量的铬对血糖及胆固醇的调节具有重要作用。铬在体内主要以三价和六价形式存在。毒理学及环境卫生学的研究已经证实,短时间大量的铬离子进入人体会引起肝细胞细胞信号转导通路的改变,从而引起酶学、形态学等不可逆的损伤,甚至凋亡。铬离子毒性与其存在的价态有极大的关系,六价铬的毒性比三价铬高约100倍。六价铬(Ⅵ)经非特异性的阴离子通道进入肝细胞,并在肝细胞内通过酶类或非酶类的方式转化为毒性较小的三价铬(Ⅲ),此过程会引起细胞的氧化应激,从而产生大量的过氧化物。产生的大量过氧化物会通过级联瀑布式的反应引起脂质过氧化及DNA损伤。而细胞为对抗此反应,会启动包括超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、谷胱甘肽还原酶(GR)及谷氨酸半胱氨酸连接酶(GCL)等的抗氧化应激系统,清除自由基,以维持细胞内环境的稳定。当前的金属对金属髋关节假体中,铬存在并释放入血的主要形式是六价的。与毒理学及环境卫生学的研究不同,MOM髋关节假体植入人体后,铬(Ⅵ)离子释放是一个缓慢且微量的过程。金属假体的腐蚀度通常为0.15-0.3μg/cm2/h或30μd,或11mg/年,血铬含量大量文献报道在2.6-26gg/L之间,且时间窗较长(当代金属对金属假体寿命一般在十年以上)。缓慢释放的微量铬(Ⅵ)是否使肝细胞产生氧化应激?肝细胞内的抗氧化应激系统的反应如何?因此,明确铬(Ⅵ)对靶器官肝脏的氧化应激的影响对于评估MOM假体的安全性具有重要意义。目的 1、构建慢性微量铬(Ⅵ)暴露小鼠慢性模拟金属对金属髋关节植入人体后引起的铬(Ⅵ)缓慢释放 2、探讨慢性微量铬(Ⅵ)暴露是否会引起小鼠肝细胞的氧化应激。 3、探讨慢性微量铬(Ⅵ)暴露是否引起小鼠肝细胞抗氧化应激系统的改变以及发生何种改变。 4、探讨金属对金属假体对远隔靶器官肝脏的安全性问题。材料和方法按照实验设计,将80只NIH小鼠随机分为4组(对照组,低、中、高剂量组),每组20只,雌雄不限。Cr03染毒组剂量分别为0mg/kg、5mg/kg、10mg/kg和20mg/kg,隔日腹腔注射1次,实验持续16周,分别于染毒4周末,第8周末,第12周末,第16周末用颈椎脱位法各处死小鼠5只,小鼠处死前进行眼球摘除取血,肝素抗凝后进行血铬含量的检测。一部分肝组织进行HE染色光镜观察,并进行透射电镜观察,余下肝组织用冰冷的生理盐水洗净,加入缓冲液后研磨成匀浆分装后迅速置于-80℃冰箱保存备用,检测肝细胞活性氧自由基水平(ROS)、脂质过氧化产物丙二醛含量(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽活性(GSH)谷胱甘肽还原酶(GR)活性及谷氨酸半胱氨酸连接酶(GCL)的表达。结果 1、0mg/kg、5mg/kg组在染毒4周后,血铬含量达峰值,随后维持在一定浓度,达到一个平台期。而对于20mg/kg组,血铬浓度持续升高,未见明显的平台期。 2、实验各组小鼠肝细胞ROS值均高于对照组,且差别具有统计学意义。并且观察到随着染毒时间的延长,低中剂量组ROS会逐渐接近一个平台期,而对20mg/kg组,ROS持续升高,未见明显的平台期。这与结果1相似。 3、在各个时间点中,20mg/kg组MDA含量较0mg/kg组升高,但差别无统计学意义。 4、与对照组相比,5mg/kg组、10mg/kg组在第4周CAT水平未见明显降低,差别无统计学意义；而在随后的各个时间点中升高明显,且差别具有统计学意义。与对照组相比,20mg/kg组随着染毒时间的延长CAT水平下降明显,且差别具有统计学意义。 5、与对照组相比,小鼠经铬染毒4周后20mg/kg组SOD降低,并具有显著性差异。8周以后随着染毒时间的延长,各组SOD活性下降明显,且差别具有统计意义。 6、与对照组相比,5mg/kg组、10mg/kg组在第4周GSH水平未见明显降低,差别无统计学意义；而在随后的各个时间点中升高明显,且差别具有统计学意义。与对照组相比,20mg/kg组随着染毒时间的延长GSH水平下降明显,且差别具有统计学意义。 7、与对照组相比在染毒的第4周5mg/kg组、10mg/kg组GR活性升高,差别具有统计学意义；20mg/kg组下降明显,且差别具有统计学意义.随着染毒时间的延长各剂量组GR水平均下降,且与对照组相比,差别具有统计学意义。 8、GCL表达的蛋白条带用image-proPlus图像分析系统对条带灰度进行扫描,扫描结果显示：5mg/kg组、10mg/kg组各个时间点GCL表达明显升高,与对照组相比,具有统计学意义。而20mg/kg组GCL表达在第8周后下降明显,差别具有统计学意义。 9、肝细胞HE染色显示：与对照组相比,5mg/kg组、10mg/kg组肝细胞未见明显异常。而20mg/kg组肝细胞在第16周可见少量的空泡变,细胞膜稍皱缩。透射电镜显示：与对照相比,高剂量染毒小鼠在第16周超微结构出现凋亡特征性改变：细胞核皱缩,染色质浓缩并发生边聚化；线粒体变的致密,峭消失,空泡化改变。结论 1、随着染毒时间的延长,慢性微量铬(Ⅵ)染毒会引起小鼠血铬含量的升高,且具有一定的浓度时间依赖性。 2、慢性微量铬(Ⅵ)暴露会引起小鼠肝细胞的氧化应激,提示金属对金属髋关节置换术后铬离子的释放会引起远隔靶器官肝脏的氧化应激。 3、慢性微量铬(Ⅵ)暴露引起小鼠肝细胞抗氧化应激系统的改变。 4、金属对金属假体对远隔靶器官肝脏的安全性尚需进一步评估。
[Abstract]:background
Since the second generation metal (Metal-on-Metal, MOM) joints were applied to the clinical B.GWeber in 1988, more than 275000 cases of Metasul joints were implanted in the human body every year, and were considered to be more suitable for young and active patients. Metal to metal hip prosthesis can be divided into metal to metal in accordance with the retention of the neck of the femur. Total hip prosthesis and surface hip prosthesis are considered to have good application prospects because of their good stiffness and strength, excellent wear resistance and corrosion resistance. However, the metal prosthesis is subjected to mechanical external forces such as bending, impact and friction, which will lead to the destruction of the inert oxide layer on the surface of the joint prosthesis. Metal ions are constantly released. It is now generally believed that after 30 years of follow-up study of the increase of the concentration of metal ions in the human body after the implantation of the MOM prosthesis, the serum cobalt and chromium metal ions in the patients with the MOM joint prosthesis are 3-5 times higher than the control group by 3-5 times.Maezawa, and 44 cases of metal to metal total hip replacement. The 7 year follow-up study found that the concentration of metal ions in the blood increased gradually in the 3 years after the operation, and the concentration of metal ions decreased in 25% of the patients in the last 4 years, but the concentration of metal ions in 16.3% of the patients was increased gradually. Chinese scholars had been followed up for 2 years in 25 patients with surface hip replacement. The results showed that the concentration of metal ions in June increased gradually and gradually declined to a stable level after June, but it was still higher than the normal value. Multi center clinical study has confirmed that compared with metal to polyethylene prosthesis, MOM produces less wear particles, but the concentration of metal ions in blood and tissue is higher around the prosthesis.
With the development of modern metallurgical technology and biomaterials, cobalt chromium alloy (with a chromium content of about 50-70%) has become the main material for metal hip prosthesis because of its good physical and mechanical properties. With the implantation of the prosthesis, the slow release of chromium will affect the human body. At present, most of the research focuses on the research. The effects of metal ions on the bone tissue, soft tissue and immune cells around the prosthesis. (1) the effects of metal ions released around the prosthesis on the surrounding soft tissue. Such as inflammatory pseudotumor. (2) aseptic loosening of the prosthesis. Prosthesis loosening is one of the most important complications of the late THR, and most scholars believe that the prosthesis is loosened. It is mainly caused by osteolysis around the prosthesis. 3. Metal ions can induce monocyte and macrophage to release a variety of cytokines, which cause the immune response. The liver is the largest detoxifying gland of the human body, and has its complex and diverse biochemical functions. The metal ions in the body, including chromium in the body, need to be produced in the liver cells. At present, there is not much research on the biological effects of chromium ions released from prosthesis on distant target organs such as liver.
Chromium is one of the essential trace elements of human body, and its content in the body decreases with age. Chromium in physiological dose plays an important role in the regulation of blood sugar and cholesterol. Chromium is mainly in the form of trivalent and six valence in the body. The changes in the signal transduction pathway of hepatocyte cells caused the irreversible damage and even apoptosis of the enzymology and morphology. The toxicity of chromium ion was greatly related to its valence state. The toxicity of six valence chromium was about 100 times higher than that of trivalent chromium. Six valence chromium (VI) entered the liver cells through the nonspecific anion channel and was in the liver cells. The process of conversion into less toxic trivalent chromium (III) by enzymes or non enzymes can cause oxidative stress of cells and produce a large number of peroxides. The large number of peroxides produced by the cascade of cascade can cause lipid peroxidation and DNA damage. Antioxidant stress systems, such as enzyme (SOD), glutathione (GSH), glutathione reductase (GR) and glutamate cysteine ligase (GCL), are used to remove free radicals to maintain the stability of the intracellular environment.
Unlike current metal to metal hip prostheses, the main form of chromium presence and release into the blood is six. Unlike toxicological and environmental hygiene studies, the release of chromium (VI) ions is a slow and trace process after MOM hip prosthesis is implanted in the human body. The corrosion degree of metal prosthesis is usually 0.15-0.3 u g/cm2/h or 30 U D, or 11mg/ years. The blood chromium content is reported in a large number of 2.6-26gg/L, and the time window is longer (the life of metal prosthesis is generally over ten years). Does the slow release of chromium (VI) cause oxidative stress in the liver cells? What is the reaction of the antioxidant stress system in the liver cells? It is important to evaluate the safety of MOM prosthesis.
1, construction of chronic trace chromium (VI) exposed chronic simulated metal to the slow release of chromium (VI) caused by metal hip joint implantation in human body.
2, to explore whether chronic trace chromium exposure can cause oxidative stress in mouse hepatocytes.
3, to explore whether chronic trace chromium exposure can cause changes in the antioxidant stress system of hepatocytes in mice and what changes occur.
4, to explore the safety of metal to metal prosthesis on distant target organs and liver.
According to the experimental design, 80 NIH mice were randomly divided into 4 groups (control group, low, middle, high dose group) with 20 rats in each group. The dose of female and male and male non limited.Cr03 group were 0mg/kg, 5mg/kg, 10mg/kg and 20mg/kg respectively. The experiment lasted for 16 weeks and the experiment lasted for 4 weeks, eighth weekend, Twelfth weekend, and sixteenth weekend with cervical dislocation. 5 mice were killed in each method. The mice were extirpated and taken blood before death, and the content of chromium was detected after anticoagulation of heparin. A part of the liver tissue was observed by HE staining light microscopy and transmission electron microscopy was carried out. The remaining liver tissue was washed with cold physiological saline, and then added to the homogenate after adding the buffer solution to the homogenate and stored in the refrigerator of -80 centigrade. The activity of active oxygen free radical (ROS), lipid peroxidation product malondialdehyde content (MDA), superoxide dismutase (SOD), glutathione activity (GSH) glutathione reductase (GR) activity and the expression of glutamate cysteine ligase (GCL) were detected.
Result
1,0mg/kg, group 5mg/kg, after 4 weeks of poisoning, the blood chromium content reached the peak, and then maintained at a certain concentration, reaching a plateau period. For group 20mg/kg, the concentration of chromium continued to rise, and no obvious platform period was found.
2, the ROS values of liver cells in all the mice were higher than those in the control group, and the difference was statistically significant. And it was observed that with the prolonged exposure time, the low medium dose group ROS gradually approached a platform period, while the ROS continued to rise in the 20mg/kg group, and no obvious platform period was found. This was similar to the result of the result.
3, at each time point, the MDA content in group 20mg/kg was higher than that in group 0mg/kg, but the difference was not statistically significant.
4, compared with the control group, group 5mg/kg and 10mg/kg were not significantly decreased at the level of CAT at fourth weeks, but there was no significant difference in the subsequent time points, and the difference was statistically significant. Compared with the control group, the CAT level of the 20mg/kg group decreased with the prolongation of the exposure time, and the difference was statistically significant.
5, compared with the control group, the SOD in the 20mg/kg group decreased after 4 weeks of chromium exposure and had significant difference after.8 weeks. With the prolongation of the time of exposure, the activity of SOD decreased significantly in each group, and the difference was statistically significant.
6, compared with the control group, group 5mg/kg and 10mg/kg were not significantly decreased at the level of GSH at fourth weeks, but there was no significant difference in the subsequent time points, and the difference was statistically significant. Compared with the control group, the GSH level of the 20mg/kg group decreased with the prolongation of the exposure time, and the difference was statistically significant.
7, compared with the control group, in the fourth week 5mg/kg group, the activity of GR in the group 10mg/kg increased, and the difference was statistically significant. The decrease of the 20mg/kg group was significant and the difference was statistically significant. With the prolonged exposure time, the GR water in each dose group decreased, and the difference was statistically significant compared with the control group.
8, the protein bands expressed by GCL were scanned by the image-proPlus image analysis system. The results showed that the GCL expression in group 10mg/kg was significantly higher at each time point in group 5mg/kg, compared with the control group, and the GCL expression in group 20mg/kg decreased obviously after eighth weeks, and the difference was statistically significant.
9, the liver cell HE staining showed that compared with the control group, the hepatocyte in the 5mg/kg group and the 10mg/kg group had no obvious abnormalities. While the 20mg/kg group had a small number of vacuoles and a slight contraction of the cell membrane in the 20mg/kg group. The transmission electron microscopy showed that the apoptotic changes in the sixteenth Zhou Chao microstructures of the high dose infected mice were characterized by the nuclear shrinkage, compared with the control. Chromatin concentrates and concentrates. Mitochondria become denser, kurtosis disappears and vacuolization changes.
conclusion
1, with the prolongation of exposure time, chronic trace chromium (VI) exposure can cause the increase of blood chromium content in mice, and has a certain concentration time dependence.
2, chronic trace chromium (VI) exposure may cause oxidative stress in the liver cells of mice, suggesting that the release of chromium ions after metal hip replacement may cause oxidative stress in the liver of the distant target organ.
3, chronic trace chromium (VI) exposure induced changes in the antioxidant stress system of hepatocytes in mice.
4, the safety of metal to metal prostheses on distant target organs and liver needs further evaluation.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R684;R114

【参考文献】