本文選題：金屬對金屬髖關(guān)節(jié)置換 + 鉻(Ⅵ)��； 參考：《南方醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景 自1988年第二代金屬對金屬(Metal-on-Metal,MOM)關(guān)節(jié)由B.GWeber應(yīng)用于臨床以來,每年有超過275000例Metasul關(guān)節(jié)在全球范圍內(nèi)被植入人體,被認(rèn)為對于年輕且活躍的病人較為適用。金屬對金屬髖關(guān)節(jié)假體按照是否保留股骨頸可分為金屬對金屬全髖關(guān)節(jié)假體及表面髖關(guān)節(jié)假體。因其具有良好的剛度和強度、優(yōu)越的抗磨損性和耐腐蝕性,被認(rèn)為具有良好的應(yīng)用前景。然而,金屬假體植入人體后不斷承受彎曲、撞擊、摩擦等機械外力作用,將導(dǎo)致關(guān)節(jié)假體表面惰性氧化層的破壞,導(dǎo)致金屬離子不斷釋放�，F(xiàn)在普遍認(rèn)為MOM假體植入術(shù)后會引起人體金屬離子濃度的升高。Sauve等經(jīng)過30年的隨訪研究發(fā)現(xiàn),使用MOM關(guān)節(jié)假體的患者其血清中鈷、鉻金屬離子濃度較對照組升高3-5倍。Maezawa,K等對44例行金屬對金屬全髖關(guān)節(jié)置換的患者進行了為期7年的隨訪研究發(fā)現(xiàn),患者術(shù)后3年血中金屬離子濃度逐漸增加,至后4年會有25%的患者金屬離子濃度有所下降,但仍會有16.3%的患者金屬離子濃度逐漸升高。我國學(xué)者曾對25例行表面髖關(guān)節(jié)置換的患者進行了2年隨訪,結(jié)果顯示患者術(shù)后6月內(nèi)金屬離子濃度逐漸上升,以后逐漸下降趨于平穩(wěn),但仍高于正常值。多中心臨床研究已證實：與金屬對聚乙烯假體相比,MOM會產(chǎn)生較少的磨損顆粒,但假體周圍、血液及組織中的金屬離子濃度較高。 隨著現(xiàn)代冶金技術(shù)及生物材料的發(fā)展,鈷鉻合金(其中鉻含量約占50-70%)因有較好的理學(xué)及機械學(xué)特性,已成為當(dāng)代金屬對金屬髖關(guān)節(jié)假體的主要材料。隨著假體的植入,鉻會不斷的緩慢釋放從而對人體產(chǎn)生影響。目前,大多數(shù)研究集中于金屬離子對假體周圍的骨組織、軟組織以及免疫細(xì)胞等的影響：①假體周圍不斷釋放的金屬離子對于周圍軟軟組織的影響。如引起炎性假瘤等。②假體無菌性松動。假體松動是THR后中、晚期最主要的并發(fā)癥之一,大部分學(xué)者認(rèn)為假體松動主要是因為假體周圍骨溶解引起。③金屬離子能夠誘導(dǎo)單核細(xì)胞及巨噬細(xì)胞釋放多種細(xì)胞因子,從而引起免疫反應(yīng)。肝臟是人體最大的解毒腺體,具有及其復(fù)雜多樣的生物化學(xué)功能。體內(nèi)包括鉻在內(nèi)的金屬離子均需在肝細(xì)胞內(nèi)進行生物學(xué)轉(zhuǎn)化。當(dāng)前,對于來源于假體的不斷釋放的鉻離子對遠隔靶器官如肝臟的生物學(xué)影響研究尚不多。 鉻是人體必需的微量元素之一,其在體內(nèi)的含量隨著年齡的增大而逐漸減少。生理劑量的鉻對血糖及膽固醇的調(diào)節(jié)具有重要作用。鉻在體內(nèi)主要以三價和六價形式存在。毒理學(xué)及環(huán)境衛(wèi)生學(xué)的研究已經(jīng)證實,短時間大量的鉻離子進入人體會引起肝細(xì)胞細(xì)胞信號轉(zhuǎn)導(dǎo)通路的改變,從而引起酶學(xué)、形態(tài)學(xué)等不可逆的損傷,甚至凋亡。鉻離子毒性與其存在的價態(tài)有極大的關(guān)系,六價鉻的毒性比三價鉻高約100倍。六價鉻(Ⅵ)經(jīng)非特異性的陰離子通道進入肝細(xì)胞,并在肝細(xì)胞內(nèi)通過酶類或非酶類的方式轉(zhuǎn)化為毒性較小的三價鉻(Ⅲ),此過程會引起細(xì)胞的氧化應(yīng)激,從而產(chǎn)生大量的過氧化物。產(chǎn)生的大量過氧化物會通過級聯(lián)瀑布式的反應(yīng)引起脂質(zhì)過氧化及DNA損傷。而細(xì)胞為對抗此反應(yīng),會啟動包括超氧化物歧化酶(SOD)、谷胱甘肽(GSH)、谷胱甘肽還原酶(GR)及谷氨酸半胱氨酸連接酶(GCL)等的抗氧化應(yīng)激系統(tǒng),清除自由基,以維持細(xì)胞內(nèi)環(huán)境的穩(wěn)定。 當(dāng)前的金屬對金屬髖關(guān)節(jié)假體中,鉻存在并釋放入血的主要形式是六價的。與毒理學(xué)及環(huán)境衛(wèi)生學(xué)的研究不同,MOM髖關(guān)節(jié)假體植入人體后,鉻(Ⅵ)離子釋放是一個緩慢且微量的過程。金屬假體的腐蝕度通常為0.15-0.3μg/cm2/h或30μd,或11mg/年,血鉻含量大量文獻報道在2.6-26gg/L之間,且時間窗較長(當(dāng)代金屬對金屬假體壽命一般在十年以上)。緩慢釋放的微量鉻(Ⅵ)是否使肝細(xì)胞產(chǎn)生氧化應(yīng)激?肝細(xì)胞內(nèi)的抗氧化應(yīng)激系統(tǒng)的反應(yīng)如何?因此,明確鉻(Ⅵ)對靶器官肝臟的氧化應(yīng)激的影響對于評估MOM假體的安全性具有重要意義。目的 1、構(gòu)建慢性微量鉻(Ⅵ)暴露小鼠慢性模擬金屬對金屬髖關(guān)節(jié)植入人體后引起的鉻(Ⅵ)緩慢釋放 2、探討慢性微量鉻(Ⅵ)暴露是否會引起小鼠肝細(xì)胞的氧化應(yīng)激。 3、探討慢性微量鉻(Ⅵ)暴露是否引起小鼠肝細(xì)胞抗氧化應(yīng)激系統(tǒng)的改變以及發(fā)生何種改變。 4、探討金屬對金屬假體對遠隔靶器官肝臟的安全性問題。 材料和方法按照實驗設(shè)計,將80只NIH小鼠隨機分為4組(對照組,低、中、高劑量組),每組20只,雌雄不限。Cr03染毒組劑量分別為0mg/kg、5mg/kg、10mg/kg和20mg/kg,隔日腹腔注射1次,實驗持續(xù)16周,分別于染毒4周末,第8周末,第12周末,第16周末用頸椎脫位法各處死小鼠5只,小鼠處死前進行眼球摘除取血,肝素抗凝后進行血鉻含量的檢測。一部分肝組織進行HE染色光鏡觀察,并進行透射電鏡觀察,余下肝組織用冰冷的生理鹽水洗凈,加入緩沖液后研磨成勻漿分裝后迅速置于-80℃冰箱保存?zhèn)溆?檢測肝細(xì)胞活性氧自由基水平(ROS)、脂質(zhì)過氧化產(chǎn)物丙二醛含量(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽活性(GSH)谷胱甘肽還原酶(GR)活性及谷氨酸半胱氨酸連接酶(GCL)的表達。 結(jié)果 1、0mg/kg、5mg/kg組在染毒4周后,血鉻含量達峰值,隨后維持在一定濃度,達到一個平臺期。而對于20mg/kg組,血鉻濃度持續(xù)升高,未見明顯的平臺期。 2、實驗各組小鼠肝細(xì)胞ROS值均高于對照組,且差別具有統(tǒng)計學(xué)意義。并且觀察到隨著染毒時間的延長,低中劑量組ROS會逐漸接近一個平臺期,而對20mg/kg組,ROS持續(xù)升高,未見明顯的平臺期。這與結(jié)果1相似。 3、在各個時間點中,20mg/kg組MDA含量較0mg/kg組升高,但差別無統(tǒng)計學(xué)意義。 4、與對照組相比,5mg/kg組、10mg/kg組在第4周CAT水平未見明顯降低,差別無統(tǒng)計學(xué)意義；而在隨后的各個時間點中升高明顯,且差別具有統(tǒng)計學(xué)意義。與對照組相比,20mg/kg組隨著染毒時間的延長CAT水平下降明顯,且差別具有統(tǒng)計學(xué)意義。 5、與對照組相比,小鼠經(jīng)鉻染毒4周后20mg/kg組SOD降低,并具有顯著性差異。8周以后隨著染毒時間的延長,各組SOD活性下降明顯,且差別具有統(tǒng)計意義。 6、與對照組相比,5mg/kg組、10mg/kg組在第4周GSH水平未見明顯降低,差別無統(tǒng)計學(xué)意義；而在隨后的各個時間點中升高明顯,且差別具有統(tǒng)計學(xué)意義。與對照組相比,20mg/kg組隨著染毒時間的延長GSH水平下降明顯,且差別具有統(tǒng)計學(xué)意義。 7、與對照組相比在染毒的第4周5mg/kg組、10mg/kg組GR活性升高,差別具有統(tǒng)計學(xué)意義；20mg/kg組下降明顯,且差別具有統(tǒng)計學(xué)意義.隨著染毒時間的延長各劑量組GR水平均下降,且與對照組相比,差別具有統(tǒng)計學(xué)意義。 8、GCL表達的蛋白條帶用image-proPlus圖像分析系統(tǒng)對條帶灰度進行掃描,掃描結(jié)果顯示：5mg/kg組、10mg/kg組各個時間點GCL表達明顯升高,與對照組相比,具有統(tǒng)計學(xué)意義。而20mg/kg組GCL表達在第8周后下降明顯,差別具有統(tǒng)計學(xué)意義。 9、肝細(xì)胞HE染色顯示：與對照組相比,5mg/kg組、10mg/kg組肝細(xì)胞未見明顯異常。而20mg/kg組肝細(xì)胞在第16周可見少量的空泡變,細(xì)胞膜稍皺縮。透射電鏡顯示：與對照相比,高劑量染毒小鼠在第16周超微結(jié)構(gòu)出現(xiàn)凋亡特征性改變：細(xì)胞核皺縮,染色質(zhì)濃縮并發(fā)生邊聚化；線粒體變的致密,峭消失,空泡化改變。 結(jié)論 1、隨著染毒時間的延長,慢性微量鉻(Ⅵ)染毒會引起小鼠血鉻含量的升高,且具有一定的濃度時間依賴性。 2、慢性微量鉻(Ⅵ)暴露會引起小鼠肝細(xì)胞的氧化應(yīng)激,提示金屬對金屬髖關(guān)節(jié)置換術(shù)后鉻離子的釋放會引起遠隔靶器官肝臟的氧化應(yīng)激。 3、慢性微量鉻(Ⅵ)暴露引起小鼠肝細(xì)胞抗氧化應(yīng)激系統(tǒng)的改變。 4、金屬對金屬假體對遠隔靶器官肝臟的安全性尚需進一步評估。
[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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R684;R114

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相關(guān)期刊論文 前1條

1 何志勇;吳海山;;金屬對金屬全髖關(guān)節(jié)表面置換術(shù)研究進展[J];臨床骨科雜志;2009年01期

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