可降解镁合金支架体外降解产物对神经细胞的安全性评价

发布时间：2018-05-22 09:37

本文选题：AZ31镁合金 + 颈内动脉支架　；参考：《中国人民解放军医学院》2016年硕士论文

【摘要】：研究背景：镁合金作为性能优越的可降解合金被广泛应用于医疗内置物材料,已在骨植入物、心血管支架方面进入临床观察阶段,而这种镁合金材料能否作为脑血管支架材料仍是未知数.目前神经介入治疗中所应用的永久性支架存在植入后对血管造成的机械性损伤、支架植入处再狭窄、且后续无法顺利行MRI检查等缺点,而可降解镁合金支架的优点在于可完全降解,能有效避免了永久性支架带来的损伤。已知镁合金在降解过程中会释放Mg离子、Al离子、以及会造成局部酸碱度的改变,而这些改变对神经系统是否会产生影响目前尚未见研究,也是镁合金支架能否运用于脑血管内重建治疗的关键。本实验就可降解镁合金支架AZ31的降解对神经细胞SY5Y的安全性进行研究.目的：明确镁合金支架AZ31对神经细胞SYSY生物学行为的影响。方法：本实验采用间接法和直接法两种评估方法。间接法：测定A1及Mg的浓度变化对SY5Y死亡率的影响.配制含不同浓度梯度MgCl2及AlCl3(如下表1、表2所示)的细胞培养液,用于培养SY5Y细胞,根据其MTT实验结果,评估A1及Mg的浓度与SY5Y细胞死亡率间的关系.收集降解过程中的降解液,每3天检测其中Al及Mg浓度,根据估测的安全浓度范围,评估降解过程中所产生的Mg、Al浓度对神经细胞是否存在细胞毒性。直接法：将收集所得降解液直接用于SY5Y细胞的培养,为了确定降解液局部引起的pH值变化对细胞生存率是否会产生影响,将pH调节至正常范围定为pH调节组,与未调节pH的降解液组对比。对照组：普通DMEM/F12培养液；实验组1(未调节pH值)：每隔三日收集的降解液处理细胞组---第1d组,第4d组,第7d组...第88d组；实验组2(调节pH值)：每隔三日收集的降解液调整pH值后处理细胞组--第1d组,第4d组,第7d组...第88d组；MTT法测定各组OD值,并计算细胞死亡率。结果间接法：SY5Y细胞的死亡率均随着Mg浓度及A1浓度的增高而上升,并且,SY5Y细胞的死亡率与Mg离子浓度呈线性相关(P0.01).Mg：体外降解的过程中,约10%天数降解产生Mg浓度低于1.8mmol/L,细胞毒性为1级,其他时间可能产生2级细胞毒性.Al：测定得出A1在降解液中浓度极低,故可以认为,AZ31镁合金可降解支架在降解过程中产生的Al浓度范围对SY5Y神经细胞是安全的.直接法：pH未调节组细胞死亡率主要与pH值相关(P0.01),pH调节组死亡率与Mg浓度相关(P0.01),两组细胞死亡率均与Al无关.不考虑pH值的影响,降解过程中约有10天,Mg对SY5Y产生2级的细胞毒性。结论1.AZ31可降解镁合金产生的降解产物Al对神经细胞是安全的,不产生细胞毒性。2.AZ31可降解镁合金产生的降解产物中Mg浓度偏高,对于神经细胞存在2级毒性反应。3.体外降解引起的pH值变化,对神经细胞存在细胞毒性.4.实验结果可以为镁合金材料改良提供依据,但不能绝对否定其体内的应用,仍需要体内试验进一步证实其安全性及可行性。
[Abstract]:Background: magnesium alloys, as degradable alloys with superior properties, have been widely used in medical implants, and have entered the clinical observation stage in bone implants and cardiovascular stents. But whether this magnesium alloy material can be used as cerebral vascular stent material is still unknown. At present, the permanent stent used in nerve interventional therapy has the disadvantages of mechanical injury to blood vessel after implantation, restenosis of stent implantation, and the failure of subsequent MRI examination. The advantage of degradable magnesium alloy scaffold is that it is completely degradable and can effectively avoid the damage caused by permanent scaffold. It is known that magnesium alloys release mg ion and Al ions during the degradation process and cause local pH changes, and whether these changes have any effect on the nervous system has not been studied. It is also the key whether magnesium alloy stents can be used in the treatment of cerebral vascular reconstruction. The safety of AZ31 degradation of degradable magnesium alloy scaffold on nerve cell SY5Y was studied in this experiment. Objective: to investigate the effect of magnesium alloy scaffold AZ31 on the biological behavior of nerve cell SYSY. Methods: indirect method and direct method were used in this experiment. Indirect method: the effects of changes of Al and mg concentrations on SY5Y mortality were determined. The cell culture medium containing different concentration gradient MgCl2 and ALCL 3 (as shown in table 1 and table 2) was used to culture SY5Y cells. The relationship between the concentration of A1 and mg and the death rate of SY5Y cells was evaluated according to the MTT results. The degradation solution was collected and the concentrations of Al and mg were detected every 3 days. According to the estimated safe concentration range, the toxicity of MgN Al concentration to nerve cells was evaluated. Direct method: the biodegradable solution collected was directly used in the culture of SY5Y cells. In order to determine whether the pH change caused by the degradation solution had an effect on cell survival, the pH was adjusted to a normal range. The results were compared with the unadjusted pH group. Control group: normal DMEM/F12 culture medium, experimental group 1 (unadjusted pH value: treated cell group every 3 days-group 1 d, group 4 d, group 7 d). Group 2 (adjusting pH value: treated cell group after adjusting pH value every 3 days-group 1 d, group 4 d, group 7 d). OD value was measured by MTT assay and cell death rate was calculated in 88 d group. Results the mortality of the cell line was increased with the increase of mg concentration and A1 concentration, and there was a linear correlation between the death rate of SY5Y cell and mg ion concentration in vitro. About 10% of the days of degradation resulted in mg concentration below 1.8 mmol / L, cytotoxicity of grade 1, other time may produce grade 2 cytotoxicity. Al: the concentration of A1 in the degradation solution was very low. It can be concluded that the concentration range of Al produced in the degradable scaffold of AZ31 magnesium alloy is safe for SY5Y nerve cells. The cell death rate in the unadjusted group was mainly related to the pH value (P 0.01) and mg concentration (P 0.01). The cell death rate was not related to Al in both groups. In the process of degradation, mg produced second-order cytotoxicity to SY5Y for about 10 days without taking into account the effect of pH value. Conclusion the degradation product Al produced by 1.AZ31 degradable magnesium alloy is safe to nerve cells. In vitro degradation caused by pH changes, there is cytotoxicity to nerve cells. 4. The experimental results can provide the basis for the improvement of magnesium alloy materials, but can not absolutely deny its application in vivo. It is still necessary to further verify the safety and feasibility of the in vivo test.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R318.08;R651.12

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