Ames试验评价聚β-羟基丁酸酯（PHB）的生物相容性
摘要
目的
提高镁合金耐蚀性的有效途径之一是在镁基体表面提供一层耐蚀的表面层作为腐蚀屏障,对基体形成有效保护。基于现代表面技术的发展以及环保的要求,微弧氧化、金属铝涂层、含氟协合涂层、气相沉积、离子注入等新技术在改善镁合金耐蚀性方面越来越受到青睐,而聚β-羟基丁酸酯涂层更是一项有良好发展前景的新技术。
聚β-羟基丁酸酯(简称PHB)是一种由微生物在不平衡生长条件下存储于细胞内的一种天然高分子聚合物,广泛存在于自然界许多原核生物中,在药物释放和组织工程方面有广泛的应用。本研究根据ISO相关标准,从材料是否致基因突变的角度,进行Ames试验,用材料浸提液对聚β-羟基丁酸酯的生物相容性进行初步研究测试,为预测该材料的遗传危害和潜在致癌性提供实验证据。
方法
1、菌株遗传性状鉴定
分别进行对组氨酸的要求,脂多糖屏障缺陷,紫外线损伤修复缺陷,抗氨苄青霉素试验,抗四环素试验和自发回变六项试验。遗传性状鉴定合格后,37℃振荡培养,使增菌液浓度达到1-3×10~9个/ml。
2、培养基
共用4种培养基按标准细菌回复突变试验(Ames)要求制作。
3、活化系统
S-9及S-9mix均按Ames原法要求制作、使用和保存。
4、剂量设计
将样品聚合物片清洗(乙醇,超声波)灭菌后,以生理盐水为浸提介质,参照GB/T16886.12-2000所规定的浸提液制备方法,以材料表面积(双面)/浸提介质为3cm~2/ml的比例,制备标准浸提液(原液),并设2倍、4倍、1/2原液4个不同浓度的浸提液;37℃±1℃,24h±2h浸提供试。
5、Ames试验
计数TA97,TA98,TA100,TA102,TA1535标准测试菌株在四个不同浓度浸提液下,37℃48h后的回变菌落数。
结果
聚β-羟基丁酸酯细菌回复突变试验(Ames),各剂量组细菌回变数小于阴性对照组细菌回变数的2倍。聚β-羟基丁酸酯细菌回复突变试验,各菌株、各剂量组无论代谢活化与否,皆为阴性试验结果。
结论
聚β-羟基丁酸酯经细菌回复突变试验(Ames)未见潜在致突变性。
Objective
One of the effective ways to improve the corrosion resistance of aluminum alloy is providing the surface of magnesium matrix with a layer of corrosion,which can be regarded as a corrosion barrier.Based on the modern surface technology,as well as the requirements of environmental protection,micro-arc oxidation,aluminum coatings, aluminum fluoride coatings,fluoride concord coating,vapor deposition,ion implantation and other new technologies in improving the corrosion resistance of aluminum-magnesium are increasingly popular.And plolyhydroxybutyrate layer is a significantly new promising technology.It,widely existing in many natural prokaryotes, is a natural polymer stored in cells,and it is formed by a kind of microorganism growing in imbalance growth condition.Plolyhydroxybutyrate is now widely used in drug delivery and tissue engineering.On the basis of ISO correlated standardization, leaching liquor of biodegradable polyhydroxybutyrate was made out to progress Salmonella typhimurium reverse mutation assay(Ames test) in this study.And the purpose of this study was to answer the question whether the biodegradable polyhydroxybutyrate had potential mutagenicity and carcinogenicity.
Methods
1、Appreciation of stain genetic determinant
Six stain genetic determinant experiments include:the requirement of histidine, discrepancy of lipopolysaccharides barrier,repair-deficiency of DUV,test of anti-AMP, test of anti-TCN and spontaneous reverse assay were carried out.After the appreciation of stain genetic determinant,incubation at 37℃,make bacterium fluid density to 1-3×10~9/ml.
2、Nutrient medium
Four kinds of nutrient mediums were made reference to Salmonella typhimurium reverse mutation assay.
3、Activation system
S-9 and S-9mix were made reference to Salmonella typhimurium reverse mutation assay.
4、Design of dosage
Clean the sample alloy plate with alcohol and supersonic,sterilizing,use normal sodium as diffusion medium,in accordance with GB/T16886.12-2000,establish standard leaching(liquorstock suspension) by means of diprosopia surface area/ diffusion medium is 3cm~2/ml.1/2,2 and 4 times of liquorstock suspension were prepared.37℃±1℃,24h±2h diffused.
5、Salmonella typhimurium reverse mutation assay
In the flat-plate incorporation test of Salmonella typhimurium,the average number of spontaneous revertants of TA97,TA98,TA100,TA102,TA1535 under 4 concentrations was calculated after incubation at 37℃for 48 hours.
Results
The number of induced revertants when compared to that of spontaneous revertants was not reproducibly higher in a dose-related manner.The result of Ames test for biodegradable polyhydroxybutyrate was negative.
Conclusion
It implies biodegradable polyhydroxybutyrate has no potential mutagenicity.
提高镁合金耐蚀性的有效途径之一是在镁基体表面提供一层耐蚀的表面层作为腐蚀屏障,对基体形成有效保护。基于现代表面技术的发展以及环保的要求,微弧氧化、金属铝涂层、含氟协合涂层、气相沉积、离子注入等新技术在改善镁合金耐蚀性方面越来越受到青睐,而聚β-羟基丁酸酯涂层更是一项有良好发展前景的新技术。
聚β-羟基丁酸酯(简称PHB)是一种由微生物在不平衡生长条件下存储于细胞内的一种天然高分子聚合物,广泛存在于自然界许多原核生物中,在药物释放和组织工程方面有广泛的应用。本研究根据ISO相关标准,从材料是否致基因突变的角度,进行Ames试验,用材料浸提液对聚β-羟基丁酸酯的生物相容性进行初步研究测试,为预测该材料的遗传危害和潜在致癌性提供实验证据。
方法
1、菌株遗传性状鉴定
分别进行对组氨酸的要求,脂多糖屏障缺陷,紫外线损伤修复缺陷,抗氨苄青霉素试验,抗四环素试验和自发回变六项试验。遗传性状鉴定合格后,37℃振荡培养,使增菌液浓度达到1-3×10~9个/ml。
2、培养基
共用4种培养基按标准细菌回复突变试验(Ames)要求制作。
3、活化系统
S-9及S-9mix均按Ames原法要求制作、使用和保存。
4、剂量设计
将样品聚合物片清洗(乙醇,超声波)灭菌后,以生理盐水为浸提介质,参照GB/T16886.12-2000所规定的浸提液制备方法,以材料表面积(双面)/浸提介质为3cm~2/ml的比例,制备标准浸提液(原液),并设2倍、4倍、1/2原液4个不同浓度的浸提液;37℃±1℃,24h±2h浸提供试。
5、Ames试验
计数TA97,TA98,TA100,TA102,TA1535标准测试菌株在四个不同浓度浸提液下,37℃48h后的回变菌落数。
结果
聚β-羟基丁酸酯细菌回复突变试验(Ames),各剂量组细菌回变数小于阴性对照组细菌回变数的2倍。聚β-羟基丁酸酯细菌回复突变试验,各菌株、各剂量组无论代谢活化与否,皆为阴性试验结果。
结论
聚β-羟基丁酸酯经细菌回复突变试验(Ames)未见潜在致突变性。
Objective
One of the effective ways to improve the corrosion resistance of aluminum alloy is providing the surface of magnesium matrix with a layer of corrosion,which can be regarded as a corrosion barrier.Based on the modern surface technology,as well as the requirements of environmental protection,micro-arc oxidation,aluminum coatings, aluminum fluoride coatings,fluoride concord coating,vapor deposition,ion implantation and other new technologies in improving the corrosion resistance of aluminum-magnesium are increasingly popular.And plolyhydroxybutyrate layer is a significantly new promising technology.It,widely existing in many natural prokaryotes, is a natural polymer stored in cells,and it is formed by a kind of microorganism growing in imbalance growth condition.Plolyhydroxybutyrate is now widely used in drug delivery and tissue engineering.On the basis of ISO correlated standardization, leaching liquor of biodegradable polyhydroxybutyrate was made out to progress Salmonella typhimurium reverse mutation assay(Ames test) in this study.And the purpose of this study was to answer the question whether the biodegradable polyhydroxybutyrate had potential mutagenicity and carcinogenicity.
Methods
1、Appreciation of stain genetic determinant
Six stain genetic determinant experiments include:the requirement of histidine, discrepancy of lipopolysaccharides barrier,repair-deficiency of DUV,test of anti-AMP, test of anti-TCN and spontaneous reverse assay were carried out.After the appreciation of stain genetic determinant,incubation at 37℃,make bacterium fluid density to 1-3×10~9/ml.
2、Nutrient medium
Four kinds of nutrient mediums were made reference to Salmonella typhimurium reverse mutation assay.
3、Activation system
S-9 and S-9mix were made reference to Salmonella typhimurium reverse mutation assay.
4、Design of dosage
Clean the sample alloy plate with alcohol and supersonic,sterilizing,use normal sodium as diffusion medium,in accordance with GB/T16886.12-2000,establish standard leaching(liquorstock suspension) by means of diprosopia surface area/ diffusion medium is 3cm~2/ml.1/2,2 and 4 times of liquorstock suspension were prepared.37℃±1℃,24h±2h diffused.
5、Salmonella typhimurium reverse mutation assay
In the flat-plate incorporation test of Salmonella typhimurium,the average number of spontaneous revertants of TA97,TA98,TA100,TA102,TA1535 under 4 concentrations was calculated after incubation at 37℃for 48 hours.
Results
The number of induced revertants when compared to that of spontaneous revertants was not reproducibly higher in a dose-related manner.The result of Ames test for biodegradable polyhydroxybutyrate was negative.
Conclusion
It implies biodegradable polyhydroxybutyrate has no potential mutagenicity.
引文
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2 Heublein B,Rohde R,Kaese V.Biocorrosion of magnesi-um alloys:a new principle in cardiovascular implant technology.Heart,2003,89:651.
3 王翰章,等.中华口腔科学[M].北京:人民卫生出版社,2000,956.
4 詹文革,生物医用钛及钛合金的研制、生产和应用[J].钛工业进展,2007,24(1):4-7.
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