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  • 祖丽皮也·阿不力克木,古丽加玛丽·阿不力克木,朱庆丰,陈彤,吴刚,易杨华,王少海.5种脱细胞鱼皮基质的制备及体外毒性[J].第二军医大学学报,2019,40(2):162-168    [点击复制]
  • Zulpiye Ablikim,Guljamal Ablikim,ZHU Qing-feng,CHEN Tong,WU Gang,YI Yang-hua,WANG Shao-hai.Preparation of five kinds of acellular fish skin matrices and in vitro toxicity analysis[J].Acad J Sec Mil Med Univ,2019,40(2):162-168   [点击复制]
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5种脱细胞鱼皮基质的制备及体外毒性
祖丽皮也·阿不力克木1,古丽加玛丽·阿不力克木2,朱庆丰1,陈彤1,吴刚1,易杨华3,王少海1*
0
(1. 海军军医大学(第二军医大学)长海医院口腔科, 上海 200433;
2. 喀什地区第一人民医院心内科, 喀什 844000;
3. 海军军医大学(第二军医大学)药学院海洋药物研究所, 上海 200433
*通信作者)
摘要:
目的 探究5种脱细胞鱼皮基质的生物安全性及相容性,筛选出符合生物相容性要求的材料。方法 以5种鱼皮(马面鱼、黑鱼、鳗鱼、鲤鱼、鲢鱼)为原料,通过脱色、脱脂、脱细胞和交联等制备工艺,制备脱细胞基质。依据国家标准(GB/T 16886.5-2017)将脱细胞鱼皮基质材料制备成稀释浓度10 g/L和标准浓度100 g/L的浸提液,用CCK-8试剂盒检测脱细胞鱼皮基质材料浸提液对人骨髓间充质干细胞的毒性。采用扫描电镜观察脱细胞鱼皮基质材料的结构,通过H-E染色和Masson染色检测制备的鱼皮基质材料是否完全脱细胞。用标准浓度100 g/L的脱细胞鱼皮基质材料浸提液与人骨髓间充质干细胞共同培养,通过溶血实验检测材料的血液相容性,采用Live/Dead染色法、TUNEL法和流式细胞术检测细胞的存活和凋亡情况。结果 在10 g/L浓度下,马面鱼及黑鱼脱细胞鱼皮基质材料浸提液中人骨髓间充质干细胞活性均>70%,其余3种材料细胞活性均<70%;在100 g/L浓度下,马面鱼脱细胞鱼皮基质材料浸提液中人骨髓间充质干细胞活性>70%,而黑鱼脱细胞鱼皮基质材料浸提液中细胞活性<70%。扫描电镜下马面鱼脱细胞鱼皮基质材料为多孔、致密结构,黑鱼脱细胞鱼皮基质材料为平滑结构;H-E和Masson染色结果显示马面鱼和黑鱼脱细胞鱼皮基质材料均为脱细胞胶原纤维结构。马面鱼及黑鱼脱细胞鱼皮基质材料浸提液溶血率分别为(1.23±0.43)%和(6.35±0.47)%(医用材料溶血率国家标准为≤5%)。人骨髓间充质干细胞在马面鱼脱细胞鱼皮基质材料浸提液中存活状况良好,而在黑鱼脱细胞鱼皮基质材料浸提液中多数发生死亡或凋亡。结论 马面鱼脱细胞鱼皮基质材料无细胞毒性、无溶血性,具有成为组织工程支架材料的潜能。
关键词:  绿鳍马面鲀  脱细胞真皮基质  间质干细胞  组织工程  支架
DOI:10.16781/j.0258-879x.2019.02.0162
投稿时间:2018-12-16修订日期:2019-02-10
基金项目:国家自然科学基金(81271177,81870808).
Preparation of five kinds of acellular fish skin matrices and in vitro toxicity analysis
Zulpiye Ablikim1,Guljamal Ablikim2,ZHU Qing-feng1,CHEN Tong1,WU Gang1,YI Yang-hua3,WANG Shao-hai1*
(1. Department of Stomatology, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China;
2. Department of Cardiology, First People's Hospital of Kashgar Prefecture, Kashgar 844000, Xinjiang Uygur Autonomous Region, China;
3. Institute of Marine Drugs, School of Pharmacy, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To explore the biological safety and compatibility of five kinds of acellular fish skin matrices, and to screen the materials meeting the criteria of biocompatibility. Methods Acellular fish skin matrices were prepared through decolorization, degreasing, decellularization and cross-linking using five kinds of fish skins leather jacket fish (Thamnaconus septentrionalis), black fish (Channa argus), eel (Anguillidae), carp (Cyprinus carpio) and silver crap (Hypophthalmichthys molitrix). According to the national standard (GB/T 16886.5-2017), different concentrations (10 g/L and 100 g/L) of extracts of acellular fish skin matrices were prepared, and the CCK-8 assay was used to detect the in vitro toxicity of extracts of acellular fish skin matrices to human bone marrow-derived mesenchymal stem cells. The surface structures of different collagen membranes were observed under scanning electron microscopy. The H-E and Masson staining were used to observe whether the fish skin matrix materials were completely acellular. The human bone marrow-derived mesenchymal stem cells were cultured with the medium containing standard concentration of 100 g/L acellular fish skin matrix material extracts. The blood compatibility of the meterial was tested by hemolysis test. Live/Dead, TUNEL and flow cytometry were used to detect the cell survival and apoptosis levels. Results CCK-8 assay showed that the viability indexes of the cells co-cultured with the leather jacket fish and black fish skin acellular matrix material extracts were both >70% at the concentration of 10 g/L, and the other three materials were all <70%. At the concentration of 100 g/L, the viability index of the cells co-cultured with the leather jacket fish skin acellular matrix material extracts was >70%, and that with the black fish was <70%. Scanning electron microscopy showed that the leather jacket fish skin acellular matrix materials had porous and compact structure inside, and black fish skin acellular matrix materials had smooth structure inside. The H-E staining and Masson staining showed that leather jacket fish and black fish skin acellular matrix materials were both acellular collagen fibers. The hemolysis rates (the national standard ≤ 5%) of leather jacket fish and black fish skin acellular matrix material extracts were (1.23±0.43)% and (6.35±0.47)%, respectively. Human bone marrow-derived mesenchymal stem cells survived well in the culture medium containing extracts of leather jacket fish skin acellular matrix material, but most cells died or apoptozed in the culture medium containing extracts of Black fish skin acellular matrix material. Conclusion The leather jacket fish decellularized skin matrix material has better biosafety and biocompatibility, and has the potential to be a scaffold material for tissue engineering.
Key words:  Thamnaconous septentrionalis  acellular dermal matrix  mesenchymal stem cells  tissue-engineering  stents