可吸收丝素生物膜对大鼠的免疫毒性
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摘要
背景:丝素生物膜的主要成分丝素蛋白是天然蛋白质,但对机体来说仍属异源蛋白,其免疫原性/毒性的强弱是决定其开发前景的重要因素。目的:通过大鼠肌肉植入实验评价可吸收丝素生物膜及其降解产物对大鼠免疫系统的毒性作用。方法:取10只Wistar大鼠(北京维通利华实验动物技术有限公司提供),右侧臀肌植入1 mm×10 mm的长条状丝素生物膜,观察植入物吸收情况,确定实验植入周期。将72只Wistar大鼠分为对照组与受试物组,每组36只,每组内雌雄各18只,受试物组在右侧臀肌植入1mm×10mm的长条状丝素生物膜,对照组不放置植入物,其余操作同受试物组,植入26周后,取其中一部分动物,进行免疫器官脏体比及组织病理学检查,以及脾淋巴细胞增殖能力、NK细胞活性、外周血T淋巴细胞及其亚群、细胞因子白细胞介素2和肿瘤坏死因子α检测;取另一部分动物,进行巨噬细胞红细胞吞噬率与吞噬指数、溶血空斑数检测。结果与结论:①丝素生物膜植入大鼠肌肉26周基本全部吸收;②在雌性或雄性Wistar大鼠中,受试物组免疫器官外观、质量和组织病理学检查未见有生物学意义的明显改变,血液学指标(血红蛋白、红细胞计数、红细胞压积、血小板计数、白细胞计数及分类)、脾淋巴细胞增殖能力、NK细胞活性、外周血T淋巴细胞及其亚群、白细胞介素2和肿瘤坏死因子α水平与对照组比较差异均无显著性意义(P>0.05); ③在雌性或雄性Wistar大鼠中,受试物组巨噬细胞红细胞吞噬率与吞噬指数、溶血空斑数与对照组比较差异均无显著性意义(P> 0.05);④结果表明,丝素生物膜未引起大鼠机体的免疫抑制或免疫刺激等免疫毒性反应,对机体免疫器官、免疫细胞和免疫分子(细胞因子)无明显影响。
BACKGROUND: Silk fibroin, the main component of silk fibroin biofilm, is a natural protein, but it is still a heterologous protein to the body. Its immunogenicity/toxicity is an important factor in determining the development prospects. OBJECTIVE: To evaluate the toxicity of absorbable silk fibroin biofilm and its degradation products on the rat immune system by muscle implantation experiments in rats. METHODS: Ten Wistar rats from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. were implanted with a long strip of 1 mm× 10 mm in the right gluteal muscle to observe the implant absorption and determine the implantation cycle. Seventy-two Wistar rats were assigned into control and experimental groups(n=36/group, 18 in either sexes). A long strip of 1 mm×10 mm was implanted into the rat right gluteal muscle in the experimental group, and the control group received no implantation. Some of the rats were taken for histological examination and calculate the organ/weight ratio, at 26 weeks postoperatively. The spleen lymphocyte proliferation ability, NK cell activity, cell classification of T lymphocytes, and levels of interleukin 2 and tumor necrosis factor α were detected. Another part of the animals was taken for macrophage phagocytosis of erythrocytes cell capacity and antibody producing cell count. RESULTS AND CONCLUSION:(1) Silk fibroin biofilm was completely absorbed after implanted into the rat muscle for 26 weeks.(2) In female or male Wistar rats, the immune organs in the experimental group showed no significant changes in the appearance, weight and histological examination. There were no significant differences in the hematological indexes(hemoglobin, red blood cell count, hematocrit, blood platelet count and white blood cell count and classification), spleen lymphocyte proliferation, NK cell activity, the ratio of T lymphocytes and their subpopulations, and the levels of interleukin 2 and tumor necrosis factor α had no significant differences between two groups(P > 0.05).(3) In female or male Wistar rats, the macrophage phagocytosis of chicken erythrocytes cell capacity and antibody producing cell count showed no significant differences between two groups(P > 0.05).(4) These results indicate that silk fibroin biofilm causes no immunosuppression or immunostimulation on immune organs, immune cells and immune molecules(cytokines) of rats.
BACKGROUND: Silk fibroin, the main component of silk fibroin biofilm, is a natural protein, but it is still a heterologous protein to the body. Its immunogenicity/toxicity is an important factor in determining the development prospects. OBJECTIVE: To evaluate the toxicity of absorbable silk fibroin biofilm and its degradation products on the rat immune system by muscle implantation experiments in rats. METHODS: Ten Wistar rats from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. were implanted with a long strip of 1 mm× 10 mm in the right gluteal muscle to observe the implant absorption and determine the implantation cycle. Seventy-two Wistar rats were assigned into control and experimental groups(n=36/group, 18 in either sexes). A long strip of 1 mm×10 mm was implanted into the rat right gluteal muscle in the experimental group, and the control group received no implantation. Some of the rats were taken for histological examination and calculate the organ/weight ratio, at 26 weeks postoperatively. The spleen lymphocyte proliferation ability, NK cell activity, cell classification of T lymphocytes, and levels of interleukin 2 and tumor necrosis factor α were detected. Another part of the animals was taken for macrophage phagocytosis of erythrocytes cell capacity and antibody producing cell count. RESULTS AND CONCLUSION:(1) Silk fibroin biofilm was completely absorbed after implanted into the rat muscle for 26 weeks.(2) In female or male Wistar rats, the immune organs in the experimental group showed no significant changes in the appearance, weight and histological examination. There were no significant differences in the hematological indexes(hemoglobin, red blood cell count, hematocrit, blood platelet count and white blood cell count and classification), spleen lymphocyte proliferation, NK cell activity, the ratio of T lymphocytes and their subpopulations, and the levels of interleukin 2 and tumor necrosis factor α had no significant differences between two groups(P > 0.05).(3) In female or male Wistar rats, the macrophage phagocytosis of chicken erythrocytes cell capacity and antibody producing cell count showed no significant differences between two groups(P > 0.05).(4) These results indicate that silk fibroin biofilm causes no immunosuppression or immunostimulation on immune organs, immune cells and immune molecules(cytokines) of rats.
引文
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[22]王延琳,马玉媛,赵雄,等.植人性医疗器械的临床前免疫毒性评价[J].医疗卫生装备,2017,38(8):117-126.
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[26]薛旸,丁婷婷,孙皎.生物材料免疫抑制效应评价的方法初探[J].中国医疗器械杂志,2009,33(1):20-22.
[27]Hilal AA,Gaylor JDS.Bioartificial liver:review of science requirements and technology.World Review ofScience,Technology,and Sustainable Development.2006;3(1):80-83.
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[30]吕志敢,郭政.肿瘤坏死因子的研究进展[J].山西医科大学学报,2006,37(3):311-314.
[2]Cha BG,Kwak HW,Park AR,et al.Structural characteristics and biological performance of silk fibroin nanofiber containing microalgae Spirulina extract.Biopolymers.2014;101(4):307-318.
[3]Luangbudnark W,Viyoch J,Laupattarakasem W,et al.Properties and biocompatibility of chitosan and silk fibroin blend films for application in skin tissue engineering.ScientificWorldJournal.2012;2012:697201.
[4]王宏昕,李敏.丝素蛋白作为组织工程生物材料的研究进展[J].中国修复重建外科杂志,2008,22(2):192-195.
[5]Wang J,Wei Y,Yi H,et al.Cytocompatibility of a silk fibroin tubular scaffold.Mater Sci Eng C Mater Biol Appl.2014;34:429-436.
[6]Calamak S,Erdo?du C,Ozalp M,et al.Silk fibroin based antibacterial bionanotextiles as wound dressing materials.Mater Sci Eng C Mater Biol Appl.2014;43:11-20.
[7]陆艳,赵霞,邵正中,等.多孔丝素材料组织相容性的初步研究[J].中国组织工程研究和临床康复,2011,15(34):6364-6367.
[8]ISO/TS l0993-20:2006,Biological evaluation of medical Devices-Part 20:Principles and methods for immunotoxicology testing of medical device.
[9]ICH:S8(Step 4)Immunoxicity Studies For Human Pharmaceutical:2005:1-11.
[10]雷静,李奕恒,刘旭昭,等.动物源I型胶原蛋白可引起BALBlc小鼠细胞免疫反应和组织免疫毒性[J].中国组织工程研究,2015,19(34):5506-5512.
[11]梅昕,马凤森,喻炎,等.高分子可降解生物材料的降解研究进展[J].材料导报,2016,30(S1):298-303.
[12]李君涛,陈周煜.可降解生物医用材料研究现状与展望[J].新材料产业,2016,18(1):32-35.
[13]李云飞.医疗器械化学性能检测的重要性分析[J].化工设计通讯,2017,43(12):169-171.
[14]陈亮,奚廷斐,王春仁.组织工程医疗产品免疫学评价研究[J].组织工程与重建外科杂志,2009,5(1):56-58.
[15]邹文,袁暾,蔡永福,等.组织诱导性材料生物安全性评价研究策略与实践[J].中国组织工程研究,2018,22(10):1534-1539.
[16]戴菁,孙旖,高婷婷,等.免疫毒性生物标志研究进展[J].毒理学杂志,2012,25(6):463-465.
[17]Pulendran B,Artis D.New paradigms in type 2 immunity.Science.2012;337(6093):431-435.
[18]Palm NW,Rosenstein RK,Medzhitov RAllergic host defences.Nature.2012;484(7395):465-472.
[19]Holsapple MP,Burns-Naas LA,Hastings KL,et al.Aproposed testing framework for developmental immunotoxicology(DIT).Toxicol Sci.2005;83(1):18-24.
[20]陈成章.免疫毒理学[M].郑州:郑州大学出版社,2008:171-174.
[21]杨晓芳,奚廷斐.医疗器械的免疫毒性评价[J].生物医学工程学杂志,2007,24(5):1191-1195.
[22]王延琳,马玉媛,赵雄,等.植人性医疗器械的临床前免疫毒性评价[J].医疗卫生装备,2017,38(8):117-126.
[23]Germolec DR.Sensitivity and predicitivity in immunotoxicity testing:immune endpoints and disease resistance.Toxicol Lett.2004;149(1-3):109-114.
[24]Luebke R,House R,Kimber I.Emanuela corsini animal and in vitro models of immunotoxicity.New York:CRC Press,2007:66-68.
[25]潘东升,范玉明,李波.新药免疫毒性评价研究进展[J].中国新药杂志,2018,27(13):1491-1495.
[26]薛旸,丁婷婷,孙皎.生物材料免疫抑制效应评价的方法初探[J].中国医疗器械杂志,2009,33(1):20-22.
[27]Hilal AA,Gaylor JDS.Bioartificial liver:review of science requirements and technology.World Review ofScience,Technology,and Sustainable Development.2006;3(1):80-83.
[28]谢从平.活化的非特异性细胞毒系统[J].国外医学(免疫学分册),1987,14(4):176.
[29]Singh J,Suruchi A.Anti TNF-a strategy:present status of this therapeutic paradigm.Indian J Pharmacol.2004;36(1):10-14.
[30]吕志敢,郭政.肿瘤坏死因子的研究进展[J].山西医科大学学报,2006,37(3):311-314.