永生化人肝细胞HepZJ的临床前急性毒性评价
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摘要
背景:肝细胞移植、肝脏组织工程与生物人工肝的研究为肝衰竭患者带来福音,然而截至目前仍未发现十分合适的种子细胞。课题组前期自行构建人永生化肝细胞系HepZJ作为新的种子细胞,并进行了临床前安全性评价研究。目的:对新型永生化人肝细胞系HepZJ进行急性毒性研究,为HepZJ应用于临床可能发生的毒副反应以及设计安全剂量提供参考依据。方法:在HepZJ细胞急性毒性研究中,将低、中、高(5×10~6,5×10~7,8.25×10~7)3个剂量组的HepZJ细胞悬液和空白对照溶液经尾静脉单次注射进入SD大鼠体内,观察大鼠的临床症状和体质量变化,注射结束后第1天以及第14天剖检进行血液学相关检验、大体病理和免疫组化检查,综合分析该细胞系的毒副反应和安全剂量。结果与结论:在HepZJ细胞急性毒性研究中,高剂量组大鼠注射结束时出现1只死亡,与对照组比较,血常规、凝血功能和血清生化部分指标差异有显著性意义(P <0.05);14 d后各项检查差异无显著性意义(P> 0.05)。低剂量组和中剂量组大鼠与对照组在临床症状、体质量变化、血液指标和大体病理形态上差异均无显著性意义。结果表明,永生化人肝细胞系HepZJ单次注射安全剂量为5×10~7/只,无明显毒副反应,剂量过大时可出现明显毒副反应,包括炎症应激、凝血障碍和肝功能损伤等,甚至死亡。因此,只有准确控制HepZJ的使用剂量,才能保证该细胞的临床应用安全性。
BACKGROUND: Hepatocyte transplantation, liver tissue engineering and biological artificial liver have brought good news to liver failure patients, but there are still no very appropriate seed cells so far. Our laboratory has built a human immortalized liver cell line named Hep ZJ as new seed cells, and a preclinical safety evaluation has been implemented.OBJECTIVE: To complete the toxicity study of the new immortalized hepatocyte cell line Hep ZJ so as to provide reference for predicting possible toxic and adverse effects and designing safe dose of HepZJ in clinical practice.METHODS: In the acute toxicity test, the Hep ZJ cell suspension of low, medium, high dose groups(5×10~6, 5×10~7, 8.25×10~7) and normal saline of control group were injected into the Sprague-Dawley rats through the caudal vein, respectively. The clinical symptoms of rats and its weight change were then observed. Anatomical examination was performed at 1 and 14 days after injection for hematological, gross pathological and immunohistochemical analyses. The adverse reactions and safety dosage of the cell line were analyzed comprehensively.RESULTS AND CONCLUSION: In the acute toxicity test of HepZJ cells, one rat in the high dose group died just after injection. Compared with the control group, parts of indexes in blood routine, clotting function and serum biochemical examination showed significant difference(P< 0.05) in the high dose group, while there was no significant difference at 14 days after injection. The experimental rats in the low dose group and medium dose group showed no obvious difference in clinical symptoms, body mass, blood indexes and gross pathological examination as compared with the control group(P > 0.05). To conclude, the safe dose of HepZJ is 5×10~7 cells for each injection, having no obvious adverse reactions. A larger dose may lead to obvious adverse reactions, such as inflammatory stress, clotting disorders, liver dysfunction and even death. Therefore, to accurately control the dose of Hep ZJ is the guarantee for the clinical safety of Hep ZJ.
BACKGROUND: Hepatocyte transplantation, liver tissue engineering and biological artificial liver have brought good news to liver failure patients, but there are still no very appropriate seed cells so far. Our laboratory has built a human immortalized liver cell line named Hep ZJ as new seed cells, and a preclinical safety evaluation has been implemented.OBJECTIVE: To complete the toxicity study of the new immortalized hepatocyte cell line Hep ZJ so as to provide reference for predicting possible toxic and adverse effects and designing safe dose of HepZJ in clinical practice.METHODS: In the acute toxicity test, the Hep ZJ cell suspension of low, medium, high dose groups(5×10~6, 5×10~7, 8.25×10~7) and normal saline of control group were injected into the Sprague-Dawley rats through the caudal vein, respectively. The clinical symptoms of rats and its weight change were then observed. Anatomical examination was performed at 1 and 14 days after injection for hematological, gross pathological and immunohistochemical analyses. The adverse reactions and safety dosage of the cell line were analyzed comprehensively.RESULTS AND CONCLUSION: In the acute toxicity test of HepZJ cells, one rat in the high dose group died just after injection. Compared with the control group, parts of indexes in blood routine, clotting function and serum biochemical examination showed significant difference(P< 0.05) in the high dose group, while there was no significant difference at 14 days after injection. The experimental rats in the low dose group and medium dose group showed no obvious difference in clinical symptoms, body mass, blood indexes and gross pathological examination as compared with the control group(P > 0.05). To conclude, the safe dose of HepZJ is 5×10~7 cells for each injection, having no obvious adverse reactions. A larger dose may lead to obvious adverse reactions, such as inflammatory stress, clotting disorders, liver dysfunction and even death. Therefore, to accurately control the dose of Hep ZJ is the guarantee for the clinical safety of Hep ZJ.
引文
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[17]Ra JC,Shin IS,Kim SH,et al.Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans.Stem Cells Dev.2011;20(8):1297-1308.
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[21]Long C,Yang J,Yang H,et al.Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant,anti?inflammatory,and anti?apoptotic activities.Mol Med Rep.2016;13(6):4697-4704.
[22]国家食品药品监督管理局药品审评中心.治疗用生物制品非临床安全性评价指导原则[EB/OL].http://www.cde.org.cn/zdyz.do?method=largePage&id=100,2010-05-06/2018-10-29.
[23]Stacey GN,Coecke S,Price AB,et al.Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.Adv Exp Med Biol.2016;856:259-297.
[24]Van Meer PJ,Graham ML,Schuurman HJ.The safety,efficacy and regulatory triangle in drug development:Impact for animal models and the use of animals.Eur J Pharmacol.2015;759:3-13.
[25]Misik J,Pavlikova R,Cabal J,et al.Acute toxicity of some nerve agents and pesticides in rats.Drug Chem Toxicol.2015;38(1):32-36.
[26]Christapher PV,Parasuraman S,Asmawi MZ,et al.Acute and subchronic toxicity studies of methanol extract of Polygonum minus leaves in Sprague Dawley rats.Regul Toxicol Pharmacol.2017;86:33-41.
[27]Xu S,Zhang Z,Chu M.Long-term toxicity of reduced graphene oxide nanosheets:Effects on female mouse reproductive ability and offspring development.Biomaterials.2015;54:188-200.
[28]Kuroda T,Yasuda S,Sato Y.Tumorigenicity studies for human pluripotent stem cell-derived products.Biol Pharm Bull.2013;36(2):189-192.
[29]De Matteis V.Exposure to Inorganic Nanoparticles:Routes of Entry,Immune Response,Biodistribution and In Vitro/In Vivo Toxicity Evaluation.Toxics.2017;5(4):E29.
[30]曾颖星,郑永霞,徐艳雯,等.肝细胞移植治疗肝病的研究进展[J].医学综述,2018,24(7):1344-1348.
[31]You S,Zhu B,Liu H,et al.Safety of Human Hepatoma Cell-Line Constructing Bioartificial Liver Supporting System Treating Patients with Liver Failure.Hepatogastroenterology.2014;61(132):933-936.
[32]Yun JW,Ahn JH,Kwon E,et al.Human umbilical cord-derived mesenchymal stem cells in acute liver injury:Hepatoprotective efficacy,subchronic toxicity,tumorigenicity,and biodistribution.Regul Toxicol Pharmacol.2016;81:437-447.
[33]国家食品药品监督管理总局药品审评中心.人体细胞治疗研究和制剂质量控制技术指导原则[EB/OL].http://www.cde.org.cn/zdyz.do?method=largePage&id=38,2008-09-04/2018-10-29.
[34]Food and Drug Administration,HHS.International Conference on Harmonisation;addendum to International Conference on Harmonisation Guidance on S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals;availability.Notice.Fed Regist.2012;77(97):29665-29666.
[35]国家食品药品监督管理总局药品审评中心.细胞制品研究与评价技术指导原则(征求意见稿)[EB/OL].http://www.cde.org.cn/news.do?method=largeInfo&id=313749,2016-12-19/2018-10-29.
[36]国家食品药品监督管理总局.《药物非临床研究质量管理规范》(国家食品药品监督管理总局令第34号)[EB/OL].http://www.gov.cn/gongbao/content/2017/content_5241929.htm,2017-08-02/2018-10-29.
[37]He J,Ruan GP,Yao X,et al.Chronic Toxicity Test in Cynomolgus Monkeys For 98 Days with Repeated Intravenous Infusion of Cynomolgus Umbilical Cord Mesenchymal Stem Cells.Cell Physiol Biochem.2017;43(3):891-904.
[38]M?kel?T,Takalo R,Arvola O,et al.Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model.Cytotherapy.2015;17(4):392-402.
[39]Lee RH,Pulin AA,Seo MJ,et al.Intravenous h MSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6.Cell Stem Cell.2009;5(1):54-63.
[40]汪溪洁,马璟.药物安全性评价新技术和新方法研究进展[J].中国医药工业杂志,2017,48(3):341-350.
[41]Kandhare AD,Bodhankar SL,Mohan V,et al.Acute and repeated doses(28 days)oral toxicity study of Vicenin-1,a flavonoid glycoside isolated from fenugreek seeds in laboratory mice.Regul Toxicol Pharmacol.2016;81:522-531.
[2]丁易涛,施晓雷.生物人工肝脏临床研究现状[J].中国研究型医院,2017,4(5):17-25.
[3]Sakiyama R,Blau BJ,Miki T.Clinical translation of bioartificial liver support systems with human pluripotent stem cell-derived hepatic cells.World J Gastroenterol.2017;23(11):1974-1979.
[4]Forbes SJ,Gupta S,Dhawan A.Cell therapy for liver disease:From liver transplantation to cell factory.J Hepatol.2015;62(1 Suppl):S157-169.
[5]Kim Y,Kang K,Yoon S,et al.Prolongation of liver-specific function for primary hepatocytes maintenance in 3D printed architectures.Organogenesis.2018;14(1):1-12.
[6]Lancett P,Williamson B,Barton P,et al.Development and Characterization of a Human Hepatocyte Low Intrinsic Clearance Assay for Use in Drug Discovery.Drug Metab Dispos.2018;46(8):1169-1178.
[7]Fang ZP,Jiang BG,Gu XF,et al.P21-activated kinase 5 plays essential roles in the proliferation and tumorigenicity of human hepatocellular carcinoma.Acta Pharmacol Sin.2014;35(1):82-88.
[8]van Wenum M,Treskes P,Tang CY,et al.Scaling-up of a HepaRGprogenitor cell based bioartificial liver:optimization for clinical application and transport.Biofabrication.2017;9(3):035001.
[9]Layton DS,Bean AG,Dodge NM,et al.Differential cytokine expression and regulation of human anti-pig xenogeneic responses by modified porcine dendritic cells.Xenotransplantation.2008;15(4):257-267.
[10]石伟,蔡端,张群华,等.异种肝细胞移植缓解大鼠急性肝功能衰竭及其排斥反应观察[J].中华肝胆外科杂志,2005,11(4):265-268.
[11]Yang G,Hong H,Torres A,et al.Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells,Neural Stem Cells and Dopaminergic Lineage.Int J Mol Sci.2018;19(9):E2788.
[12]Steens J,Klein D.Current Strategies to Generate Human Mesenchymal Stem Cells In Vitro.Stem Cells Int.2018;2018:6726185.
[13]Hansel MC,Gramignoli R,Blake W,et al.Increased reprogramming of human fetal hepatocytes compared with adult hepatocytes in feeder-free conditions.Cell Transplant.2014;23(1):27-38.
[14]Crombleholme TM,Langer JC,Harrison MR,et al.Transplantation of fetal cells.Am J Obstet Gynecol.1991;164(1 Pt 1):218-230.
[15]Zhang Y,Shi J,Liu S.Establishment and Characterization of a Telomerase-Immortalized Sheep Trophoblast Cell Line.Biomed Res Int.2016;2016:5808575.
[16]卢扬洲,黎少,姜华,等.新型永生化人肝细胞系HepZJ的安全性研究[J].药物评价研究,2018,41(6):1062-1067.
[17]Ra JC,Shin IS,Kim SH,et al.Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans.Stem Cells Dev.2011;20(8):1297-1308.
[18]王琼,汤钱球,张小云,等.炎症和凝血指标在脓毒血症诊断和预后评估中价值分析[J].临床血液学杂志(输血与检验),2018,31(5):750-753.
[19]Thorell SE,Nash MJ,Thachil J.Clinical implications of clotting screens.Int J Lab Hematol.2015;37(1):8-13.
[20]Eschbach D,Horst K,Sassen M,et al.Hypothermia does not influence liver damage and function in a porcine polytrauma model.Technol Health Care.2018;26(2):209-221.
[21]Long C,Yang J,Yang H,et al.Attenuation of renal ischemia/reperfusion injury by oleanolic acid preconditioning via its antioxidant,anti?inflammatory,and anti?apoptotic activities.Mol Med Rep.2016;13(6):4697-4704.
[22]国家食品药品监督管理局药品审评中心.治疗用生物制品非临床安全性评价指导原则[EB/OL].http://www.cde.org.cn/zdyz.do?method=largePage&id=100,2010-05-06/2018-10-29.
[23]Stacey GN,Coecke S,Price AB,et al.Ensuring the Quality of Stem Cell-Derived In Vitro Models for Toxicity Testing.Adv Exp Med Biol.2016;856:259-297.
[24]Van Meer PJ,Graham ML,Schuurman HJ.The safety,efficacy and regulatory triangle in drug development:Impact for animal models and the use of animals.Eur J Pharmacol.2015;759:3-13.
[25]Misik J,Pavlikova R,Cabal J,et al.Acute toxicity of some nerve agents and pesticides in rats.Drug Chem Toxicol.2015;38(1):32-36.
[26]Christapher PV,Parasuraman S,Asmawi MZ,et al.Acute and subchronic toxicity studies of methanol extract of Polygonum minus leaves in Sprague Dawley rats.Regul Toxicol Pharmacol.2017;86:33-41.
[27]Xu S,Zhang Z,Chu M.Long-term toxicity of reduced graphene oxide nanosheets:Effects on female mouse reproductive ability and offspring development.Biomaterials.2015;54:188-200.
[28]Kuroda T,Yasuda S,Sato Y.Tumorigenicity studies for human pluripotent stem cell-derived products.Biol Pharm Bull.2013;36(2):189-192.
[29]De Matteis V.Exposure to Inorganic Nanoparticles:Routes of Entry,Immune Response,Biodistribution and In Vitro/In Vivo Toxicity Evaluation.Toxics.2017;5(4):E29.
[30]曾颖星,郑永霞,徐艳雯,等.肝细胞移植治疗肝病的研究进展[J].医学综述,2018,24(7):1344-1348.
[31]You S,Zhu B,Liu H,et al.Safety of Human Hepatoma Cell-Line Constructing Bioartificial Liver Supporting System Treating Patients with Liver Failure.Hepatogastroenterology.2014;61(132):933-936.
[32]Yun JW,Ahn JH,Kwon E,et al.Human umbilical cord-derived mesenchymal stem cells in acute liver injury:Hepatoprotective efficacy,subchronic toxicity,tumorigenicity,and biodistribution.Regul Toxicol Pharmacol.2016;81:437-447.
[33]国家食品药品监督管理总局药品审评中心.人体细胞治疗研究和制剂质量控制技术指导原则[EB/OL].http://www.cde.org.cn/zdyz.do?method=largePage&id=38,2008-09-04/2018-10-29.
[34]Food and Drug Administration,HHS.International Conference on Harmonisation;addendum to International Conference on Harmonisation Guidance on S6 Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals;availability.Notice.Fed Regist.2012;77(97):29665-29666.
[35]国家食品药品监督管理总局药品审评中心.细胞制品研究与评价技术指导原则(征求意见稿)[EB/OL].http://www.cde.org.cn/news.do?method=largeInfo&id=313749,2016-12-19/2018-10-29.
[36]国家食品药品监督管理总局.《药物非临床研究质量管理规范》(国家食品药品监督管理总局令第34号)[EB/OL].http://www.gov.cn/gongbao/content/2017/content_5241929.htm,2017-08-02/2018-10-29.
[37]He J,Ruan GP,Yao X,et al.Chronic Toxicity Test in Cynomolgus Monkeys For 98 Days with Repeated Intravenous Infusion of Cynomolgus Umbilical Cord Mesenchymal Stem Cells.Cell Physiol Biochem.2017;43(3):891-904.
[38]M?kel?T,Takalo R,Arvola O,et al.Safety and biodistribution study of bone marrow-derived mesenchymal stromal cells and mononuclear cells and the impact of the administration route in an intact porcine model.Cytotherapy.2015;17(4):392-402.
[39]Lee RH,Pulin AA,Seo MJ,et al.Intravenous h MSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6.Cell Stem Cell.2009;5(1):54-63.
[40]汪溪洁,马璟.药物安全性评价新技术和新方法研究进展[J].中国医药工业杂志,2017,48(3):341-350.
[41]Kandhare AD,Bodhankar SL,Mohan V,et al.Acute and repeated doses(28 days)oral toxicity study of Vicenin-1,a flavonoid glycoside isolated from fenugreek seeds in laboratory mice.Regul Toxicol Pharmacol.2016;81:522-531.