永生化人肝细胞HepZJ的临床前急性毒性评价

Pre-clinical acute toxicity of an immortalized human hepatocyte cell line HepZJ

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.