体外培养人胸腺组织切片移植至裸鼠肌肉后的非临床安全性评价OA

OBJECTIVE To evaluate the toxicological safety and effects of human thymus tissue slices cultured in vitro and transplanted into the muscle of nude mice.METHODS ① The BALB/c nude mice were divided into a control group and a transplantation group.Each group underwent acute toxicity tests at 2 weeks post-operation,subacute toxicity tests at 16 weeks post-operation,and chronic toxicity tests at 26 weeks post-operation.The control group did not receive human thymus tissue slices,while the transplantation group had approximately 7.0 mg of human thymus tissue slices that were prepared in vitro implanted into the quadriceps femoris.The recovery of the transplantation site was observed post-operation,and the body weight,body temperature,and mental state of the nude mice were moni-tored.The proportions of clusters of differentiation 3(CD3)+T-cell receptor-β(TCR-β)+T lymphocytes in the peripheral blood of nude mice were monitored by flow cytometry.② Peripheral blood was collected from nude mice at 2,16,and 26 weeks post-transplantation to detect liver and kidney function-related indicators in the serum,including albumin,triglycerides,urea nitrogen,creatinine,total cholesterol,and total protein content,as well as the activities of aspartate aminotransferase and alanine aminotransferase.The levels of inflammatory factors interleukin-6(IL-6),IL-1β,and tumor necrosis factor-α(TNF-α)in the serum were measured by ELISA.The organ coefficients of the heart,liver,spleen,lung,kidney,brain,and intestine(small intestine and large intestine)were calculated.HE staining was used to evaluate the pathological changes in the heart,liver,spleen,lung,kidney,brain,and transplantation site.③ RT-qPCR was used to detect the mRNA expression levels of IL-6,IL-1β,IL-10 and TNF-α in the tissue at the transplantation site of nude mice.④ Immunohistochemical(IHC)staining was used to detect the pro-tein expressions of human thymic epithelial cell markers keratin 5(KRT5),KRT8 and KRT14 at the transplantation site,and RT-qPCR was used to detect the expression of human KRT5,KRT8 and KRT14 genes in the heart,liver,spleen,lung,kidney and brain.RESULTS ① Within 2 weeks of trans-plantation,the wounds at the transplantation site of nude mice completely healed without swelling,inflammatory exudate,or infection.Within 2 weeks of transplantation,the body temperature of nude mice in the transplantation group showed no significant changes compared with the control group.From 2 to 26 weeks post-operation,there were no significant changes in body weight or mental state scores between the transplantation group and the control group.Comped with the control group,the proportion of CD3+TCR-β+T lymphocytes in the transplantation group increased at 22 and 26 weeks after surgery.② At 2,16,and 26 weeks post-operation,there were no significant changes in liver and kidney function indicators in the serum of the transplantation group compared with the control group,including albumin,triglycerides,urea nitrogen,creatinine,total cholesterol,and total protein content,as well as the activities of aspartate aminotransferase and alanine aminotransferase,and the levels of TNF-α,IL-1β,and IL-6.There were no significant changes in the organ coefficients of the heart,liver,spleen,lung,kidney,brain,and intestine in the transplantation group.No pathological changes,such as abnormal hyperplasia,tumorigenesis or chronic inflammation,were found in the organs and at the transplantation site of the transplantation group.③ Compared with the control group,there were no significant changes in the mRNA expression levels of IL-6,IL-1β and TNF-α at the transplantation site of the transplantation group.IHC staining showed the expressions of KRT5,KRT8,and KRT14 proteins at the transplantation site,and RT-qPCR detected the mRNA expression of human thymic epithelial cell identity genes KRT5,KRT8 and KRT14 only at the transplantation site,with no expression in other tissues.CONCLUSION The transplantation of human thymus tissue slices into the quadriceps femoris of nude mice does not cause local or systemic acute or chronic inflammation,nor does it induce toxic side effects or functional damage to internal organs,and the transplanted thymus tissue slices remain long stable at the surgical site,suggesting that the heterotopic transplantation of human thymus tissue slices in vivo is a safe and non-toxic immune reconstitution strategy.