扩增活化的淋巴细胞在小鼠体内的生物分布评价

Biological distribution study of expanded activated lymphocytes in C57BL/6 mice

目的:研究免疫细胞治疗产品EAL细胞在小鼠体内的分布特征,为其临床应用提供安全性依据。方法:活体成像检测:采用C57BL/6小鼠,分阴性对照组和供试品组,阴性对照组(尾静脉注射,iv)给予生理盐水,供试品组给予DiR标记的EAL细胞,iv单次给药,于3 h及1,3,6,9,15和21 d 7个时间点,麻醉动物后进行活体成像,检测DiR标记的细胞在小鼠体内的分布。流式细胞仪检测:采用C57BL/6小鼠,分阴性对照组和供试品组,阴性对照组给予非转基因鼠源的EAL细胞,供试品组给予GFP转基因鼠源的EAL细胞,iv单次给药,于3 h及1,3,6,9,15和21 d 7个时间点麻醉解剖,依次取外周血、脾脏、肝脏、肺脏、胸腺、淋巴结、肾脏、骨髓,流式细胞仪检测GFP^+EAL细胞在上述组织器官中所占的比例及分布数量。结果:EAL细胞注射进入动物体内后,其分布水平在给药后3 h至给药后3 d处于较高水平,随时间延长分布水平逐步降低,在给药后d 21可以降低至约峰值的65%以上,分布较高的脏器为肝脏、肺脏、脾脏、淋巴结和外周血液,其他器官有少量分布;同时,流式细胞检测及活体成像检测结果具有较好的一致性。结论:EAL细胞回输小鼠后,在体内不会广泛分布和长期存续,具有较好的临床前安全性。

Objective: To investigate the biodistribution of EAL and to provide safety basis for its clinical application. Methods: In vivo imaging detection: C57 BL/6 mice were randomized into a negative control group which was given normal saline( tail vein injection) and EAL group which was administrated with EAL intravenously.In vivo bioluminescence imaging: The distribution of the DiR marked cells in mice was detected after mice were anesthetized using living imaging apparatus at 3 h and 1,3,6,9,15,21 d separately. Flow cytometry( FCM):C57 BL/6 mice were randomized into a negative control group and experimental group. The negative control group was given EAL cells derived from non-transgenic mice,and the experimental group was given EAL cells derived from GFP transgenic mice( i. v. single dose). After mice were anesthetized at 3 h and 1,3,6,9,15 and 21 d,lymph nodes were taken firstly. Then,thymuses were taken after completely blood collection. Heart were exposed and perfused with Sodium Chloride 0. 9%. Finally,spleen,liver,lung,kidney and bone marrow were taken oneby one. By flow cytometer,the proportion and amount of GFP + EAL were detected in these tissues. Results:After,high levels of EAL cells were found in liver,lungs,spleen,lymph nodes,and peripheral blood 3 h to 3 d after the EAL cells were injected into the mice. After day 3,EAL levels decreased gradually with time,and fell to about 65% of the peak value at day 21 after administration. EALs in kidney,bone marrow and thymus remained a relatively low level at each detection point of time. The results of the living imaging and flow cytometry were comparable.Conclusion: After EALs cells were transferred back to the mice,they were not widely distributed or long-lasting in the body,which had good preclinical safety.