摘要
目的:观察67Ga-Oxine标记间充质干细胞的可行性,检测经静脉移植的间充质干细胞在脊髓损伤鼠体内的迁移和分布。方法:实验于2004-01/2005-01在北京大学干细胞研究中心完成。①从人脐带血分离培养间充质干细胞,体外进行67Ga-Oxine和Hoechst33342标记。②SD大鼠40只随机分为两组,实验组(脊髓损伤大鼠20只)和对照组(正常大鼠20只)均经尾静脉注射相同标记细胞悬液。③分别于1d和1周后取材,新鲜组织(脊髓、肺、肝、脾、肾、骨髓、静脉血)称湿重后伽玛计数仪检测其放射性,计算肺、肝、脾、肾、骨髓分别与静脉血单位质量放射性计数的比值,两组比较分析;实验组损伤脊髓和对照组相同部位脊髓分别与其临近正常脊髓放射性计数的比值之间比较。相应组织做冷冻切片(包括骨髓涂片),荧光显微镜观察Hoechst33342标记细胞在各组织内的分布。结果:40只SD大鼠全部进入结果分析。①体外间充质干细胞的67Ga-Oxine标记率为82.5%,放射活性为15.26MBq/106细胞,标记48h后细胞存活率>95%。②移植1d和1周后肺、肝、脾、肾、骨髓与静脉血单位质量放射性计数的比值,实验组与对照组对比差异均无显著性(P>0.05)。实验组损伤脊髓和对照组相同部位脊髓分别与其临近正常脊髓放射性计数的比值之间比较,1d后差异无显著性(P>0.05),1周后实验组明显高于对照组(1.441±0.038,1.003±0.022,P<0.01)。③相应组织连续冷冻切片荧光显微镜观察显示移植初期Hoechst33342标记细胞主要集中于肺部,而后在肝、脾、骨髓逐渐增加可持续到1周后。移植1d后脊髓未见明显标记细胞,1周后实验组损伤脊髓部位标记细胞明显增加并相对聚集于灰质。结论:间充质干细胞可在体外应用67Ga-Oxine成功标记,为核素显像示踪移植间充质干细胞的相关研究提供了依据。经静脉移植的间充质干细胞可以迁移到损伤脊髓,为应用间充质干细胞移植治疗脊髓损伤提供了一种可能的无创性移植方法。
AIM： To explore the feasibility of ^67Ga-Oxine labeling of mesenchymal stem cells （MSCs）, and determine MSCs distribution and migration after intravenous transplantation in spinal cord-injured rats. METHODS： The experiment was conducted in the Stem Cells Research Center of Peking University between January 2004 and January 2005. （1） MSCs were isolated from human umbilical cord blood and labeled with 67Ga-Oxine and Hoechst33342 in vitro. （2） Forty SD rats were randomly divided into 2 groups： Experiment group （subjected to spinal cord injury, n=20） and control group （normal rats, n=20）, which were injected the same ^67Ga-Oxine labeled MSCs suspension into tail vein. （3）Rats were killed at 1 day and 1 week after transplantation. The wet weights and radioactivity of various fresh tissues （spinal cord, lung, liver, spleen, kidney, bone marrow, and venous blood） were measured with Gamma Count Meter to calculate the ratios of radioactivity in unit weight of lung, liver, spleen, kidney and bone marrow to venous blood, respectively. The ratios of radioactivity of injured spinal cord and the normal spinal cord at the same site of the control group to adjacent normal spinal cord were also determined, respectively. The ratios were compared between the two groups. The various tissues were sectioned on a freezing microtome to observe the distribution of Hoechst33342-1abeling cells in each tissues by Olympus fluorescent microscope. RESULTS： All the 40 rats were involved in the result analysis; （1）MSCs labeling efficiency with ^67Ga-Oxine was 82.5% in vitro, resulting in an radioactivity of approximately 15.26 MBq/10^6 cells; The cells survival rate was 〉 95% tested at 48 hours after labeling. （2）The ratios of radioactivity in unit weight of lung, liver, spleen, bone marrow and kidney to venous blood respectively in the experiment group had no significant difference compared with the control group at 1 day and 1 week after transplantation （P 〉 0.05）. The ratios of radioactivity of injured spinal cord to adjacent normal spinal cord of the experiment group had no obvious differences at 1 day after transplantation （P 〉 0.05）, but were significantly higher at 1 week after transplantation compared with the control group （1.441±0.038, 1.003±0.022, P 〈 0.01）. （3）Fluorescent observation of sections showed that high initial MSCs with Hoechst33342 labeling occurred in the lungs and increased in the livers, spleens and bone marrows, which could last for 1 week. Appreciable accumulation of cells with label were not found in the spinal cord at 1 day after transplantation, but occurred in the injured spinal cord of the experiment group and mainly gathered in gray matter 1 week later. CONCLUSION： ^67Ga-Oxine radiolabelling of MSCs is feasible, and can provides evidence for radionuclide imaging to track implanted MSCs. MSCs may migrate to the injured spinal cord after intravenous transplantation, which provides a non-invasive method for treatment of spinal cord injury with MSCs transplantation.
出处
《中国临床康复》
CSCD
北大核心
2006年第25期30-33,i0002,共5页
Chinese Journal of Clinical Rehabilitation
基金
国家高技术研究发展计划(2002AA205081)
北京市科技计划项目(H020220010390)~~
