In vivo distribution of luciferase gene-labeled bone marrow mesenchymal stem cells infused through different approaches

doi:10.3969/j.issn.2095-4344.2017.05.004

Abstract

Abstract:

BACKGROUND: Most bone marrow mesenchymal stem cells are infused intravenously and have very low efficiency of homing to the bone marrow. However, cell infusion via the femoral approach is little reported.
OBJECTIVE: To explore the distribution of luciferase gene modified red fluorescent protein transgenic bone marrow mesenchymal stem cells in vivo through different infusion routes.
METHODS: Luciferase gene modified bone marrow mesenchymal stem cells at different gradients (5×10⁶, 1×10⁶, 1×10⁵, 1×10⁴) were seeded or injected into the in vitro pore plate or free femurs to observe the fluorescence imaging and select the best concentration of cells. Luciferase gene modified bone marrow mesenchymal stem cells at the best cell concentration were injected into the mice via the femur and the tail vein, respectively. The distribution of fluorescence and cell number in the mice were explored by using bioluminescence, pathological examination, flow cytometry and quantitative PCR.
RESULTS AND CONCLUSION: Ex vivo fluorescence intensity of luciferase gene modified bone marrow mesenchymal stem cells was positively correlated with the cell concentration; fluorescent cells in vivo appeared in the femur first and then quickly spread to the lungs in the femur group, while fluorescent cells in the tail vein group spread to the lungs quickly after cell infusion. Fluorescent cells could be seen in the spleen, liver and other organs 24 hours later in the two groups. The distribution and migration of cells in mice could be observed successfully by bioluminescence; 5 minutes after cell infusion, the lungs of mice in the two groups began to emit fluorescence that could spread to the liver, spleen and other tissues 24 hours later, and the fluorescence intensity reached its peak after 15 minutes. The distribution of bone marrow mesenchymal stem cells in mice had no significant difference between the femur group and the tail vein group. To conclude, cell injection through the bone marrow cavity and tail vein fails to promote the homing of bone marrow mesenchymal stem cells to the bone marrow.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Tissue Engineering, Stem Cells, Femur

CLC Number:

R394.2

Sun Xiao-wei, Huang Hao, Zhou Yong-jun, Chen Xiao-li, Qiao Peng-xin, Zou Chun, Zhang Qiu-xia, Jiang Qian-li. In vivo distribution of luciferase gene-labeled bone marrow mesenchymal stem cells infused through different approaches[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 676-681.

Figures/Tables 2

2.1 实验动物数量分析纳入小鼠18只，均进入结果分析，无死亡和感染，最终计入结果分析的数量为18只。 2.2 RFP-BMSCs和NeoR-Luc-RFP-BMSCs生长情况 ①RFP-BMSCs：接种6 h后部分细胞已经贴壁，接种24 h后贴壁细胞呈多角形、短棒形或者圆形。第2天换液后贴壁细胞迅速增殖，呈纺锤形或梭形，部分细胞形成集落，增殖加快，形成大小不等的集落，集落融合成片，折光性强，细胞形态多为纺锤形或者多角形，接种3 d时细胞80%-90%融合，铺满瓶底，开始传代。传代培养24 h细胞完全贴壁生长，分布均匀，呈梭形，传代至第3代时，细胞形态均一，呈辐射状或者漩涡状；②NeoR-Luc-RFP- BMSCs生长情况同RFP-BMSCs。 2.3 细胞浓度梯度实验结果小动物活体成像仪结果显示：5×108 L-1细胞浓度孔没有荧光显示，其他3个细胞浓度孔(2.5×1011 L-1，5×1010 L-1，5×109 L-1)均可以产生荧光，并且随着细胞浓度的增加，荧光强度逐步增加，单独加细胞孔和单独加底物孔无荧光显示(图1)。 2.4 体外游离股骨中NeoR-Luc-RFP-BMSCs的荧光显像情况通过小动物活体成像仪可见，在实验组，2.5×1011 L-1组的股骨中央可见较强荧光，输注5×1010 L-1组的股骨远端可见微弱荧光，5×1010 L-1组的股骨内未见荧光显示；在空白组，股骨内未见荧光显示(图2)。说明使用生物发光法观察股骨中的荧光所需最佳细胞浓度2.5×1011 L-1。 2.5 不同途径NeoR-Luc-RFP-BMSCs的体内输注实验结果生物发光实验结果：注射NeoR-Luc-RFP-BMSCs与荧光素底物混合悬液5 min后，在股骨组，荧光首先出现在股骨注射部位，后迅速扩散至双肺；在尾静脉组，可见小鼠双肺处首先出现荧光，且两组小鼠肺部荧光强度无明显差异(图3)，15 min后双肺荧光强度达到高峰，股骨处荧光逐渐减弱。两组小鼠脏器病理切片结果：共聚焦显微镜观察结果显示：细胞输注后1 h股骨组和尾静脉组小鼠的肺脏出现大量红色荧光细胞，24 h后肝脏、脾脏亦可见红色荧光细胞出现，两组病理切片上红色荧光分布未见明显差异(图4)。流式细胞术结果：在细胞注射后1，24 h，两组小鼠肝、脾、肺和骨髓内RFP+细胞比例差异无显著性意义(表2)。 2.6 实时荧光定量PCR检测小鼠组织及器官中的RFP+细胞的表达在细胞注射后1，24 h，股骨组和尾静脉组小鼠组织、脏器中RFP+细胞的表达差异无显著性意义(表3)。"

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