CM-Dil体外标记骨髓间充质干细胞及其在烧伤大鼠模型肠组织内的示踪

Labeling bone marrow mesenchymal stem cells by CM-Dil and tracking in intestine tissue of burned rat models

目的:探讨氯甲基苯甲酰胺(CM-Dil)对骨髓间充质干细胞(BMSCs)的体外标记及其在烧伤大鼠模型肠组织内的示踪能力。方法:采用贴壁培养法分离、纯化、扩增BMSCs。碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)和维甲酸(RA)诱导BMSCs向神经元样细胞分化,免疫细胞化学法检测神经元表面标志神经细胞特异性标志微管相关蛋白-2(MAP-2)和神经元特异核蛋白(NeuN)的表达。4、6和8 mg·L-1CM-Dil标记BMSCs,CCK-8法检测不同浓度CM-Dil对细胞的毒性作用,流式细胞仪检测其标记率。选用成年雄性Wistar大鼠,制备烧伤大鼠模型,经球后静脉注射1×107个CM-Dil标记的BMSCs。大鼠烧伤2周及6个月后取肠组织,制备冰冻切片,用激光共聚焦显微镜观察BMSCs在大鼠肠组织内的定植情况。结果:免疫细胞化学结果表明,BMSCs诱导分化的神经元样细胞表达NeuN和MAP-2。CCK-8法结果表明,8 mg·L-1CM-Dil组细胞毒性显著高于对照组(P<0.05),4和6 mg·L-1CM-Dil与对照组比较差异无统计学意义(P>0.05)。4 mg·L-1CM-Dil标记BMSCs对细胞无毒性,标记率达93.9%,细胞形态无改变。冰冻切片激光共聚焦显微镜观察,CM-Dil标记的BMSCs在大鼠烧伤后2周及6个月的肠组织内定植。结论:CM-Dil可以体外标记BMSCs,可以用于BMSCs在烧伤大鼠模型肠组织组织内的示踪研究。

Objective To explore the cell labeling ability of CM-Dil on bone marrwow mesenchymal stem cells（BMSCs） in vitro as well as its tracking capability in intestine tissues of burned rats.Methods BMSCs were isolated,purified and expanded by the method of adherent culture,and were induced to differentiate into neuron-like cells by basic fibroblast growth factor（bFGF）,epidermal growth factor（EGF） and retinoic acid（RA）.The surface markers of neuron-like cells differentiated from BMSCs,microtubule-associated protein-2（MAP-2） and neuron specific nuclear protein（NeuN） were detected by immunocytochemistry.The BMSCs were labeled with CM-Dil at the doses of 4,6,8 mg·L^-1.Then the cytotoxic effect was determined by CCK-8 assay,the labeling rate was examined using flow cytometry.107 BMSCs labeled by CM-Dil were administrated by retro-orbital intravenous injection after burned models of Wistar rats were prepared.The intestine tissues were obtained and fixed on the 2 weeks and 6 months after the burning,then the engraftment of the labeled BMSCs were observed using a laser scanning confocal microscope.Results The nueon-like cells differentiated from BMSCs expressed neuron markers,NeuN and MAP-2.The results of CCK-8 assay showed that compared with control group,the cell viability in 8 mg·L^-1 group was significantly decreased（P〈0.05）.There were no significantly differences between 4,6 μg·mL-1 groups and control group.No significant adverse effects on BMSCs were observed during CM-Dil labeling at the dose of 4 mg·L-1,and the labeling rate was as high as 93.9%.The morphological changes of the BMSCs were not shown.Frozen sections of intestine tissues were performed on the 2 weeks and 6 months after burning,the CM-Dil labeled BMSCs were engrafted at the rat intestine tissues.Conclusion CM-Dil is capable of labeling BMSCs in vitro,thus could be utilized as tracking of BMSCs in the intestine tissue of the burned rats.