脑缺血再灌注损伤后BMSCs不同示踪方式的比较

Comparison of different ways to trace BMSCs after cerebral ischemia-reperfusion injury

目的比较脑缺血再灌注损伤后,不同示踪方式观察骨髓间充质干细胞(BMSCs)移植后的效果。方法BMSCs复苏培养后,分别采用5-溴脱氧尿嘧啶核苷(BrdU)、PKH26进行标记,并且与绿色荧光蛋白(GFP)转染细胞,分别对脑缺血再灌注模型大鼠脑组织进行移植。将75只SPF级雄性SD大鼠随机均分为假手术组,模型组,BrdU、PKH26、GFP示踪组。采用线栓改良法制备大鼠左侧大脑中动脉局灶性脑缺血再灌注损伤模型。脑缺血再灌注24h后,假手术组、模型组分别采用脑立体定位仪经脑实质植入10μL生理盐水;BrdU、PKH26、GFP示踪组分别植入10μL的BMSCs/BrdU、BMSCs/PKH26、BMSCs/GFP。采用神经行为学评分、TTC染色、脑组织含水量法进行模型鉴定及疗效判定,采用焦油紫染色进行神经元计数,并在荧光显微镜下计数标记细胞阳性率。结果移植前细胞BrdU、PKH26、GFP标记率无明显差异。移植4周后3示踪组大鼠神经行为学评分、脑梗死体积、脑组织含水量均显著低于模型组,神经元计数显著高于模型组;神经行为学评分、脑梗死体积高于假手术组,脑组织含水量、神经元计数与假手术组差异无统计学意义;3示踪组间上述指标差异均无统计学意义。GFP示踪组的荧光标记细胞阳性率高于BrdU、PKH26示踪组。结论脑缺血再灌注损伤中,GFP示踪方式效果更为持久,优于BrdU和PKH26。

Objective To compare different ways to trace bone marrow mesenchymal stem cells （BMSCs） after being transplanted in cerebral ischemia-reperfusion injury. Methods Male SD rats of SPF grade were randomly divided into sham group, model group （ischemia-reperfusion,IR）, BrdU tracing group, PKH26 tracing group and GFP tracing group. Focal cerebral ischemia-reperfusion model was established by blocking middle cerebral artery. 24 hours after cerebral ischemia-reperfusion injury, 10μL BMSCs that were labeled respectively by BrdU, PKH26, GFP were added respectively into BrdU, PKH26 and GFP tracing group while equal volum of normal saline was added into sham group and model group. Model and transplanting cells efficacy was determined by neural behavioral score, TTC staining and brain water content;Neurons were counted using tar violet staining;The number of transplant cells in the transplanting site was assessed by fluorescence microscopy. Results Before transplanting, there was no significant difference among BrdU, PKH26 and GFP group in cell labeled efficacy. By contrast, neural behavioral score, brain infarct volume and brain tissue water content were significantly lower in all three tracing groups than that in model group 4 weeks after transplantation while neuron counts were markedly higher. There was no significant difference of above parameters among the three tracing groups. However, the number of traced transplanting cells in damaging area in GFP group is significantly higher than that in BrdU group and PKH26 group. Conclusion In cerebral ischemia-reperfusion injury, the tracing effect of GFP last longer, therefore it is significantly more effective than BrdU and PKH26.