ADSC移植对大鼠大脑中动脉闭塞脑梗死模型梗死灶周围DCC和NF-200表达的影响

Effect of ADSC transplantation on the expression of DCC and NF-200 in peri-infarct cortex of rat model of middle cerebral artery occlusion

目的分析脂肪来源干细胞(ADSC)移植对大鼠大脑中动脉闭塞脑梗死模型梗死灶周围神经丝蛋白200(NF-200)和结直肠癌缺失蛋白(DCC)表达的影响。方法取36只SD大鼠,其中30只用于构建脑梗死大鼠模型,随机分为假手术组(不插入线栓,注入生理盐水)、脑梗死组(插入线栓,注入生理盐水)、移植组(插入线栓,注入ADSC悬液),三组各10只;剩余6只用于分离培养ADSC。分别于造模后第1、2周,采用荧光显微镜观察ADSC在大鼠脑组织中的迁移情况;通过免疫印迹法测定NF-200和DCC蛋白在大鼠脑梗死病灶周围皮质中的表达情况;采用免疫荧光法测定NF-200和DCC在大鼠脑梗死病灶周围皮质中的分布情况。激光扫描共聚焦显微镜下观察DCC蛋白在梗死灶周围皮质中的定位。结果经荧光显微镜观察可见,移植组脑组织中可见PKH-26标记的ADSC,且多见于脑梗死周围皮质中。经免疫印迹法检测可知,相比假手术组,脑梗死组造模后第1、2周脑梗死病灶周围皮质中NF-200蛋白表达明显下调,DCC蛋白表达明显上调(P<0.05);与假手术组比较,移植组造模后第1周脑梗死病灶周围皮质中NF-200蛋白表达明显下调(P<0.05),造模后第1、2周DCC蛋白表达明显上调(P<0.05);移植组与假手术组造模后第2周NF-200表达的比较,并无显著差异(P>0.05);相比脑梗死组,移植组造模后第1、2周脑梗死病灶周围皮质中NF-200和DCC蛋白表达水平均显著上调(P<0.05)。经免疫荧光组织化学染色法检测可知,脑梗死组与移植组脑梗死病灶周围皮质中NF-200和DCC蛋白的表达较假手术组更加明显,且移植组更加明显;NF-200蛋白在大鼠脑梗死病灶周围皮质中主要定位于神经元轴突中,DCC蛋白呈现条索状表达。激光扫描共聚焦显微镜下观察可见,DCC蛋白主要定位于大鼠脑梗死病灶周围皮质中NF-200阳性的神经元轴突中。结论通过颈动脉注射途径移植的ADSC可移至大鼠脑梗死病灶周围皮质中,且其促进神经元轴突再生修复的作用机制可能与诱导脑梗死病灶周围皮质NF-200和DCC表达密切相关。

Objective To analyze the effect of adipose-derived stem cells(ADSC)transplantation on the expression of neurofilament-200(NF-200)and deleted in colorectal cancer(DCC)in peri-infarct cortex of rats with middle cerebral artery occlusion.Methods Thirty-six SD rats were randomly divided into three groups:sham operation group(no thread embolus was inserted,normal saline was injected),cerebral infarction group(thread embolus was inserted,normal saline was injected),and transplantation group(thread embolus was inserted,ADSC suspension was injected),10 rats in each group;the remaining six rats were used for isolation and culture of ADSC.The migration of ADSC in rat brain tissue was observed under fluorescence microscopy at the first and second week after model establishment.The expression of NF-200 and DCC protein in peri-infarct cortex was measured by Western blot analysis.The distribution of NF-200 and DCC in peri-infarct cortex was measured by immunofluorescence.Localization of DCC protein in the cortex around the infarct was observed using a laser scanning confocal microscope.Results The ADSCs labeled with PKH-26 were found in the brain tissues of the transplantation group by fluorescence microscopy,and most of them were located in the peri-infarct cortex.The expression level of NF-200 in the transplantation group was significantly lower than that in the sham group at the first week after model establishment,the expression of DCC was significantly higher at the first and second model establishment(P<0.05),but there was no significant difference in the expression of NF-200 in the transplantation group and the sham group at 2nd week after modeling(P>0.05).The expression level of NF-200 and DCC in the transplantation group was significantly higher than that in the cerebral infarction group(P<0.05).Immunofluorescence histochemical staining showed that the expressions of NF-200 and DCC protein in the peri-infarct cortex in cerebral infarction group and transplantation group were more obvious than those in the sham group,and the expression of NF-200 protein in the peri-infarct cortex of rats was mainly located in the axons of neurons,and DCC protein showed strip-like expression.Laser scanning confocal microscopy showed that DCC protein was mainly located in the axons of NF-200 positive neurons in the peri-infarct cortex of rats.Conclusion The ADSCs can migrate to the peri-infarct cortex after transplantation via carotid artery,and may strengthen the axonal regeneration and repairment by promoting the expression of DCC and NF-200 in the peri-infarct cortex.