大鼠体神经-内脏神经吻合再生后脊髓前角异体神经元的分离和凝集素的表达变化及作用  

ISOLATION OF FOREIGN NEURONS IN CORNU ANTERIUS MEDULLAE SPINALIS AFTER ARTIFICAL SOMATIC-AUTONOMIC NERVE ANASTOMOSIS OF RATS AND STUDY OF EXPRESSION CHANGES OF AGRIN

作　　者：海波[1] 刘宇[2] 陈朝晖[1] 李伟[1] 钟伟[1] 肖传国[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院泌尿外科,武汉430022 [2]郑州大学基础医学院解剖学教研室,郑州450052

出　　处：《解剖学报》2007年第5期515-519,共5页Acta Anatomica Sinica

基　　金：国家重点基础研究发展计划(973计划)资助项目(2003CB515300)

摘　　要：目的研究大鼠体神经-内脏神经吻合以及体神经-体神经吻合再生后,凝集素(agrin)在脊髓前角运动神经元中的表达及其差异,探讨异体神经再生中参与突触形成和维持的相关因子。方法建立大鼠体神经-内脏神经吻合动物模型和体神经-体神经吻合动物模型,通过荧光染料逆行追踪、标记L4脊髓前角支配盆神经节的运动神经元(异体神经元)。流式细胞仪分选被标记细胞,用实时定量PCR测定分选细胞中agrin的mRNA水平。用荧光染料顺行追踪结合免疫荧光标记盆神经节处的新突触。结果模型鼠吻合再生4个月后,体神经能够再生并替代内脏神经长入盆神经节,并最终形成新的突触结构。在激光共焦显微镜下观察到了这种结构。应用荧光染料逆行追踪结合流式细胞分选技术能满意分离出L4脊髓前角的运动神经元。实时定量PCR测得agrin在各组分选神经元中均有表达,模型组中agrin表达水平较模型对照组和正常组有少许降低(P<0.05)。结论异体神经再生长入盆节后能够形成新的突触结构,agrin参与形成和维持这种突触结构,其表达量的少许下调,可能与支配的靶器官改变而引起相应内环境的改变有关。Objective To study the changes of agrin expression in spinal cord motor neurons after somatic-autonomic ventral root （VR） anastomosis and somatic-somatic VR anastomosis, and the factors related to the synapse formation of foreign nerve regeneration. Methods Artifical somatic-automomic reflex pathway and somatic nerve self-anastomosis were established in rats. Retrograde tracing was carried out to label the motor neurons, and the neurohs were isolated by flow cytometry. Real-time PCR was used to investigate the agrin mRNA expression of the neurons. Anteregrade tracing and technique were employed to label the new synapses of pelvic ganglia （PG）. Results After 4 months of surgery, the somatic nerve can regenerate into PG successfully and form new synapses observed under laser scanning confocal microscope. The neurons involved in foreign nerve regeneration can be isolated successfully by retrograde tracing and flow cytometry. The expression of agrin in L4-L6 VRs anastomosis group was lower than that of the control group of L4-L4 VR self-anastomosis （P 〈 0.05） and the normal group （ P 〈 0.05）. Conclusion The somatic nerve could regenerate into PG and form new synapses. The differences of agrin expression between somatic-autonomic and somatic-somatic nerve regeneration may be related to the changes of target organ.

关 键 词：体神经 内脏神经 运动神经元 凝集素 实时定量PCR 流式细胞分选 四甲基罗丹明 大鼠 

分 类 号：R322.85[医药卫生—人体解剖和组织胚胎学]