【作者】 马建敏；

【导师】 刘德明；

【作者基本信息】 江西医学院 ， 人体解剖与组织胚胎学， 2005， 硕士

【摘要】 目的:将具有免疫隔离效应的海藻酸钠微囊包被异种雪旺细胞,并进行移植用于脊髓损伤的治疗研究,通过免疫组织化学方法观察脊髓损伤后及进行微囊化神经组织细胞移植后星形胶质细胞中胶质纤维酸性蛋白(glia fibrillary acidic protein,GFAP)表达的变化。方法:无菌条件下取家兔双侧坐骨神经制成组织细胞悬液,低速离心后与1.5%海藻酸钠溶液混合并喷入20mmol/L 的氯化钡溶液中制成微囊化坐骨神经组织细胞。130 只成年Sprague-Dawley (SD)大鼠随机分为正常组、单纯损伤组、细胞悬液组和微囊组四组:正常组为10 只;其他三组每组各40 只,大鼠于T10右侧半横断伤后,分别植入明胶海绵、明胶海绵吸附的坐骨神经组织细胞悬液及明胶海绵吸附的微囊化坐骨神经组织细胞,术后3、7、14 及28 天,每组各10 只大鼠,4%多聚甲醛经心-升主动脉灌注后取出损伤部位脊髓标本;正常组大鼠则取相应节段脊髓。冰冻切片后行HE 染色观察组织学变化,行免疫组织化学染色观察GFAP 表达的变化。结果:脊髓损伤后神经元数量减少,部分神经元萎缩变小,细胞边界模糊不清,并有大量的巨噬细胞浸润,灰质中有囊腔变化;术后第14 天时炎性反应减轻,胶质细胞增生、肥大,而在微囊组胶质细胞增生较轻;第28 天时微囊组神经元形态基本恢复正常。脊髓损伤后第3 天GFAP 免疫组化染色即出现阳性,星形胶质细胞增大,高峰在损伤后7 天持续至14 天,微囊组与单纯损伤组、细胞悬液组比较差异显著(P <0.01);至第28 天时则减少,但仍高于正常组。脊髓损伤后,随时间的推移,GFAP 阳性细胞每平方毫米细胞数、阳性细胞体积密度(Vv)、数密度(Nv)、粒子平均体积(—V )和表面积密度(Sv)均增加。其中阳性细胞数、Sv、Vv、Nv 和—V 在第7 天时,微囊组与对照组、细胞悬液组比较均有明显降低,差异有显著性(P <0.05);到第28 天时微囊组的阳性细胞数、Vv、Nv、Sv 和—V 都低于另两组(P <0.01)。结论: 微囊化异种坐骨神经组织细胞移植能抑制损伤脊髓GFAP 的表达,减轻由反应性胶质化所形成的胶质瘢痕。更多还原

【Abstract】 Objective: To study the recovery of function of injured spinal cord by xenotransplantation of microencapsulatrd Schwann cells,we observed the changes of GFAP after spinal cord injured and the changes after transplantation of microencapsulated sciatic nerve’s tissue/cells by using immohistochemistry method.Methods: Bilateral sciatic nerves of rabbits were dissected under sterile conditions and made into nerve’s tissue/cells suspension. Following centrifugation, the tissue/cells were mixed with 1.5% sodium alginate solution. The mixture was then extruded using a droplet generation device into 20mmol/L barium chloride solution to form barium-alginate capsules. A total of 130 adult Sprague-Dawley rats were randomly divided into four groups: Group A,B,C and D. Ten rats of Group D were normal control. Forty animals in each group of C, B and A were performed a lateral hemisection of spinal cord at left T10 level,then gelatin sponge alone, with nerve’s tissue/cells suspension and with microencapsulated nerve’s tissue/cells were implanted into the injured area respectively. 3,7,14 and 28 days postinjury, 10 rats of each group in B, C and D as well as the normal rats were anesthetized and perfused transcardially with 4% paraformaldehyde. The thoracic cord including the injured site and the spinal cord of the normal rats at corresponding level were removed, freezed,Samples were cut and processed using standard procedures for hematoxylin-eosin(HE) and Nissl staining. Using appropriate antibodies, sections were also stained immunohisto-chemically for glia fibrillary acidic protein(GFAP).Results: After SCI the number of the neurons reduced and the part of neurons contracted,the boundary of cell was obscure,there were a number of macrophagocytes surrounding injury distract,small cavity was observed in the gray matter of spinal cord. On the 14th day we observed the state of hypertrophy and hyperplasia of astrocytes. These changes were not seen evidently in Group A. On the 28th day the form of neurons in Group A generally resume normal appearance. GFAP positive reaction was observed on the 3st day postinjury and rose to its peak on the 14th day, which in Group A was inferior to that in Group B or C(P <0.01).The expression of GFAP decreased on the 28th day, but still there was superior than normal group.The expression of GFAP showed an increasing tendency after SCI, the number of positive cell ,the Volume Density (Vv),the Number Density (Nv), the Surface Density (Sv) and the average volume of cell(—V) were raised. On the 7th day the number of positive cell、Sv、Vv、Nv and —V in Group A differed significantly from it in Group B or C until the 14th day postoperatively(P <0.05). Compared to that in Group B or C,all the Sterological parameters were lower in Group A on the 28th day(P <0.01). Conclusion: The results indicate that xenotransplantation of micro-encapsulated sciatic nerve’s tissue/cells can decrease the express of GFAP in astrocytes of injured spinal cord,and reduce glial scar caused by reactive astrogliosis.更多还原