许旺细胞移植促进大鼠脑胆碱能神经纤维损伤后的修复

Repair of cholinergic nerve fiber injury by Schwann cell transplantation in rats

目的:利用显微移植技术,观察体外培养纯化的鼠坐骨神经许旺细胞悬液植入损伤的脑隔-海马纤维束后对胆碱能神经纤维的修复情况。方法:2004-06/2005-01在中国医科大学完成。①选取清洁级SD大鼠72只,随机数字表法分为许旺细胞即时移植组、许旺细胞延迟移植组、模型对照组,24只/组。另选用SD乳鼠1只用于坐骨神经许旺细胞悬液的制备。②3组大鼠均建立隔-海马纤维束损伤模型。造模后,许旺细胞即时移植组于中线旁1.8mm、脑皮层下4.5mm、前囟后1.8mm和2.0mm两处立即注射许旺细胞悬液,每处注射量为10μL,持续5min,许旺细胞延迟移植组造模后缝合头皮,回笼继续饲养,6d后再行许旺细胞悬液移植治疗,步骤同上。模型对照组造模后仅即时注入RPMI1640培养液。各组大鼠回笼饲养,分别于细胞植入后的1,2,4,6周处死,6只/时相点。③于上述各时相点麻醉大鼠,断头取脑,预行免疫组织化学染色的标本置于体积分数为0.1甲醛中固定,免疫组织化学染色检测低亲和力神经生长因子受体的表达;预行乙酰胆碱脂酶纤维染色的脑组织先在40g/L多聚甲醛灌注液中固定6h,然后置于体积分数为0.15的磷酸蔗糖液中4℃过夜,观察脑海马损伤区乙酰胆碱酯酶能神经纤维再生情况。移植后第4周,对各组距损伤区分别为0.5mm,1.5mm,2.5mm层面海马内的乙酰胆碱酯酶阳性纤维数量进行定量分析。结果:72只大鼠均进入结果分析。①许旺细胞移植后低亲和力神经生长因子受体的表达:模型对照组无表达低亲和力神经生长因子受体的许旺细胞出现;许旺细胞即时移值组2周后出现低亲和力神经生长因子受体的明显表达,4周达高峰,6周时程度略有下降;许旺细胞延迟移值组表现与即时移值组相似。②脑海马损伤区乙酰胆碱酯酶能神经纤维再生情况及相关定量分析:乙酰胆碱酯酶能再生神经纤维分布于损伤区内皮层下隔区、移植的许旺细胞团内及其周围,增生的纤维呈束样向海马内延伸。移植后第4周,许旺细胞即时移植组和延迟移植组海马CA1区、CA3区和齿状回内各层面的乙酰胆碱酯酶纤维密度均明显高于模型对照组(t=3.70,P<0.05);且许旺细胞延迟移植组3个区域各层面乙酰胆碱酯酶纤维密度均明显高于即时移植组(t=3.44,P<0.05)。结论:脑损伤后植入的许旺细胞可在受体内存活,表达可结合神经生长因子的低亲和力神经生长因子受体,促进胆碱能神经纤维再生。此外,于脑损伤后稍晚期植入的许旺细胞可能更有利于胆碱能神经纤维的再生。

AIM： Using fiber transplantation with microsurgery, the purpose of the present study is to observe the regeneration of acetylcholinesterase （AchE） fibers in the brain by transplanting rats sciatic nerves Schwann cells （SCs） suspension, which is cultivated in vitro andsublimed in the lesioned septo-hippecampal chclinergic pathway. METHODS： The experiment was accomplished in China Medical University between June 2004 and January 2005.（1） Totally 72 SD rats of cleaning grade were randomly divided into 3 groups： early transplantation （SCs were transplanted at the lesion time）, late transplantation （SCs were transplanted at 6 days pest-damaging） and control groups, every group had 24 rats. Another one SD neonate rat was used for making sciatic nerves SCs suspension. （2）These three groups＇ rats were all induced to build lesioned septo-hippocampal model. Then rats of early transplantation group were injected with sciatic nerves SCs suspension immediately at 1.8 mm away from midline, 4,5 mm below brain cortex, 1.8 and 2.0 mm behind anterior fontanel, the injection dosage of every location was 10 μL for 5 continuous minutes. White the rats of late transplantation group were sutured scalp and retumed into cage to keep on being fed, then these rats were given SCs suspension transplantation 6 days later, with the same approach as above. The control rats were only injected with RPMI 1640 culture medium instantly. All groups＇ rats returned the cage to be sequential feed and be executed respectively at weeks 1, 2, 4, 6 after transplantation, every time point for 6 rats. （3） Rats were anaesthetized and decollated at different time points above. The samples of each rat were put into formaldehyde that volume fraction was 0.1 for fixation, and immunohistochemistry stain was used to measure expression of Iow-addinity nerve growth factor receptor （LNGFr）. The brain tissues taken for AchE fiber stain were fixed in 40 g/L pelyformaldehyde perfusion liquid for 6 hours, and then put into phosphate sucrose that volume fraction was 0.15 at 4 % for one night： The regeneration of AchE fibers in brain hippocampal lesion site was observed. At 4 weeks pest-transplantation, the amount of the AChE electropesitive fiber in each group was quantificationally analyzed at 0.5 mm, 1.5 mm and 2.5 mm from the lesion. RESULTS： All 72 SD rats were taken into result analysis.（1）The transplanted SCs expressed LNGFr： In control group, there was no SCs expressed LNGFr. In early transplantation group, the expressions of LNGFr were obviously visible at 2 weeks post-transplantation, reached peak at 4 weeks post-transplantation, and began to lower down at 6 weeks post-transplantation. The early transplantation group and later transplantation group had the similar expressions.（2）In the brain hippocampal lesion, the regeneration and the correlative quantitative analysis of AchE fiber： AchE-regeneration fibers presented in the endoderm of lesion site, around and in transplanted SCs. Hyperplastic fibers stretched into hippocampi. At 4 week post-transplantation, the AChE in the CA1, CA3 and dentate gyrus region revealed a significantly greater fiber density in the early transplantation group and later transplantation group compared with the control group （t =3.70, P 〈 0.05）, and that of late transplantation group presented a higher fiber density than the early transplantation group （t =3.44, P 〈 0.05）. CONCLUSION： After brain trauma, the transplanted SCs can survive in the receptor and express LNGFr which can attach nerve growth factor and promote regeneration of AchE fibers. Moreover, the late transplantation of SCs is much more advantageous to promote regeneration of AchE fibers.