摘要
目的:观察体外分离、培养、纯化的许旺细胞对脱细胞神经移植物修复神经损伤的促进作用,为临床应用组织工程化神经修复周围神经缺损提供实验依据。方法:实验于2004-04/2005-04在中国医科大学组织工程实验室完成。纳入普通级雄性Wistar大鼠24只,体质量180～220g。随机分为实验组、空白对照组、自体移植对照组共3组,每组8只。分笼饲养。另纳入5～8d龄Sprague-Dawley乳鼠40只,取其双侧坐骨神经与臂丛神经。实验组大鼠右后腿用种植许旺细胞的ARSN桥接人为造成的神经缺损。空白对照组大鼠右后腿用单纯ARSN桥接人为造成的神经缺损。酶反复消化法与差速贴壁法分离和培养许旺细胞;许旺细胞与10mm长的脱细胞神经移植物共培养后,应用外科手术移植到大鼠坐骨神经缺损处,自体移植对照组大鼠右后腿用单纯切断的神经上下颠倒原位桥接人为造成的神经缺损。13周取材通过透射电镜和扫描电镜观察神经纤维的再生情况。结果:Wistar大鼠24只全部进入结果分析,没有脱失。①实验组较空白对照组的再生有髓神经纤维均匀,轴索略粗,髓鞘厚度有差别,部分神经纤维髓鞘还未形成板层结构,许旺细胞包绕神经纤维,轴索内微丝微管结构较清晰,无髓神经纤维直径较小,不均匀分布在有髓神经纤维之间,由一层纤维结缔包裹形成纤维束,实验组较自体移植对照组的髓鞘略薄,自体移植组再生神经纤维均较粗,许旺细胞包裹轴突形成排列规则、电子密度较高的髓鞘,髓鞘厚薄基本相同。②实验组可见大量许旺细胞呈双极梭形,并且首尾相连排列成链状或网状特征性分布。许旺细胞还可在神经纤维上的胶原丝上附着。③实验组有髓神经纤维数、髓鞘厚度、G率(有髓纤维总面积/神经干总面积)接近自体移植对照组,差异无显著性意义[(5344±592),(5514±373);(3.13±0.16),(3.19±0.25)μm;(48.43±3.62)%,(57.11±2.28)%;P>0.05];与空白对照组比较差异有显著性意义[(5344±592),(3191±236);(3.13±0.16),(1.28±0.33)μm;(48.43±3.62)%,(31.05±4.19)%;P<0.05]。结论:与许旺细胞共培养的脱细胞神经移植物可加快宿主轴索再生,促进神经损伤修复,是临床修复神经缺损的可行办法。
AIM： To observe the effers of in vitro isolated Sehwann cells （Ses） cocultured with aeellular nerve grafts on improving repair of nerve injury, and provide experimental evidence for clinical application of tlssue-engineered nerve in repairing peripheral neurological defect.
METHODS： The experiment was conducted at the Tissue Engi,eering Laboratory of China Medical University between April 2004 and April 2005. Twenty-four healthy male Wistar rats of 180-220 g were randomized into three groups with 8 rats iu each group： experimental group, blank control group and autograft group. All the rats were bred iu separated cages. In addition, 40 Sprague-Dawkey rats aged 5-8 days were collected to fetch bilateral m.iatic nerve at,I In＇achial plexus nerve. Humau-eaused nem＇ologi- cal detect was cotmected with right hind legs of rats in experimeutal group by ARSN bridge, which was used for implanting Scs. while connected with simple ARSN in right hind legs of rats in blank eontrol group. Scs were isolated and enzymatically digested with collagenase. then co-cultured with 10 uun-long aeellular nerve grafts, faittlly implanted surgically inlo rat sci atic nerve defect. In the autograft group, sinlply-sheared nerve reverse in sita bridge was applied to implant human-caused neurological detect into right hind legs of rats. The regeneration of nerve fiber was analyzed at the 13th week with transmission electrum microscope and seamming electron microsrope.
RESULTS： Totally 24 Wistar rats were involved into the result analysis without any drop.①Compared with blank control group, regenerated myelinated nerve fibers （MNF） were more even in the experimental group, with the thicker axon. And thickness differed in myelin sheath, which of part nerve fibers did not shape as plain structure. Cultured Scs coated nerve fibers. In the axis-cylinder, the microfilament and microtubule constituted distinctly. The unmyelinated nerve fibers of short diameters, distributed unevenly between MNF, and developed into fibrous bands with single layer of fibers. In the autograft group, myelin sheath and regenerated MNF were thicker, and axis-cylinder coated by Scs came into regnlately-arranged myelin sheathe of higher electron density and identical thickness.②In the experimental group, there were a large amount of Scs shaped dipolar fusiform and displayed the chain or net arraying feature at a head-tail connection. Moreover, Scs were attached to collagen of nerve fibers.③ There was insignificant difference in the number of MNF, thickness of myelin sheath and G rate （total volume of myelinated nerve/total volume of nerve trunk） between experimental group and auto.graft group [（5 344±592）, （5 514±373）; （3.13±0.16）, （3.19±0.25） μm; （48.43±3.62）%, （57.11±2.28）%, P〉0.05]; But difference was significant between blank control group and autograft group [（5 344±592）, （3 191±236）; （3.13±0.16）, （1.28±0.33） μm; （48.43±3.62）%,（31.05±4.19）%;P〈0.05].
CONCLUSION： Scs co-cultured with acellnlar nerve grafts can quicken the regeneration of host axis-cylinder and promote rehabilitation of injured sciatic nerve in rats, so as to offer a novel approach for the repair of injured peripheral nerve in clinic.
出处
《中国临床康复》
CSCD
北大核心
2006年第29期19-21,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助项目(30070775)
辽宁省教育厅高校科研资助项目(2005L537)~~
