雪旺细胞填充高分子神经导管修复大鼠坐骨神经缺损的实验研究
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摘要
背景资料
应用可降解的高分子复合材料制备神经导管修复神经缺损已经成为周围神经损伤后修复的重要研究方向。神经导管一方面能使神经营养因子在导管内积聚,浓度增高,另一方面引导神经缺损两端准确对合,同时导管的植入操作简单,可以减少手术对神经的损伤。神经导管修复神经损伤的效果主要与两方面有关:一是神经导管的材料种类及理化特点;二是神经导管形成的再生微环境包括其中的体液环境、基质环境和细胞环境。对于后者,研究主要集中在神经营养因子(neurotrophic factors,NTFs),众多学者尝试将外源性NTFs引入神经导管,以期尽可能有效地促进轴突再生,但外源性NTFs导入的浓度和释放的途径仍需进一步研究,同时最有益于神经再生所必须的营养因子的数量和种类也待进一步明确。雪旺细胞(Schwann cells,SCs)是周围神经的主要结构和功能细胞,它能分泌各种神经营养因子,产
浙江大学医学院硕士学位论文岔雪旺细胞充填高分子神经导管修复大鼠坐骨神经缺损的实验研究
生细胞外基质和细胞勃附因子,已有许多研究确认了雪旺细胞具有显
著的促进神经再生作用。与浙江大学高分子研究所共同研制的聚酷-
聚碳酸酷高分子神经导管经初步的动物试验,具有良好的通透性和生
物相容性及适宜的降解时间,可促进神经纤维的再生和功能修复,但
其修复的质量仍较正常神经为差,将体外培养的雪旺细胞悬液填充导
管内以进一步提高修复效果,具有理论上的可行性,故设计本实验,
评价填充雪旺细胞的高分子材料导管修复神经缺损的效果。
研究目的
评价雪旺细胞填充高分子材料导管修复大鼠坐骨神经缺损的效
果。
方法
1采用大鼠坐骨神经预变性的方法培养纯化雪旺细胞,免疫组化
鉴定。
2取45只SD大鼠随机分成3组,A、B、C组各15只,切除
部分右后肢坐骨神经使缺损距离在10mm一12mm之间,套上导管。A
组导管内填充雪旺细胞及DMEM培养液,B组内填充DMEM培养液,
C组为空导管组。
3术后观察大鼠的行走步态,并在20周处死大鼠前测定坐骨神经
功能指数(Seiatie九nction index,sFx)。
4 20周时处死大鼠,观察坐骨神经生长的情况,测定神经传导
速度(nerve eonductive veloeity,NCV)及肌肉湿重(包括健侧和实验
浙江大学医学院硕士学位论文爸雪旺细胞充填高分子神经导管修复大鼠坐骨神经缺损的实验研究
侧)o坐骨神经标本取横向及纵向截面切片,显微镜下观察神经再生
的情况,并对再生神经纤维数量进行计数统计。比较A组中的健侧与
实验侧的神经传导速度、肌肉湿重及神经纤维数量。
5部分标本做电镜观察髓鞘生长情况。
6数据采用SPSS10.0统计软件进行统计学分析,以P<0.05为有
显著性差异。
结果
1细胞鉴定:细胞培养后经免疫组化鉴定,雪旺细胞胞体及其
突起呈棕褐色,阳性细胞占细胞总数的90%。
2大体观察:饲养过程中无大鼠死亡,大鼠患肢屈曲挛缩情况
及拖曳步态在4周以后有所改善。大鼠20周处死后,未见导管残留,
导管桥接部分增粗,未见桥接段上下神经萎缩,周围有血管增生、粘
连现象。
3坐骨神经功能指数:三组比较A组与B组、A组与C均有显
著性差异(P<0 .001),B组与C组无显著性差异(P>0 .05)。
4肌肉湿重恢复率(实验侧与健侧的比值):A组与B组、A组
与C相互比较有显著性差异(P<0 .01,P<0 .05,),B组与C组差异无统计
学意义(P>0.05)。比较A组实验侧与健侧的肌肉湿重有显著性差异
(P(0.001)。
5电生理观察:三组NCV相互比较,A组与B组、A组与C
组均有显著性差异(P<0.01),B组与C组无显著性差异(P>0 .05)。比较
It was an attractive idea to use absorbable macromolecule nerve conduits to repair peripheral nerve gaps. There were many advantanges with nerve conduits to repairing nerve gaps. First neurotrophic factors could be concentrated within nerve conduits. Second fibre-bridge to anastomose two parts of nerve gaps could be formed ecxactly. Third operation of nerve conduits could be transplanted easily. Two factors were related to the effect of repair peripheral nerve injury with nerve conduits. one were category, physical and chemical character of materials. Another were microenvironment in nerve conduits ,which include fluid, extracellular matrix(ECM) and cells. Research of microenvironment were
focused on the neurotrophic factors (NTFs). Many studies have been done to use nerve conduits filled with exogenetic NTFs with the intention of promote axonal regeneration to the greatest extent efficiently. However concentration of exogenetic NTFs and the means of releasing NTFs still need to be studied. Meanwhile the types and volume of NTFs are most beneficial to peripheral nerve regeneration still need further investigation. Schwann cells are structural and functional cells of peripheral nerve, which can excrete all kinds of NTFs and generate ECM and cellular adhesion molecules. Several lines of investigation have confirmed the marked effects of Shwann cells on promote peripheral nerve regeneration. According to our early research, polyester-polycarbonate nerve conduits made by Zheijang University Macromolecule Institution and us had good permeability, compatibility and degradate time. The conduits could promote peripheral nerve regeneration and functional repair, but the quality of regenerate nerve was still inferior to normal nerve. It is possible theoretically that polyester-polycarbonate nerve conduits filled with Schwann cells cultured in vitro can improve effects of repairing nerve gaps. So we design this experiment that use polyester-polycarbonate nerve conduits filled with Schwann cells to repair nerve gaps in order to define the effects of this conduits. Objective
To evaluate effects of polyester-polycarbonate nerve conduits filled with Schwann cells on repairing sciatic nerve gaps of rats. Materials and MethodsUsing the method of predegeneration to cluture Schwann cell and identified through immoumocytochemical stain. Forty - five adult SD rats were divided into three groups randomly, with 15 in each group. A gap of 10mm~12mm of right sciatic nerve was removed and then bridged with polyester-polycarbonate nerve conduits. In group A, nerve conduit was filled with Schwann cells and DMEM culture fluid. In group B, nerve conduit was filled with DMEM culture fluid. In group C, nerve conduit was empty. After operation walk manner of rats was observed at stated intervals time. They were put to death 20 weeks later. Before death they were mensured sciatic function index(SFI). After death, nerve regeneration was observed. Nerve conductive velocity(NCV) and wet weight of tibial muscle(including experimental and normal side) are mensured. Specimens of sciatic nerve were made according to transverse section and vertical section. Regeneration nerve fiber and myelinated axons were observed and takend count of with microscope. Part of Specimens were examined to know status about myelin sheaths by electron microscope. The data of SFI, NCV, wet weight of tibial muscle and nerve fiber were analysed with statistical software SPSS10.0 (statistically significant
difference P<0.05). Results1 Identification of cells Identification of cells in immoumocytochemical stain, body and protuberance of Schwann cells present dark-brown, positive cells is about 90% of the total cells.2 Observation During experimental time, no rats died and improvement of flexing and contractive limb were found after 4 weeks. Nerve conduits have completely collapsed after 20 weeks, The bridge of conduits and nerves had enlarged. There weren't atrophy of top and bottom segment of nerves. There were many increasing vessels and much conglutination ar
应用可降解的高分子复合材料制备神经导管修复神经缺损已经成为周围神经损伤后修复的重要研究方向。神经导管一方面能使神经营养因子在导管内积聚,浓度增高,另一方面引导神经缺损两端准确对合,同时导管的植入操作简单,可以减少手术对神经的损伤。神经导管修复神经损伤的效果主要与两方面有关:一是神经导管的材料种类及理化特点;二是神经导管形成的再生微环境包括其中的体液环境、基质环境和细胞环境。对于后者,研究主要集中在神经营养因子(neurotrophic factors,NTFs),众多学者尝试将外源性NTFs引入神经导管,以期尽可能有效地促进轴突再生,但外源性NTFs导入的浓度和释放的途径仍需进一步研究,同时最有益于神经再生所必须的营养因子的数量和种类也待进一步明确。雪旺细胞(Schwann cells,SCs)是周围神经的主要结构和功能细胞,它能分泌各种神经营养因子,产
浙江大学医学院硕士学位论文岔雪旺细胞充填高分子神经导管修复大鼠坐骨神经缺损的实验研究
生细胞外基质和细胞勃附因子,已有许多研究确认了雪旺细胞具有显
著的促进神经再生作用。与浙江大学高分子研究所共同研制的聚酷-
聚碳酸酷高分子神经导管经初步的动物试验,具有良好的通透性和生
物相容性及适宜的降解时间,可促进神经纤维的再生和功能修复,但
其修复的质量仍较正常神经为差,将体外培养的雪旺细胞悬液填充导
管内以进一步提高修复效果,具有理论上的可行性,故设计本实验,
评价填充雪旺细胞的高分子材料导管修复神经缺损的效果。
研究目的
评价雪旺细胞填充高分子材料导管修复大鼠坐骨神经缺损的效
果。
方法
1采用大鼠坐骨神经预变性的方法培养纯化雪旺细胞,免疫组化
鉴定。
2取45只SD大鼠随机分成3组,A、B、C组各15只,切除
部分右后肢坐骨神经使缺损距离在10mm一12mm之间,套上导管。A
组导管内填充雪旺细胞及DMEM培养液,B组内填充DMEM培养液,
C组为空导管组。
3术后观察大鼠的行走步态,并在20周处死大鼠前测定坐骨神经
功能指数(Seiatie九nction index,sFx)。
4 20周时处死大鼠,观察坐骨神经生长的情况,测定神经传导
速度(nerve eonductive veloeity,NCV)及肌肉湿重(包括健侧和实验
浙江大学医学院硕士学位论文爸雪旺细胞充填高分子神经导管修复大鼠坐骨神经缺损的实验研究
侧)o坐骨神经标本取横向及纵向截面切片,显微镜下观察神经再生
的情况,并对再生神经纤维数量进行计数统计。比较A组中的健侧与
实验侧的神经传导速度、肌肉湿重及神经纤维数量。
5部分标本做电镜观察髓鞘生长情况。
6数据采用SPSS10.0统计软件进行统计学分析,以P<0.05为有
显著性差异。
结果
1细胞鉴定:细胞培养后经免疫组化鉴定,雪旺细胞胞体及其
突起呈棕褐色,阳性细胞占细胞总数的90%。
2大体观察:饲养过程中无大鼠死亡,大鼠患肢屈曲挛缩情况
及拖曳步态在4周以后有所改善。大鼠20周处死后,未见导管残留,
导管桥接部分增粗,未见桥接段上下神经萎缩,周围有血管增生、粘
连现象。
3坐骨神经功能指数:三组比较A组与B组、A组与C均有显
著性差异(P<0 .001),B组与C组无显著性差异(P>0 .05)。
4肌肉湿重恢复率(实验侧与健侧的比值):A组与B组、A组
与C相互比较有显著性差异(P<0 .01,P<0 .05,),B组与C组差异无统计
学意义(P>0.05)。比较A组实验侧与健侧的肌肉湿重有显著性差异
(P(0.001)。
5电生理观察:三组NCV相互比较,A组与B组、A组与C
组均有显著性差异(P<0.01),B组与C组无显著性差异(P>0 .05)。比较
It was an attractive idea to use absorbable macromolecule nerve conduits to repair peripheral nerve gaps. There were many advantanges with nerve conduits to repairing nerve gaps. First neurotrophic factors could be concentrated within nerve conduits. Second fibre-bridge to anastomose two parts of nerve gaps could be formed ecxactly. Third operation of nerve conduits could be transplanted easily. Two factors were related to the effect of repair peripheral nerve injury with nerve conduits. one were category, physical and chemical character of materials. Another were microenvironment in nerve conduits ,which include fluid, extracellular matrix(ECM) and cells. Research of microenvironment were
focused on the neurotrophic factors (NTFs). Many studies have been done to use nerve conduits filled with exogenetic NTFs with the intention of promote axonal regeneration to the greatest extent efficiently. However concentration of exogenetic NTFs and the means of releasing NTFs still need to be studied. Meanwhile the types and volume of NTFs are most beneficial to peripheral nerve regeneration still need further investigation. Schwann cells are structural and functional cells of peripheral nerve, which can excrete all kinds of NTFs and generate ECM and cellular adhesion molecules. Several lines of investigation have confirmed the marked effects of Shwann cells on promote peripheral nerve regeneration. According to our early research, polyester-polycarbonate nerve conduits made by Zheijang University Macromolecule Institution and us had good permeability, compatibility and degradate time. The conduits could promote peripheral nerve regeneration and functional repair, but the quality of regenerate nerve was still inferior to normal nerve. It is possible theoretically that polyester-polycarbonate nerve conduits filled with Schwann cells cultured in vitro can improve effects of repairing nerve gaps. So we design this experiment that use polyester-polycarbonate nerve conduits filled with Schwann cells to repair nerve gaps in order to define the effects of this conduits. Objective
To evaluate effects of polyester-polycarbonate nerve conduits filled with Schwann cells on repairing sciatic nerve gaps of rats. Materials and MethodsUsing the method of predegeneration to cluture Schwann cell and identified through immoumocytochemical stain. Forty - five adult SD rats were divided into three groups randomly, with 15 in each group. A gap of 10mm~12mm of right sciatic nerve was removed and then bridged with polyester-polycarbonate nerve conduits. In group A, nerve conduit was filled with Schwann cells and DMEM culture fluid. In group B, nerve conduit was filled with DMEM culture fluid. In group C, nerve conduit was empty. After operation walk manner of rats was observed at stated intervals time. They were put to death 20 weeks later. Before death they were mensured sciatic function index(SFI). After death, nerve regeneration was observed. Nerve conductive velocity(NCV) and wet weight of tibial muscle(including experimental and normal side) are mensured. Specimens of sciatic nerve were made according to transverse section and vertical section. Regeneration nerve fiber and myelinated axons were observed and takend count of with microscope. Part of Specimens were examined to know status about myelin sheaths by electron microscope. The data of SFI, NCV, wet weight of tibial muscle and nerve fiber were analysed with statistical software SPSS10.0 (statistically significant
difference P<0.05). Results1 Identification of cells Identification of cells in immoumocytochemical stain, body and protuberance of Schwann cells present dark-brown, positive cells is about 90% of the total cells.2 Observation During experimental time, no rats died and improvement of flexing and contractive limb were found after 4 weeks. Nerve conduits have completely collapsed after 20 weeks, The bridge of conduits and nerves had enlarged. There weren't atrophy of top and bottom segment of nerves. There were many increasing vessels and much conglutination ar
引文
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3. Hudson TW, Evans GR, Schmidt CE. Engineering strategies for peripheral nerve repair. Orthop Clin North Am, 2000,31 (3):485-98.
4. Jenq CB, Coggeshall RE. Nerve Regeneration Through Holey Silicone Tubes. Brain Res, 1985,361:233-41.
5. Braun S, Croizat B, Lagrange MC, et al. Neurotrophins increase motoneurons' ability to innervate skeletal muscle fibers in rat spinal cord human muscle cocultures.J Neurol Sci, 1996,136(1-2):17-23.
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2002,18(2):362-72.
8. Fawcett JW, Keynes RJ. Peripheral nerve regeneration.Annu Rev Neurosci,.1990,13:43-60. Review.
9.陈高,张建民,胡华,等.高分子神经导管的理化特性对周围神经再生影响,浙江大学学报医学版,2004,33(4):306-10.
10. Gerburg K, Hisham F, Welfgang F, et al.In vivo predegeneration of peripheral nerves: an effective technique to obtain Schwann cells for nerve conduits. J Neu-rosci Methods, 1999, 89: 17-24.
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2. Bellamkonda R, Aebischer P. Tissue engineering in the nerveous system.In: Bronzino JD, editor. Biomedical engineering hand-book. Boca Raton, Publisher: CRC Press, 1995, 1754-1773.
3. Hudson TW, Evans GR, Schmidt CE. Engineering strategies for peripheral nerve repair. Orthop Clin North Am, 2000,31 (3):485-98.
4. Jenq CB, Coggeshall RE. Nerve Regeneration Through Holey Silicone Tubes. Brain Res, 1985,361:233-41.
5. Braun S, Croizat B, Lagrange MC, et al. Neurotrophins increase motoneurons' ability to innervate skeletal muscle fibers in rat spinal cord human muscle cocultures.J Neurol Sci, 1996,136(1-2):17-23.
6. Henderson CE, Camu W, Mettling C, et al. Neurotrophins promote motor ne-uron survival and are present in embryonic limb bud. Nature, 1 993,363(6426) :266-70.
7. Rutkowski GE, Heath CA. Development of a Bioartificial Nerve Graft. I. Design Based on a Reaction-Diffusion Model. Biotechnol Prog,
2002,18(2):362-72.
8. Fawcett JW, Keynes RJ. Peripheral nerve regeneration.Annu Rev Neurosci,.1990,13:43-60. Review.
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