去细胞神经支架修复周围神经缺损的研究
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摘要
周围神经缺损在战争及和平时期都很常见，目前临床上仍采用自体神经
游离移植的方法修复周围神经缺损，虽然其疗效比较肯定，但由于切取供移
植的自体皮神经不可避免地造成新的感觉障碍，而且存在供区受限的问题，
所以开发理想的神经桥接材料仍是周围神经外科的重要课题，具有十分重要
的临床意义。虽然在非神经自体组织移植、神经组织工程和异体神经等方面
的研究都取得了一定的进展，但至今尚未开发出能够代替自体神经移植的理
想方法。
本论文用形态学与功能相结合的方法对一种新型生物支架材料（去细胞
神经支架）单独应用，以及与外源性生长因子联合应用修复同种异体神经缺
损的效果进行了评价；并进一步对去细胞神经支架能否用于异种移植进行了
初步研究。实验分为以下6个部分：
1．用改良的化学程序对兔胫神经进行处理，制取去细胞的神经支架材
料。组织学染色证明获得的去细胞神经支架内无任何细胞结构，免疫组织化
学方法显示去细胞神经支架由含有丰富laminin的管状结构组成。支架本身外
观完整，具有一定的韧性和抗牵拉能力，可操作性强，符合对神经桥接材料
的一般要求。
— —
2·实验采用神经电生理功能学指标、抗神经微丝免疫组织化学染色、美
兰髓鞘染色、透射电镜及计算机图象分析等技术，对去细胞神经支架异体移
植修复周围神经＊、ZCm和 3Cm长度缺损的远期神经再生进行了量化检
测。免疫组化染色观察发现再生神经纤维比较流畅地通过了不同长度的移植
体。各组动物的展趾功能和电生理功能均有不同程度的恢复。从动作电位幅
值恢复率、传导速度恢复率等功能学指标和再生有髓纤维数量、髓鞘厚度等
形态学量化指标比较来看，随着支架长度的延长神经再生效果减低，3。m支
架的神经再生效果虽劣于自体神经移植组，但仍有、定的功能恢复。
3、选用 bFGF经皮下导管多次注射的方法与去细胞异体神经支架联合修
复周围神经缺损。首先研究不同浓度和剂量的外源性 bFGF对去细胞支架移
植后早期神经再生的影响。实验采用抗神经微丝免疫组化染色观察各组神经
纤维再生的距离，抗S－100免疫组化染色观察雪旺氏细胞的分布，碱性磷酸
酶染色观察血管再生。发现高浓度 bFGF组门 和 500 AMl）的
神经再生距离、血管再生密度在术后10天明显大于盐水对照组。而低浓度
6厂GF组（50 AUAUhal和mo AU／ml）与盐水对照组无明显差别。此结果提示，
在支架移植后早期应用一定浓度和剂量的 bFGF能明显提高再生轴突在支架
内的生长速度。
4·进一步研究成纤维细胞生长因子和去细胞异体神经支架联合修复神经
缺损的远期效果。实验用 3cm支架修复异体神经缺损，实验组术后每日经皮
下埋置的导管将1毫升500 AU恤的 bFGF注射到移植体周围，对照组注射
等量生理盐水。术后20周时，进行展趾功能检查和动作电位、传导速度、肌
张力等电生理检测，以及髓鞘染色、电镜观察和计算机图象分析。实验组动
物功能恢复、再生有髓纤维的数量和成熟度等指标明显优于生理盐水对照组，
有比较理想的恢复。提示外源性 bFGF局部应用能明显提高去细胞神经支架修
·5·
第四军医大学整形外科博士学位论文
一
复神经缺损的功能效果，去细胞异体神经支架结合外源性生长因子能较满意
修复一定长度的神经缺损，是有希望的一种方法。
5．目的是探索去细胞神经支架材料用于异种移植的可行性。取预溃变和
新鲜的兔腔神经形成去细胞神经支架材料，异种移植修复大鼠坐骨神经
15rum缺损。术后 6个月，首先进行坐骨神经功能指数测定和疼痛试验，然
后用美兰染色、免疫组化、透射电镜等方法对吻合口、移植体中央和远侧神
经段的再生神经纤维进行形态学观察，并对再生有髓纤维的数量、直径、髓
鞘厚度等进行量化分析。自体神经移植组、溃变支架组和支架组3个组比较，
坐骨神经功能指数无显著性差异，均有较好恢复；自体移植组与溃变支架组
再生有髓纤维数无显著差异，二者均高于正常对照和支架组；自体移植组移
植体中央再生有髓纤维髓鞘厚度大于溃变支架组和支架组，但在远侧端3组
无差别；3组再生神经有髓纤维的直径和大于spin有髓纤维数无显著性差
异。结果提示去细胞神经支架能够在不使用任何免疫抑制措施的条件下，用
于异种移植良好修复大鼠乃rum周围神经缺损。
6．目的是观察CD4＋、CDS＋细胞在去细胞神经支
Peripheral nerve defects may occur frequently both hi war and peace periods, which can result in serious motor and sensory disabilities of the injured extremities. The autologous nerve grafting is still the standard method to repair a nerve gap clinically. For the reason that new sensory disturbance is unavoidable when an autologous cutaneous nerve having been harvested, it is very important to develop a new effective material instead of autologous nerve graft to repair the nerve defects.
In this paper, a new developed biomaterial called acellular nerve scaffolds (ANS) was evaluated using morphometry combining with neurophisiological technique to show the efficacy of nerve regeneration when bridging an allogenic nerve gap alone or enhanced by exogenous bFGF. Also the possibility of ANS to repair a xenogeneic nerve defect was explored. There are six parts of work as
follows.
1. An improved chemical procedure was utilized to obtain acellular nerve scaffolds of the rabbit tibial nerve. The extracted ANSs were quite good in physical character and easy to be handled as being transplanted. Histology and immunohistochemistry were used to evaluate the results of extracting procedure, which showed that it is cell free in an acellular nerve constituted by numerous parallel lamina tubes.
2.The aim of part two was to evaluate the effectiveness of acellular allogeneic nerve graft to bridge peripheral nerve defects. Various length of rabbit tibial nerve gaps were repaired by 1 cm, 2cm and 3 cm long acellular nerve scaffolds respectively and the nerve regeneration was evaluated by functional measurement combining with morphological observation. Nerve defects were successfully repaired with functional recovery in all experimental groups twenty weeks postoperatively. The results of nerve regeneration in the 1 cm-group and the 2cm-group were satisfactory; however, in the 3 cm-group nerve regeneration was inferior to autologous nerve grafting.
3.The early mechanism of promoting effect by bFGF on nerve regeneration following ANS grafting was explored, and the effects of various concentration of bFGF on axon growth were compared. Immunohistochemistry staining of neurofilament-160 and S-100 was used to show the length of axonal growth and Schwann cell infiltration 10 days after the surgery. The average distance of regenerated nerve fibers in the high dose groups (lOOOAU/ml and 500 AU/ml ) was longer than that in the normal saline control group; but there was no difference between the low dose groups (250 AU/ml and 100 AU/ml) and the
control group.
4.The purpose of this part was to evaluate whether exogenous bFGF enhanced the long term effect of nerve regeneration after nerve repair with ANSs extracted by detergent. In the experimental group 3 cm long allogeneic ANSs were used to bridge the rabbit tibial nerve defects, and 1ml of 500 IU bFGF solution was given from the second day after surgery with the schedule once per day for 2 weeks and twice a week for another 6 weeks. In the control group normal saline solution was given as the same course as in the experimental group. The functional recovery and the maturity of regenerated myelinated nerve fibers in the experimental group were markedly superior to that in the control group in 20 weeks after surgery. So, it seems that allogeneic ANSs combining with exogenous some growth factors might be an alternative of autologous nerve grafting to bridge nerve defects.
5. The aim was to explore the possibility of using xenogeneic acellular nerve scaffolds to bridge peripheral nerve defects. Two kinds of ANS, extracted from either a fresh rabbit tibial nerve or a predegenerated one, were transplanted to bridge 15 mm rat sciatic nerve gaps. Rats treated with orthotopic transplantation and unrepaired ones were as control respectively. Six months after the grafting, functional recovery was evaluated by gait analysis, pinch test, morphological and morphometric analysis. The sciatic nerve function index (SFI) was -30.7%?.8% in rats treated with xenogeneic acellular nerve,
游离移植的方法修复周围神经缺损，虽然其疗效比较肯定，但由于切取供移
植的自体皮神经不可避免地造成新的感觉障碍，而且存在供区受限的问题，
所以开发理想的神经桥接材料仍是周围神经外科的重要课题，具有十分重要
的临床意义。虽然在非神经自体组织移植、神经组织工程和异体神经等方面
的研究都取得了一定的进展，但至今尚未开发出能够代替自体神经移植的理
想方法。
本论文用形态学与功能相结合的方法对一种新型生物支架材料（去细胞
神经支架）单独应用，以及与外源性生长因子联合应用修复同种异体神经缺
损的效果进行了评价；并进一步对去细胞神经支架能否用于异种移植进行了
初步研究。实验分为以下6个部分：
1．用改良的化学程序对兔胫神经进行处理，制取去细胞的神经支架材
料。组织学染色证明获得的去细胞神经支架内无任何细胞结构，免疫组织化
学方法显示去细胞神经支架由含有丰富laminin的管状结构组成。支架本身外
观完整，具有一定的韧性和抗牵拉能力，可操作性强，符合对神经桥接材料
的一般要求。
— —
2·实验采用神经电生理功能学指标、抗神经微丝免疫组织化学染色、美
兰髓鞘染色、透射电镜及计算机图象分析等技术，对去细胞神经支架异体移
植修复周围神经＊、ZCm和 3Cm长度缺损的远期神经再生进行了量化检
测。免疫组化染色观察发现再生神经纤维比较流畅地通过了不同长度的移植
体。各组动物的展趾功能和电生理功能均有不同程度的恢复。从动作电位幅
值恢复率、传导速度恢复率等功能学指标和再生有髓纤维数量、髓鞘厚度等
形态学量化指标比较来看，随着支架长度的延长神经再生效果减低，3。m支
架的神经再生效果虽劣于自体神经移植组，但仍有、定的功能恢复。
3、选用 bFGF经皮下导管多次注射的方法与去细胞异体神经支架联合修
复周围神经缺损。首先研究不同浓度和剂量的外源性 bFGF对去细胞支架移
植后早期神经再生的影响。实验采用抗神经微丝免疫组化染色观察各组神经
纤维再生的距离，抗S－100免疫组化染色观察雪旺氏细胞的分布，碱性磷酸
酶染色观察血管再生。发现高浓度 bFGF组门 和 500 AMl）的
神经再生距离、血管再生密度在术后10天明显大于盐水对照组。而低浓度
6厂GF组（50 AUAUhal和mo AU／ml）与盐水对照组无明显差别。此结果提示，
在支架移植后早期应用一定浓度和剂量的 bFGF能明显提高再生轴突在支架
内的生长速度。
4·进一步研究成纤维细胞生长因子和去细胞异体神经支架联合修复神经
缺损的远期效果。实验用 3cm支架修复异体神经缺损，实验组术后每日经皮
下埋置的导管将1毫升500 AU恤的 bFGF注射到移植体周围，对照组注射
等量生理盐水。术后20周时，进行展趾功能检查和动作电位、传导速度、肌
张力等电生理检测，以及髓鞘染色、电镜观察和计算机图象分析。实验组动
物功能恢复、再生有髓纤维的数量和成熟度等指标明显优于生理盐水对照组，
有比较理想的恢复。提示外源性 bFGF局部应用能明显提高去细胞神经支架修
·5·
第四军医大学整形外科博士学位论文
一
复神经缺损的功能效果，去细胞异体神经支架结合外源性生长因子能较满意
修复一定长度的神经缺损，是有希望的一种方法。
5．目的是探索去细胞神经支架材料用于异种移植的可行性。取预溃变和
新鲜的兔腔神经形成去细胞神经支架材料，异种移植修复大鼠坐骨神经
15rum缺损。术后 6个月，首先进行坐骨神经功能指数测定和疼痛试验，然
后用美兰染色、免疫组化、透射电镜等方法对吻合口、移植体中央和远侧神
经段的再生神经纤维进行形态学观察，并对再生有髓纤维的数量、直径、髓
鞘厚度等进行量化分析。自体神经移植组、溃变支架组和支架组3个组比较，
坐骨神经功能指数无显著性差异，均有较好恢复；自体移植组与溃变支架组
再生有髓纤维数无显著差异，二者均高于正常对照和支架组；自体移植组移
植体中央再生有髓纤维髓鞘厚度大于溃变支架组和支架组，但在远侧端3组
无差别；3组再生神经有髓纤维的直径和大于spin有髓纤维数无显著性差
异。结果提示去细胞神经支架能够在不使用任何免疫抑制措施的条件下，用
于异种移植良好修复大鼠乃rum周围神经缺损。
6．目的是观察CD4＋、CDS＋细胞在去细胞神经支
Peripheral nerve defects may occur frequently both hi war and peace periods, which can result in serious motor and sensory disabilities of the injured extremities. The autologous nerve grafting is still the standard method to repair a nerve gap clinically. For the reason that new sensory disturbance is unavoidable when an autologous cutaneous nerve having been harvested, it is very important to develop a new effective material instead of autologous nerve graft to repair the nerve defects.
In this paper, a new developed biomaterial called acellular nerve scaffolds (ANS) was evaluated using morphometry combining with neurophisiological technique to show the efficacy of nerve regeneration when bridging an allogenic nerve gap alone or enhanced by exogenous bFGF. Also the possibility of ANS to repair a xenogeneic nerve defect was explored. There are six parts of work as
follows.
1. An improved chemical procedure was utilized to obtain acellular nerve scaffolds of the rabbit tibial nerve. The extracted ANSs were quite good in physical character and easy to be handled as being transplanted. Histology and immunohistochemistry were used to evaluate the results of extracting procedure, which showed that it is cell free in an acellular nerve constituted by numerous parallel lamina tubes.
2.The aim of part two was to evaluate the effectiveness of acellular allogeneic nerve graft to bridge peripheral nerve defects. Various length of rabbit tibial nerve gaps were repaired by 1 cm, 2cm and 3 cm long acellular nerve scaffolds respectively and the nerve regeneration was evaluated by functional measurement combining with morphological observation. Nerve defects were successfully repaired with functional recovery in all experimental groups twenty weeks postoperatively. The results of nerve regeneration in the 1 cm-group and the 2cm-group were satisfactory; however, in the 3 cm-group nerve regeneration was inferior to autologous nerve grafting.
3.The early mechanism of promoting effect by bFGF on nerve regeneration following ANS grafting was explored, and the effects of various concentration of bFGF on axon growth were compared. Immunohistochemistry staining of neurofilament-160 and S-100 was used to show the length of axonal growth and Schwann cell infiltration 10 days after the surgery. The average distance of regenerated nerve fibers in the high dose groups (lOOOAU/ml and 500 AU/ml ) was longer than that in the normal saline control group; but there was no difference between the low dose groups (250 AU/ml and 100 AU/ml) and the
control group.
4.The purpose of this part was to evaluate whether exogenous bFGF enhanced the long term effect of nerve regeneration after nerve repair with ANSs extracted by detergent. In the experimental group 3 cm long allogeneic ANSs were used to bridge the rabbit tibial nerve defects, and 1ml of 500 IU bFGF solution was given from the second day after surgery with the schedule once per day for 2 weeks and twice a week for another 6 weeks. In the control group normal saline solution was given as the same course as in the experimental group. The functional recovery and the maturity of regenerated myelinated nerve fibers in the experimental group were markedly superior to that in the control group in 20 weeks after surgery. So, it seems that allogeneic ANSs combining with exogenous some growth factors might be an alternative of autologous nerve grafting to bridge nerve defects.
5. The aim was to explore the possibility of using xenogeneic acellular nerve scaffolds to bridge peripheral nerve defects. Two kinds of ANS, extracted from either a fresh rabbit tibial nerve or a predegenerated one, were transplanted to bridge 15 mm rat sciatic nerve gaps. Rats treated with orthotopic transplantation and unrepaired ones were as control respectively. Six months after the grafting, functional recovery was evaluated by gait analysis, pinch test, morphological and morphometric analysis. The sciatic nerve function index (SFI) was -30.7%?.8% in rats treated with xenogeneic acellular nerve,
引文
1．谢卫国，朱家恺．非神经组织移植修复周围神经缺损．见：朱家恺主编．周围神经外科进展．广州：广东高等教育出版社，1991年，第1版，P66-84．
2．朱家恺．周围神经外科学．三环出版社，1991；277．
3. Chiu DTW, et al. Autogenous vein graft as a conduit for nerve regeneration. Surgery 1982,91:226-232.
4．洪光祥，王发斌，黄省秋，等．自体静脉移植修复周围神经缺损实验研究．中华显微外科杂志，1991；14(4)：220-2．
5．丛国辉，鲁开化．不同性质骨骼肌复合材料桥接周围神经缺损的比较研究．中华显微外科杂志，1995；18(4)：275-6．
6．韩岩，鲁开化，马福成，等．含有Ⅰ、Ⅳ型胶原的静脉管腔对周围神经缺损修复作用的影响．中华显微外科杂志，1999；22(3)：198-200．
7．利春叶，曹代成．碱性成纤维细胞生子因子结合静脉套接法修复周围神经缺损的实验研究．中国修复重建外科杂志，2000；14(1)：14-16．
8．韩岩，鲁开化．游离神经片段延长静脉桥接周围神经缺损长度的实验研究．中华外科杂志，1993；31(12)：726-9．
9. Tang JB. Vein conduits with interposition of nerve tissue for peripheral nerve defects. J Reconstr Microsurg. 1995;11(1):21-6.
10. Brunelli GA, Battiston B, Vigasio A, et al. Bridging nerve defects with combined skeletal muscle and vein conduits. Microsurgery. 1993;14(4):247-51
11. Tang JB, Gu YQ, Song YS. Repair of digital nerve defect with autogenous vein graft during flexor tendon surgery in zone 2. J Hand Surg Br. 1993;18(4):449-53.
12. Tang JB. Group fascicular vein grafts with interposition of nerve slices for long ulnar nerve defects: report of three cases. Microsurgery. 1993;14(6):404-8.
13. Walton RL, Brown RE, Matory WE, et al. Autogenous vein graft repair of digital nerve defects in the finger: a retrospective clinical study. Plast Reconstr Surg 1989,84:944-949.
14. Chiu-DT. Autogenous venous nerve conduits. A review. Hand Clin. 1999,15(4):667-71.
15．罗永湘，等．静脉桥接正中神经、尺神经缺损四例的教训．中华显微外科杂志，1990；13：229．
16. Tang JB, Shi D, Zhou H. Vein conduits for repair of nerves with a prolonged gap or in unfavourable conditions: an analysis of three failed cases. Microsurgery. 1995;16(3):133-7.37.
17．尹维田，等．静脉桥接治疗周围神经缺损．中华显微外科杂志，1990；13(1)：13-14．
18．洪光祥，王发斌，黄省秋，等．自体静脉移植修复周围神经缺损的疗效观察．中华手外科杂志，1993；9(1)：10-12．
19. Suematsu N, Atsuta Y, Hirayama T. Vein gaft for repair of peripheral nerve gap. J Reconstr Microsurg 1988,4:313-318.
20. Chiu DTW, et al. Comparative electrophysiologic vein grafts as nerve conduits: an experimental study. J Reconstr Microsurg 1988,4:303-310.
21. Chiu DTW and Strauch B. A prospective clinical evaluation of autogenous vein grafts used as a nerve conduit for distal sensory nerve defects of 3 cm or less. Plast Reconstr Surg 1990,86:928-934.
22. Rice DH and Berstein FD. The use of autogenous vein for nerve grafting. Otolaryngol Head Neck Surg 1984,92:410.
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