GDNF基因修饰的人工神经对大鼠坐骨神经缺损的修复作用及机理
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摘要
周围神经缺损是临床常见的病症, 其可由机械性、热性、化学性、先天性或病理性等因素所引起, 如果未能得到及时修复或修复不佳，将导致运动、感觉功能的丧失和疼痛性神经炎的发生，给病患者带来疼痛和残疾。针对周围神经缺损的治疗，目前的外科手段主要是采取自体神经移植，这一方法长期以来被认为是一种“黄金标准”方法，但由于供体的来源的限制、供区的永久性失神经支配以及供受区神经的匹配等问题使其应用范围受到严重限制。随着材料科学及生物学技术的进展，组织工程化人工神经成为自体神经组织很有希望的替代品。其核心成分主要包括支架材料和支持细胞。尽管相关的研究显示许多令人鼓舞的进展和希望，但目前的人工神经移植体尚不能达到与自体神经移植相似的效果。
为此本课题引入基因转染技术，对种植的雪旺氏细胞进行胶质细胞源性神经营养因子（GDNF）基因修饰以增加其合成及分泌神经营养因子的水平,然后应用所获取的GDNF基因修饰的雪旺氏细胞结合生物可降解材料(聚乳酸-聚羟基乙酸共聚物)及细胞外基质凝胶构建基因修饰的人工神经复合体,用于修复大鼠坐骨神经缺损。以探讨一种自体神经移植的替代方法及策略。
本课题的研究主要包括以下三个部分，所取得的主要结果及结论如下：
第一部分 组织工程人工神经诱导管的制作及其性能研究
1) 本研究首先应用组织工程技术,以构成比不同的聚乳酸-聚羟基乙酸共聚物
[poly(DL-lactide-co-glycolide) PLGA] [(85:15)或(50:50)]为原料成功制备了人工神经诱导管。以0.2MPBS（PH7.4）为人工降解液，观测PLGA(85:15)和PLGA(50:50)管12周体外降解期间吸水率、失重率及生物降解率变化，通过扫描电镜观察其微结构变化；并采用肌肉包埋方法，观测PLGA(85:15)和PLGA(50:50)管8周体内降解期间生物降解率变化。结果显示：
① 体内外降解条件下,PLGA(50:50)管吸水率、失重率及生物降解率显著高于PLGA(85:15)管；
② 两种材料样品体内生物降解率显著高于体外降解率；
③ 扫描电镜观察显示，随降解时间延长，材料样品表现为特征性的虫蚀样破坏并
进行性加重，以PLGA（50:50）管尤为明显。
这些结果提示：共聚物的构成比以及降解环境是影响聚乳酸－聚羟基乙酸共聚物生物降解性能的重要因素。
2) 在此基础上，我们进一步采用肌肉包埋、雪旺氏细胞直接接种以及坐骨神经缺损桥接预实验的方法观察两种构成比不同的PLGA管[（85:15）或（50:50）]的生物相容性及其神经再生诱导作用。结果显示：
① 肌肉包埋条件下,PLGA早期诱发以淋巴细胞及成纤维细胞为主的轻度的非特异性炎性反应,持续到10－12周时基本消退；
② 采用细胞直接接种的方法,观察到雪旺氏细胞在PLGA膜上能良好的生长并发生增殖；
③ 体内神经桥接情况下，硅胶管促发有明显的纤维组织增生，并形成包囊包裹于管周,而两种PLGA管组未见类似现象；
④ 与经典的硅胶管组相似,PLGA(85:15)管能够顺利完成坐骨神经缺损桥接作用，再生神经电生理及组织学评价显示两组无明显差异。而PLGA(50:50)管体内神经桥接4周时即出现破裂，不能为坐骨神经缺损提供良好的桥接支持作用。
以上这些结果提示，与硅胶管及PLGA(50:50)相比，PLGA(85:15)是一种异物反应小、生物相容性佳，生物降解速率合适的周围神经组织工程材料。
第二部分 pLXSN-GDNF的鉴定、包装及转染
1）对获赠的携带胶质细胞源性神经营养因子的逆转录病毒载体质粒(pLXSN-GDNF)进行测序鉴定。结果证实pLXSN-GDNF结构正确，GDNF序列完整；
2）采用脂质体包裹的方法，用pLXSN-GDNF重组逆转录病毒载体质粒转染包装细胞PA317，获得G418抗性细胞，RT-PCR证实其培养上清含有重组逆转录病毒，用NIH3T3细胞测定病毒滴度为104－105CFU/ml；
3) 用含重组逆转录病毒的上清感染雪旺氏细胞，获得G418抗性细胞SCsGDNF，PCR鉴定提示GDNF目的片段整合到细胞基因组；RT-PCR的结果证实因病毒感染而整合于细胞基因组的外源性基因具转录功能，且GDNFmRNA的表达水平比未感染的雪旺氏细胞（SCs）显著提高（P<0.01）。Western Blot证实SCsGDNFGDNF蛋白含量显著高于SCs (P<0.01)。这些结果提示重组逆转录病毒（pLXSN-GDNF）的基因转染显著上调了雪旺氏细胞GDNF蛋白及GDNFmRNA表达水平。
第三部分 GDNF基因修饰的人工神经复合体的体外构建及体内桥接
坐骨神经缺损的动物实验
1） 应用体外获取的GDNF基因修饰的雪旺氏细胞结合细胞外基质凝胶及PLGA管成功构建了GDNF基因修饰的人工神经复合体；
2）采取Hoechst33342对种植细胞（SCs或SCsGDNF）进行预标记的方法，在人工神经复合体大鼠神经桥接实验中示踪细胞的分布及存活情况。结果显示异体移植的雪旺氏细胞（SCs或SCsGDNF）在受体组织内存活时间可达4周左右。
3）通过GDNF基因修饰的人工神经复合体桥接大鼠坐骨神经缺损的实验，结果发现：
① 坐骨神经损伤后，GDNF蛋白及GDNFmRNA的表达水平，不仅在损伤神经干显著提高，而且在脊髓前角运动神经元及其周围胶质细胞也显著上调。这一结果提示运动神经元不仅通过靶源性神经营养因子的逆行性运输，而且可能
Peripheral nerve defects are a common clinical condition that can result from mechanical, thermal, chemical, congenital or pathological etologies. Failure to repair these damaged nerves can result in loss of muscle function, impaired sensations, or painful neuropathies. Current surgical strategies for repair of critical nerves involve autograft transfers of normal nerves from uninjured parts of the body. However, this "gold standard" treatment is frequently limited by tissue availability, risk of disease spread, secondary deformities, and differences in tissue structure and size. One possible alternative to autologous nerve replacement is the development of engineered artificial nerve grafts consisting mainly of a scaffold and support cells.
Despite advances in the field of tissue engineering, results to date with artificial nerve grafts have failed to equal nerve regeneration achieved with autologous nerve grafts. In order to enhance nerve regeneration and find an alternative to autografts, we modified Schwann cells to increase GDNF expression using a gene transfer technique. We then combined GDNF expressing Schwann cells with extracellular matrix gel and a biodegradable guidance conduit made of poly(DL-lactide-co-glycolide) (PLGA) to construct an artificial nerve complex and used this construct to bridge sciatic nerve lesions in adult Wistar rats. Our studies were focused on three key aspects of graft construction and function and the main results are indicated below.
Part I: Characterization of the stability and biocompatibility of PLGA conduits.
1) Artificial nerve conduits were successfully fabricated using two different copolymer ratios of PLGA(85:15 and 50:50)(lactide:glycolide).
2) Degradation of PLGA(85:15) and PLGA(50:50) conduits were examined in vitro for 12 weeks in phosphate buffered saline (PBS) at 37℃, pH 7.4 and in vivo for up to 8 weeks following implantation into the rat dorsal muscle.
A) PLGA conduits constructed with a 50:50 copolymer ratio exhibited greater
changes in weight, water absorption, and morphology and more extensive degradation in vitro than conduits constructed with an 85:15 polymer ratio.
B) Rates of degradation for both types of conduits were more rapid in vivo than in vitro.
3) Biocompatibility of PLGA grafts were assessed by implanting PLGA into dorsal muscle, culturing Schwann cells in PLGA film and assessing integrity of conduits bridging sciatic nerve lesions.
A) PLGA caused only minor nonspecific inflammatory reactions characterized by infiltration of small numbers of lymphocytes and fibroblasts at early time points.
B) Schwann cells grew and proliferated well on PLGA films.
C) Similar to silicone tube implanted control groups, PLGA(85:15) conduits provided a stable support for axon regeneration for up to 12 weeks, while PLGA(50:50) collapsed 4 weeks after transplantation.
Conclusions: Biocompatibility and degradation rate assessments indicate that PLGA(85:15) conduits are suitable for facilitating nerve repair and are significantly more stable than conduits made with equal co-polymer ratios.
Part II: Characterization, package, and transfer of recombinant retroviral vector (pLXSN-GDNF)
1) The pLXSN-GDNF construct, (a gift of Ruan Huaizheng ) was characterized by DNA sequencing and compared to gene bank data to confirm expression of proper sequences.
2) PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes. Recombinant retrovirus particles were then harvested from culture media of G418 resistant transfected cells and analyzed using RT-PCR. Virus titers in supernatants were 104-105 CFU/ml.
3) Rat Schwann cells were then exposed to supernatants containing recombinant retrovirus particles. Transfected Schwann cells were identified by G418 resistance and PCR of genomic DNA. PCR and Western blot analysis showed that both GDNF RNA and protein were upregulated in transfected Schwann cells as compared to untransfected cells.
Conclusions: Transfection with pLXSN-GDNF retr
为此本课题引入基因转染技术，对种植的雪旺氏细胞进行胶质细胞源性神经营养因子（GDNF）基因修饰以增加其合成及分泌神经营养因子的水平,然后应用所获取的GDNF基因修饰的雪旺氏细胞结合生物可降解材料(聚乳酸-聚羟基乙酸共聚物)及细胞外基质凝胶构建基因修饰的人工神经复合体,用于修复大鼠坐骨神经缺损。以探讨一种自体神经移植的替代方法及策略。
本课题的研究主要包括以下三个部分，所取得的主要结果及结论如下：
第一部分 组织工程人工神经诱导管的制作及其性能研究
1) 本研究首先应用组织工程技术,以构成比不同的聚乳酸-聚羟基乙酸共聚物
[poly(DL-lactide-co-glycolide) PLGA] [(85:15)或(50:50)]为原料成功制备了人工神经诱导管。以0.2MPBS（PH7.4）为人工降解液，观测PLGA(85:15)和PLGA(50:50)管12周体外降解期间吸水率、失重率及生物降解率变化，通过扫描电镜观察其微结构变化；并采用肌肉包埋方法，观测PLGA(85:15)和PLGA(50:50)管8周体内降解期间生物降解率变化。结果显示：
① 体内外降解条件下,PLGA(50:50)管吸水率、失重率及生物降解率显著高于PLGA(85:15)管；
② 两种材料样品体内生物降解率显著高于体外降解率；
③ 扫描电镜观察显示，随降解时间延长，材料样品表现为特征性的虫蚀样破坏并
进行性加重，以PLGA（50:50）管尤为明显。
这些结果提示：共聚物的构成比以及降解环境是影响聚乳酸－聚羟基乙酸共聚物生物降解性能的重要因素。
2) 在此基础上，我们进一步采用肌肉包埋、雪旺氏细胞直接接种以及坐骨神经缺损桥接预实验的方法观察两种构成比不同的PLGA管[（85:15）或（50:50）]的生物相容性及其神经再生诱导作用。结果显示：
① 肌肉包埋条件下,PLGA早期诱发以淋巴细胞及成纤维细胞为主的轻度的非特异性炎性反应,持续到10－12周时基本消退；
② 采用细胞直接接种的方法,观察到雪旺氏细胞在PLGA膜上能良好的生长并发生增殖；
③ 体内神经桥接情况下，硅胶管促发有明显的纤维组织增生，并形成包囊包裹于管周,而两种PLGA管组未见类似现象；
④ 与经典的硅胶管组相似,PLGA(85:15)管能够顺利完成坐骨神经缺损桥接作用，再生神经电生理及组织学评价显示两组无明显差异。而PLGA(50:50)管体内神经桥接4周时即出现破裂，不能为坐骨神经缺损提供良好的桥接支持作用。
以上这些结果提示，与硅胶管及PLGA(50:50)相比，PLGA(85:15)是一种异物反应小、生物相容性佳，生物降解速率合适的周围神经组织工程材料。
第二部分 pLXSN-GDNF的鉴定、包装及转染
1）对获赠的携带胶质细胞源性神经营养因子的逆转录病毒载体质粒(pLXSN-GDNF)进行测序鉴定。结果证实pLXSN-GDNF结构正确，GDNF序列完整；
2）采用脂质体包裹的方法，用pLXSN-GDNF重组逆转录病毒载体质粒转染包装细胞PA317，获得G418抗性细胞，RT-PCR证实其培养上清含有重组逆转录病毒，用NIH3T3细胞测定病毒滴度为104－105CFU/ml；
3) 用含重组逆转录病毒的上清感染雪旺氏细胞，获得G418抗性细胞SCsGDNF，PCR鉴定提示GDNF目的片段整合到细胞基因组；RT-PCR的结果证实因病毒感染而整合于细胞基因组的外源性基因具转录功能，且GDNFmRNA的表达水平比未感染的雪旺氏细胞（SCs）显著提高（P<0.01）。Western Blot证实SCsGDNFGDNF蛋白含量显著高于SCs (P<0.01)。这些结果提示重组逆转录病毒（pLXSN-GDNF）的基因转染显著上调了雪旺氏细胞GDNF蛋白及GDNFmRNA表达水平。
第三部分 GDNF基因修饰的人工神经复合体的体外构建及体内桥接
坐骨神经缺损的动物实验
1） 应用体外获取的GDNF基因修饰的雪旺氏细胞结合细胞外基质凝胶及PLGA管成功构建了GDNF基因修饰的人工神经复合体；
2）采取Hoechst33342对种植细胞（SCs或SCsGDNF）进行预标记的方法，在人工神经复合体大鼠神经桥接实验中示踪细胞的分布及存活情况。结果显示异体移植的雪旺氏细胞（SCs或SCsGDNF）在受体组织内存活时间可达4周左右。
3）通过GDNF基因修饰的人工神经复合体桥接大鼠坐骨神经缺损的实验，结果发现：
① 坐骨神经损伤后，GDNF蛋白及GDNFmRNA的表达水平，不仅在损伤神经干显著提高，而且在脊髓前角运动神经元及其周围胶质细胞也显著上调。这一结果提示运动神经元不仅通过靶源性神经营养因子的逆行性运输，而且可能
Peripheral nerve defects are a common clinical condition that can result from mechanical, thermal, chemical, congenital or pathological etologies. Failure to repair these damaged nerves can result in loss of muscle function, impaired sensations, or painful neuropathies. Current surgical strategies for repair of critical nerves involve autograft transfers of normal nerves from uninjured parts of the body. However, this "gold standard" treatment is frequently limited by tissue availability, risk of disease spread, secondary deformities, and differences in tissue structure and size. One possible alternative to autologous nerve replacement is the development of engineered artificial nerve grafts consisting mainly of a scaffold and support cells.
Despite advances in the field of tissue engineering, results to date with artificial nerve grafts have failed to equal nerve regeneration achieved with autologous nerve grafts. In order to enhance nerve regeneration and find an alternative to autografts, we modified Schwann cells to increase GDNF expression using a gene transfer technique. We then combined GDNF expressing Schwann cells with extracellular matrix gel and a biodegradable guidance conduit made of poly(DL-lactide-co-glycolide) (PLGA) to construct an artificial nerve complex and used this construct to bridge sciatic nerve lesions in adult Wistar rats. Our studies were focused on three key aspects of graft construction and function and the main results are indicated below.
Part I: Characterization of the stability and biocompatibility of PLGA conduits.
1) Artificial nerve conduits were successfully fabricated using two different copolymer ratios of PLGA(85:15 and 50:50)(lactide:glycolide).
2) Degradation of PLGA(85:15) and PLGA(50:50) conduits were examined in vitro for 12 weeks in phosphate buffered saline (PBS) at 37℃, pH 7.4 and in vivo for up to 8 weeks following implantation into the rat dorsal muscle.
A) PLGA conduits constructed with a 50:50 copolymer ratio exhibited greater
changes in weight, water absorption, and morphology and more extensive degradation in vitro than conduits constructed with an 85:15 polymer ratio.
B) Rates of degradation for both types of conduits were more rapid in vivo than in vitro.
3) Biocompatibility of PLGA grafts were assessed by implanting PLGA into dorsal muscle, culturing Schwann cells in PLGA film and assessing integrity of conduits bridging sciatic nerve lesions.
A) PLGA caused only minor nonspecific inflammatory reactions characterized by infiltration of small numbers of lymphocytes and fibroblasts at early time points.
B) Schwann cells grew and proliferated well on PLGA films.
C) Similar to silicone tube implanted control groups, PLGA(85:15) conduits provided a stable support for axon regeneration for up to 12 weeks, while PLGA(50:50) collapsed 4 weeks after transplantation.
Conclusions: Biocompatibility and degradation rate assessments indicate that PLGA(85:15) conduits are suitable for facilitating nerve repair and are significantly more stable than conduits made with equal co-polymer ratios.
Part II: Characterization, package, and transfer of recombinant retroviral vector (pLXSN-GDNF)
1) The pLXSN-GDNF construct, (a gift of Ruan Huaizheng ) was characterized by DNA sequencing and compared to gene bank data to confirm expression of proper sequences.
2) PA317 cells were transfected with recombinant retroviral vector pLXSN-GDNF using liposomes. Recombinant retrovirus particles were then harvested from culture media of G418 resistant transfected cells and analyzed using RT-PCR. Virus titers in supernatants were 104-105 CFU/ml.
3) Rat Schwann cells were then exposed to supernatants containing recombinant retrovirus particles. Transfected Schwann cells were identified by G418 resistance and PCR of genomic DNA. PCR and Western blot analysis showed that both GDNF RNA and protein were upregulated in transfected Schwann cells as compared to untransfected cells.
Conclusions: Transfection with pLXSN-GDNF retr
引文
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