PLGA-Laminin复合神经导管诱导周围神经生物学修复的实验研究
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摘要
目前临床上主要采用外膜缝合法修复神经,其缺点为没有束间方向的控制,即再生的神经纤维错向生长,难以保证精确对合而影响神经的再生;外膜虽对合,轴突有可能回缩,留有间隙导致瘢痕形成,影响神经功能的恢复。导管法修复神经比其它方法具有明显的优点。神经导管不仅仅是作为神经再生的临时通道,更重要的是为周围神经功能性再生提供适宜的微环境,故单纯改进导管自身的设计远不能满足神经的功能性再生。因此,可以从仿生设计的角度考虑,改进神经导管内微环境的设计出发,以神经基底膜成分为基础,用含有生物活性分子的材料制成复合神经导管,不仅可以模拟细胞外基质的环境,提高材料对雪旺细胞(SC)的黏附性,从而最大限度地促进神经功能性再生。
本实验对聚乳酸-羟基乙酸共聚物(PLGA)材料进行体外MTT细胞毒性实验和细胞相容性实验。通过观察其生物学性状,检测特异性表面标志,为下一步在体内修复周围神经提供理论依据。在体外实验基础上,用PLGA制成神经导管,并在导管内表面制成多层层黏连蛋白(LN)活性膜结构,在大鼠坐骨神经上构建神经再生室,分成T1和T2两组,T1组:(T1L)左侧坐骨神经,外膜缝合法;(T1R)右侧坐骨神经,PLGA桥接法(3mm间隙);T2组:(T2L)左侧坐骨神经,层黏连蛋白修饰后的PLGA桥接法(3mm间隙);(T2R)右侧坐骨神经,PLGA桥接法(3mm间隙)。通过特殊染色来观察再生轴突的组织形态学和超微结构的变化,通过S-100蛋白和Gap-43蛋白免疫组化实验、脊髓Tunnel凋亡实验、小腿三头肌形态学观察、蛋白相对量检测和神经电生理图的分析来评价不同方法修复周围神经的效果,为临床应用提供理论依据。
通过上述实验研究,我们认为,PLGA材料对SC毒性低;PLGA表面具有良好的细胞相容性和细胞黏附性;在体外,外源性LN能促进SC的黏附及生长,为其应用于动物实验提供了理论依据;PLGA-LN复合神经导管能促进轴突的再生及成熟;PLGA-LN复合神经导管能减少脊髓前角神经细胞的凋亡,对运动神经元有保护作用;PLGA-LN复合神经导管能较早地促进靶器官的神经再支配。
Peripheral nerve injuries are very common in clinic. Epineurium suture is still widely used, which could not provide accurate direction for regeneration axons and lead to the formation of scar to effect functional recovery. Nerve guide duct is better than other methods, therefore we design the study, which includes cell toxicity and compatibility experiment in Vitro. Based on these, PLGA-LN compounded nerve guide duct is used to repair sciatic nerves compared with epineurium suture and common PLGA nerve guide duct to evaluate the results of different methods for repairing nerves.There were two groups,T1 group and T2 group,T1 group:T1L,epineurium suture;T1R, PLGA;T2 group: T2L, PLGA-LN; T2R, PLGA.
When the concentration of the fluid with PLGA reached 10mg/ml,the growth rate of RSC96 was above 80%,which indicated PLGA had lower toxicity. RSC96 was cultivated on PLGA membrane and TCPs, RSC 96 was fusiform shaped in PLGA group, whose shape was similar to TCPs group,which indicated PLGA had better cell adhesion and compatibility. RSC 96 was fusiform shaped in PLGA group, whose shape was similar to TCPs group, which indicated PLGA have better cell adhesion and compatibility. RSC96 was cultivated on PLGA membrane and LN membrane, after 48 hours, S-100 immunofluorescence was applied, which indicated LN group was better than PLGA group. Exogenous laminin can promote adhesion and growth of SC.
The number of proximal myelinated fibers was no obvious difference brfore 12 weeks, T2L group was better than other groups at 24 weeks.The thickness of sheath of T2L group wass better than other groups at 8 weeks for the proximal part. And the distal part was quite different from the proximal one. The number of myelinated fibers and thickness of sheath of T2L group was better than other groups at 8 weeks. The special staining disclosed that T2L group in which the regenerated axons aligned regularly and the sheath was mature was better than other groups. We can conclude that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers.
TSCI of T2L group in which muscle fibers were aligned regularly and closely was better than other three groupsat 12 weeks and 24weeks (P<0.05),muscle fibers were aligned regularly and not closely in T1R and T2R groups, muscle fibers were aligned irregularly and not closely in T1L group. We concluded that the atrophy degree of T2L group was lighter than other groups because it got earlier reinnervation .
In normal situation, S-100 distributes conditionedly in peripheral nerves, there is dynamic balance among synthesis、excretion and transport, destruction of balance induces decrease of S-100 necessarily.We found that there was almost no expression at 1 week, increased a little at 2 weeks, the express of T2L group was better than other groups at 8 weeks(P<0.05),the expression of S-100 trended stable after this.We concluded that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers.
The express of T2L group was better than other groups at 2 weeks(P<0.05), we found that expression of Gap-43 increased obviously, then attenuated, the expression of Gap-43 trended stable after 10 weeks. The relative quantity of Real-time PCR of Gap-43 also identified the point. The number of apoptosis of motor neuron of cornu anterior medullae spinalis reached the highest at 2 weeks,T2L group was less than the other groups(P<0.05),then attenuated at 1 day and trended stable after 8 weeks.We concluded that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers and protect motor neuron of cornu anterior medullae spinalis .
The change of ultrastrucutre in 4 groups identified that T2L group was better than other groups at 8 weeks including both CV and Amp(P<0.05), We concluded that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers to promote stimulation conduction.
As above-mentioned, we concluded that PLGA with better compatibility and adhesion has lower toxicity for SC, exogenous LN can promote growth and adhesion of SC in Vitro. We also concluded that PLGA-LN compounded nerve guide duct can promote regeneration and maturation of axons ,decrease the apoptosis number of cornu anterior medullae spinalis and protect motor neuron of cornu anterior medullae spinalis , reinnervation of target organs.
本实验对聚乳酸-羟基乙酸共聚物(PLGA)材料进行体外MTT细胞毒性实验和细胞相容性实验。通过观察其生物学性状,检测特异性表面标志,为下一步在体内修复周围神经提供理论依据。在体外实验基础上,用PLGA制成神经导管,并在导管内表面制成多层层黏连蛋白(LN)活性膜结构,在大鼠坐骨神经上构建神经再生室,分成T1和T2两组,T1组:(T1L)左侧坐骨神经,外膜缝合法;(T1R)右侧坐骨神经,PLGA桥接法(3mm间隙);T2组:(T2L)左侧坐骨神经,层黏连蛋白修饰后的PLGA桥接法(3mm间隙);(T2R)右侧坐骨神经,PLGA桥接法(3mm间隙)。通过特殊染色来观察再生轴突的组织形态学和超微结构的变化,通过S-100蛋白和Gap-43蛋白免疫组化实验、脊髓Tunnel凋亡实验、小腿三头肌形态学观察、蛋白相对量检测和神经电生理图的分析来评价不同方法修复周围神经的效果,为临床应用提供理论依据。
通过上述实验研究,我们认为,PLGA材料对SC毒性低;PLGA表面具有良好的细胞相容性和细胞黏附性;在体外,外源性LN能促进SC的黏附及生长,为其应用于动物实验提供了理论依据;PLGA-LN复合神经导管能促进轴突的再生及成熟;PLGA-LN复合神经导管能减少脊髓前角神经细胞的凋亡,对运动神经元有保护作用;PLGA-LN复合神经导管能较早地促进靶器官的神经再支配。
Peripheral nerve injuries are very common in clinic. Epineurium suture is still widely used, which could not provide accurate direction for regeneration axons and lead to the formation of scar to effect functional recovery. Nerve guide duct is better than other methods, therefore we design the study, which includes cell toxicity and compatibility experiment in Vitro. Based on these, PLGA-LN compounded nerve guide duct is used to repair sciatic nerves compared with epineurium suture and common PLGA nerve guide duct to evaluate the results of different methods for repairing nerves.There were two groups,T1 group and T2 group,T1 group:T1L,epineurium suture;T1R, PLGA;T2 group: T2L, PLGA-LN; T2R, PLGA.
When the concentration of the fluid with PLGA reached 10mg/ml,the growth rate of RSC96 was above 80%,which indicated PLGA had lower toxicity. RSC96 was cultivated on PLGA membrane and TCPs, RSC 96 was fusiform shaped in PLGA group, whose shape was similar to TCPs group,which indicated PLGA had better cell adhesion and compatibility. RSC 96 was fusiform shaped in PLGA group, whose shape was similar to TCPs group, which indicated PLGA have better cell adhesion and compatibility. RSC96 was cultivated on PLGA membrane and LN membrane, after 48 hours, S-100 immunofluorescence was applied, which indicated LN group was better than PLGA group. Exogenous laminin can promote adhesion and growth of SC.
The number of proximal myelinated fibers was no obvious difference brfore 12 weeks, T2L group was better than other groups at 24 weeks.The thickness of sheath of T2L group wass better than other groups at 8 weeks for the proximal part. And the distal part was quite different from the proximal one. The number of myelinated fibers and thickness of sheath of T2L group was better than other groups at 8 weeks. The special staining disclosed that T2L group in which the regenerated axons aligned regularly and the sheath was mature was better than other groups. We can conclude that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers.
TSCI of T2L group in which muscle fibers were aligned regularly and closely was better than other three groupsat 12 weeks and 24weeks (P<0.05),muscle fibers were aligned regularly and not closely in T1R and T2R groups, muscle fibers were aligned irregularly and not closely in T1L group. We concluded that the atrophy degree of T2L group was lighter than other groups because it got earlier reinnervation .
In normal situation, S-100 distributes conditionedly in peripheral nerves, there is dynamic balance among synthesis、excretion and transport, destruction of balance induces decrease of S-100 necessarily.We found that there was almost no expression at 1 week, increased a little at 2 weeks, the express of T2L group was better than other groups at 8 weeks(P<0.05),the expression of S-100 trended stable after this.We concluded that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers.
The express of T2L group was better than other groups at 2 weeks(P<0.05), we found that expression of Gap-43 increased obviously, then attenuated, the expression of Gap-43 trended stable after 10 weeks. The relative quantity of Real-time PCR of Gap-43 also identified the point. The number of apoptosis of motor neuron of cornu anterior medullae spinalis reached the highest at 2 weeks,T2L group was less than the other groups(P<0.05),then attenuated at 1 day and trended stable after 8 weeks.We concluded that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers and protect motor neuron of cornu anterior medullae spinalis .
The change of ultrastrucutre in 4 groups identified that T2L group was better than other groups at 8 weeks including both CV and Amp(P<0.05), We concluded that LN from PLGA-LN can promote proliferation of SC and accelerate regeneration and maturation of myelinated fibers to promote stimulation conduction.
As above-mentioned, we concluded that PLGA with better compatibility and adhesion has lower toxicity for SC, exogenous LN can promote growth and adhesion of SC in Vitro. We also concluded that PLGA-LN compounded nerve guide duct can promote regeneration and maturation of axons ,decrease the apoptosis number of cornu anterior medullae spinalis and protect motor neuron of cornu anterior medullae spinalis , reinnervation of target organs.
引文
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