Zhongguo xiu fu chong jian wai ke za zhi = Zhongguo xiufu chongjian waike zazhi = Chinese journal of reparative and reconstructive surgery · 2009
[Effects of neurotrophic factor 3 gene modified SC on sciatic nerve regeneration in rats].
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Dong Yuzhen, Chen Zhenbing, Hong Guangxiang
Аннотация
OBJECTIVE: To investgate the effects of neurotrophic factor 3 (NT-3) genes modified SC on facilitating nerve regeneration and protecting neuronal survival after the sciatic nerve transection in rats.
METHODS: The double sciatic nerves were harvested from 3-day-old Wistar rats and the SCs were separated, cultured and purified with double enzyme digestion and adherent culture. The third generation purified SCs were used. The NT-3 cDNA gene was transfected into cultured SCs by using cationic liposome. The NT-3 expression were identified by ELISA after 1, 2, 4 and 8 weeks. The plasmids expressing NT-3 genes were transfected into SCs with lipofectamine. The purity of SCs were detecting before and after modified with NT-3. The nerve-grafting complexes were constructed by SCs (3 x 10(7)/mL) modified NT-3, third generation SCs (3 x 10(7)/mL), NT-3 gene, respectively. And the nerve-grafting complexes were combined with ECM gel and PLGA conduit. Forty-eight adult SD rats were made the models of the right sciatic nerve defect (10 mm). According to the nerve-grafting complexes which were repaired the sciatic nerve defects, the models were divided into 4 groups randomly (n=12): group A (ECM gel and PLGA conduits), group B (SC, ECM gel and PLGA conduits), group C (NT-3 gene, ECM gel and PLGA conduits) and group D (NT-3 modified SC, ECM gel and PLGA conduits). At 2, 4, 6, 8 and 12 weeks after operation, the nerve gross were observed. Electrophysiological examination, histological observation and transmission electron microscope observation were performed at 12 weeks after operation.
RESULTS: The concentrations of NT-3 protein were 0.39 +/- 0.25, 0.76 +/- 0.22, 1.06 +/- 0.38 and 1.61 +/- 0.35 at 1, 2, 4 and 8 weeks after operation; showing statistically significant differences (P < 0.05). The purity of SCs was 94.7% +/- 2.1% and 95.6% +/- 2.5% before and after modified with NT-3, respectively; showing a statistically significant difference (P < 0.05). The feet of injury rats began inflammation and ulcer, which healed at 12 weeks in group D, followed by groups C and B, but which was serious in group A gradually. The observations of gross, sections under microscope and transmission electron microscope at 12 weeks showed the regeneration of defect nerve was best in group D, followed by groups C and B, and group A was worst. There were statistically significant differences (P < 0.05) in latent period, amplitude, motor nerve conduction velocity, the number and thickness of axon, the diameter of nerve fiber, the percentage of the nerve tissue area between group A and groups B, C, D, between groups B, C and group D at 12 weeks. At 12 weeks after operation, the transmission electron microscope showed observation the maturation of medullary sheath was best in group D, followed by groups C and B, and group A was worst.
CONCLUSION: The nerve-grafting complex of NT-3 genes modified SCs could repair injured nerve. The competence is superior to SCs and neurotrophic factors.
METHODS: The double sciatic nerves were harvested from 3-day-old Wistar rats and the SCs were separated, cultured and purified with double enzyme digestion and adherent culture. The third generation purified SCs were used. The NT-3 cDNA gene was transfected into cultured SCs by using cationic liposome. The NT-3 expression were identified by ELISA after 1, 2, 4 and 8 weeks. The plasmids expressing NT-3 genes were transfected into SCs with lipofectamine. The purity of SCs were detecting before and after modified with NT-3. The nerve-grafting complexes were constructed by SCs (3 x 10(7)/mL) modified NT-3, third generation SCs (3 x 10(7)/mL), NT-3 gene, respectively. And the nerve-grafting complexes were combined with ECM gel and PLGA conduit. Forty-eight adult SD rats were made the models of the right sciatic nerve defect (10 mm). According to the nerve-grafting complexes which were repaired the sciatic nerve defects, the models were divided into 4 groups randomly (n=12): group A (ECM gel and PLGA conduits), group B (SC, ECM gel and PLGA conduits), group C (NT-3 gene, ECM gel and PLGA conduits) and group D (NT-3 modified SC, ECM gel and PLGA conduits). At 2, 4, 6, 8 and 12 weeks after operation, the nerve gross were observed. Electrophysiological examination, histological observation and transmission electron microscope observation were performed at 12 weeks after operation.
RESULTS: The concentrations of NT-3 protein were 0.39 +/- 0.25, 0.76 +/- 0.22, 1.06 +/- 0.38 and 1.61 +/- 0.35 at 1, 2, 4 and 8 weeks after operation; showing statistically significant differences (P < 0.05). The purity of SCs was 94.7% +/- 2.1% and 95.6% +/- 2.5% before and after modified with NT-3, respectively; showing a statistically significant difference (P < 0.05). The feet of injury rats began inflammation and ulcer, which healed at 12 weeks in group D, followed by groups C and B, but which was serious in group A gradually. The observations of gross, sections under microscope and transmission electron microscope at 12 weeks showed the regeneration of defect nerve was best in group D, followed by groups C and B, and group A was worst. There were statistically significant differences (P < 0.05) in latent period, amplitude, motor nerve conduction velocity, the number and thickness of axon, the diameter of nerve fiber, the percentage of the nerve tissue area between group A and groups B, C, D, between groups B, C and group D at 12 weeks. At 12 weeks after operation, the transmission electron microscope showed observation the maturation of medullary sheath was best in group D, followed by groups C and B, and group A was worst.
CONCLUSION: The nerve-grafting complex of NT-3 genes modified SCs could repair injured nerve. The competence is superior to SCs and neurotrophic factors.