电针对周围神经损伤大鼠神经生长导向因子的影响研究
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摘要
目的
以周围神经损伤再生修复的关键环节——轴突再生为切入点,动态观察电针对实验性坐骨神经损伤大鼠相应神经组织中神经生长导向因子Slit1、Slit2、 Netrin-1及其mRNA的影响,探讨电针促进周围神经损伤再生修复的可能性机制,为电针治疗周围神经损伤提供科学的实验依据。
方法
选用SD大鼠72只,雌雄各半,随机分成空白对照组,模型对照组和电针治疗组,每组24只。除空白对照组外,模型对照组与电针治疗组动物采用右后肢坐骨神经锐性横断后即刻端对端外膜缝合的方法造模。造模后第2日,电针治疗组取患侧腧穴“环跳”、“足三里”,常规针刺后进行电针治疗,正极接“环跳”,负极接“足三里”,采用疏密波,频率为5Hz,电流强度以肌肉出现轻微抽动为度,每天1次,每次15分钟,7天1个疗程,连续治疗3个疗程：模型对照组、空白对照组不做任何治疗。分别于第1-3疗程治疗结束后每组取8只动物进行行为学(SFI)检测和取材。采用免疫组化法观察电针对损伤的坐骨神经组织中雪旺细胞(S100蛋白)和神经生长导向因子Slit1、Slit2、Netrin-1的影响；采用RT-PCR法观察电针对损伤的坐骨神经和相应脊髓组织中神经生长导向因子Slit1mRNA、Slit2mRNA、Netrin-1mRNA的影响。
结果
1.行为学(SFI)动态检测结果显示：每一疗程结束后,模型对照组SFI都明显低于空白对照组(P<0.01)；而电针治疗组SFI都明显高于模型对照组(P<0.01)。
2.S100蛋白表达结果显示：坐骨神经组织中,第一疗程结束后,模型对照组S100蛋白表达高于空白对照组(P<0.01)；第1疗程结束后,模型对照组低于空白对照组(P<0.01)；第3疗程结束后,模型对照组高于空白对照组(P>0.05)。整个过程电针治疗组S100蛋白表达持续高于模型对照组,第1疗程结束后,有显著性差异(P<0.05),第2、3疗程后有非常显著性差异(P<0.01)。
3. Slit1、SlitlmRNA表达结果显示：模型对照组坐骨神经和相应脊髓组织中Slit1、Slit1mRNA表达在第1疗程后达到高峰,之后逐渐降低,但仍高于空白组(P<0.01)。电针治疗组坐骨神经和相应脊髓组织中Slit1、Slit1mRNA也在第1疗程后达到高峰,之后逐渐降低,且都高于模型对照组(P<0.01,P<0.05)。
4. Slit2、Slit2mRNA表达结果显示：模型对照组坐骨神经和相应脊髓组织中Slit2、Slit2mRNA表达在第1疗程后达到高峰,之后逐渐降低,但仍高于空白组(P<0.01)。电针治疗组坐骨神经和相应脊髓组织中Slit2、Slit2mRNA也在第1疗程后达到高峰,之后逐渐降低,且都高于模型对照组(P<0.01)。
5. Netrin-1、Netrin-1mRNA表达结果显示：模型对照组坐骨神经和相应脊髓组织中Netrin-1、Netrin-1mRNA表达在第1疗程后达到高峰,之后逐渐降低,但仍高于空白组(P<0.01,P<0.05)。电针治疗组坐骨神经和相应脊髓组织中Netrin-1、Netrin-1mRNA也在第1疗程后达到高峰,之后逐渐降低,且都高于模型对照组(P<0.01)。
结论
1.电针可促进实验性周围神经损伤模型大鼠神经功能的恢复。
2.电针能促进周围神经损伤相应神经组织中神经生长导向因子Slit1、Slit2、 Netrin-1及Slit1mRNA、Slit2mRNA、Netrin-1mRNA的表达,且每个神经生长导向因子及其mRNA的表达水平增强趋势同步。
3.电针可能通过促进损伤的周围神经组织中雪旺细胞增殖,为神经再生修复奠定重要的基础,以及影响神经生长导向因子的表达等营造有利于神经再生修复的微环境,实现对周围神经损伤再生修复的促进作用。
Objective
Take axonal regeneration,the key link of regenerative repair of peripheral nerve injury, as the breakthrough point, we dynamically observed of the influence electroacupuncture on the nerve growth guidance factors in relative nerve tissue in experimental sciatic nerve injured rats to study the likely mechanism for promoting regenerative repairing of peripheral nerve injury, and to provide the scientific experimental evidence for electroacupuncture treatment of peripheral nerve injury.
Methods
72SD rats, half male and half female, were randomly divided into blank control group, model control group and electroacupuncture treatment group randomly with each group24rats. Except the blank control group, the left two groups were molding by end to end or end adventitial suture after sharp transection of sciatic nerve of the right hind leg. The second day after molding, the electro-acupuncture treatment group choosed points "huantiao" and "zusanli", and were treated by electroacupuncture after conventional acupuncture, the positive pole connected to "huantiao" and the negative pole connected to "zusanli", using rarefaction-dense wave, the frequency of5Hz,current intensity was as set as the slight twitch of muscle, once a day with each time15minutes,7days per course of treatment, continuous three courses of treatment; There were not any treatment in model control group and blank control group. take8animals in each group for the SFI detection and tissue sampling and trimming from rats after the end of1to3course of treatment. Observing the influence of the electroacupuncture on Schwann and the nerve growth guidance factors:Slit1, slit2, netrin-1of the injured sciatic nerve tissue by immunohistochemistry, and observing the influence of the electroacupuncture on the nerve growth guidance factors: SlitlmRNA, slit2mRNA, netrin-1mRNA of the injured sciatic nerve and relative spinal cord tissue and by RT-PCR.
Results
1.Dynamic observation of result of SFI shows:after each treatment course, the SFI of model control group was obviously lower than blank control group(P<0.01);while the SFI of electroacupuncture treatment group was obviously higher than model control group(P<0.01).
2.The S100protein expression shows:in the injured sciatic nerve tissue, after the first course, the S100protein expression of model control group was higher than blank control group(P<0.01),after the second course, model group was lower than blank control group(P<0.01), after the third course, model control group was higher than blank control group(P>0.05). Electroacupuncture group was higher than model control group in the whole course, and after the first course, there were significant differences(P<0.05),even greater differences in the second and third course(P<0.01).
3.The expression results of Slitl and SlitlmRNA shows:the result of Slitland SlitlmRNA in injured sciatic nerve and its relative spinal cord tissue in model group reached the peak after the first course, and gradually decreased and higher than blank control group(P<0.01). Slit1and Slit1mRNA in injured sciatic nerve and its relative spinal cord tissue in electroacupuncture group also reached the peak after the first course, and gradually decreased while higher than model group(P<0.01,P<0.05).
4.The expression results of Slit2and Slit2mRNA shows:the result of Slit2and Slit2mRNA in injured sciatic nerve and its relative spinal cord tissue in model group reached the peak after the first course, and gradually decreased but higher than blank control group(P<0.01). Slit2and Slit2mRNA in injured sciatic nerve and its relative spinal cord tissue in electroacupuncture group also reached the peak after the first course, and gradually decreased while higher than model group(P<0.01).
5.The expression results of Netrin-land Netrin-1mRNA shows:the result of Netrin-land Netrin-1mRNA in injured sciatic nerve and its relative spinal cord tissue in model group reached the peak after the first course, and gradually decreased but higher than blank'control group(P<0.01,P<0.05). Netrin-land Netrin-1mRNA in injured sciatic nerve and its relative spinal cord tissue in electroacupuncture group also reached the peak after the first course, and gradually decreased while higher than model group(P<0.01).
Conclusion
1.Electroacupuncture can promote the recovery of nerve function of experimental peripheral injured rats.
2. Electroacupuncture can promote the expression of nerve growth guidance fact-ors (Slit1/Slit2/Netrin-1and Slit1mRNA/Slit2mRNA/Netrin-1mRNA) of the periphe-ral nerve injury tissue. And the expression level of each factor and its'mRNA are enhanced in sync.
3.Electroacupuncture promote the increasing of Scs in peripheral nerve tissue, and lay an important foundation for nerve regeneration and repair; Electroacupuncture may influence the expression of the nerve growth guidance factors and Swchann cells increasing to make a favorable microenvironment for nerve recovery, and carry out the promotion function on regeneration and repair of peripheral injury.
以周围神经损伤再生修复的关键环节——轴突再生为切入点,动态观察电针对实验性坐骨神经损伤大鼠相应神经组织中神经生长导向因子Slit1、Slit2、 Netrin-1及其mRNA的影响,探讨电针促进周围神经损伤再生修复的可能性机制,为电针治疗周围神经损伤提供科学的实验依据。
方法
选用SD大鼠72只,雌雄各半,随机分成空白对照组,模型对照组和电针治疗组,每组24只。除空白对照组外,模型对照组与电针治疗组动物采用右后肢坐骨神经锐性横断后即刻端对端外膜缝合的方法造模。造模后第2日,电针治疗组取患侧腧穴“环跳”、“足三里”,常规针刺后进行电针治疗,正极接“环跳”,负极接“足三里”,采用疏密波,频率为5Hz,电流强度以肌肉出现轻微抽动为度,每天1次,每次15分钟,7天1个疗程,连续治疗3个疗程：模型对照组、空白对照组不做任何治疗。分别于第1-3疗程治疗结束后每组取8只动物进行行为学(SFI)检测和取材。采用免疫组化法观察电针对损伤的坐骨神经组织中雪旺细胞(S100蛋白)和神经生长导向因子Slit1、Slit2、Netrin-1的影响；采用RT-PCR法观察电针对损伤的坐骨神经和相应脊髓组织中神经生长导向因子Slit1mRNA、Slit2mRNA、Netrin-1mRNA的影响。
结果
1.行为学(SFI)动态检测结果显示：每一疗程结束后,模型对照组SFI都明显低于空白对照组(P<0.01)；而电针治疗组SFI都明显高于模型对照组(P<0.01)。
2.S100蛋白表达结果显示：坐骨神经组织中,第一疗程结束后,模型对照组S100蛋白表达高于空白对照组(P<0.01)；第1疗程结束后,模型对照组低于空白对照组(P<0.01)；第3疗程结束后,模型对照组高于空白对照组(P>0.05)。整个过程电针治疗组S100蛋白表达持续高于模型对照组,第1疗程结束后,有显著性差异(P<0.05),第2、3疗程后有非常显著性差异(P<0.01)。
3. Slit1、SlitlmRNA表达结果显示：模型对照组坐骨神经和相应脊髓组织中Slit1、Slit1mRNA表达在第1疗程后达到高峰,之后逐渐降低,但仍高于空白组(P<0.01)。电针治疗组坐骨神经和相应脊髓组织中Slit1、Slit1mRNA也在第1疗程后达到高峰,之后逐渐降低,且都高于模型对照组(P<0.01,P<0.05)。
4. Slit2、Slit2mRNA表达结果显示：模型对照组坐骨神经和相应脊髓组织中Slit2、Slit2mRNA表达在第1疗程后达到高峰,之后逐渐降低,但仍高于空白组(P<0.01)。电针治疗组坐骨神经和相应脊髓组织中Slit2、Slit2mRNA也在第1疗程后达到高峰,之后逐渐降低,且都高于模型对照组(P<0.01)。
5. Netrin-1、Netrin-1mRNA表达结果显示：模型对照组坐骨神经和相应脊髓组织中Netrin-1、Netrin-1mRNA表达在第1疗程后达到高峰,之后逐渐降低,但仍高于空白组(P<0.01,P<0.05)。电针治疗组坐骨神经和相应脊髓组织中Netrin-1、Netrin-1mRNA也在第1疗程后达到高峰,之后逐渐降低,且都高于模型对照组(P<0.01)。
结论
1.电针可促进实验性周围神经损伤模型大鼠神经功能的恢复。
2.电针能促进周围神经损伤相应神经组织中神经生长导向因子Slit1、Slit2、 Netrin-1及Slit1mRNA、Slit2mRNA、Netrin-1mRNA的表达,且每个神经生长导向因子及其mRNA的表达水平增强趋势同步。
3.电针可能通过促进损伤的周围神经组织中雪旺细胞增殖,为神经再生修复奠定重要的基础,以及影响神经生长导向因子的表达等营造有利于神经再生修复的微环境,实现对周围神经损伤再生修复的促进作用。
Objective
Take axonal regeneration,the key link of regenerative repair of peripheral nerve injury, as the breakthrough point, we dynamically observed of the influence electroacupuncture on the nerve growth guidance factors in relative nerve tissue in experimental sciatic nerve injured rats to study the likely mechanism for promoting regenerative repairing of peripheral nerve injury, and to provide the scientific experimental evidence for electroacupuncture treatment of peripheral nerve injury.
Methods
72SD rats, half male and half female, were randomly divided into blank control group, model control group and electroacupuncture treatment group randomly with each group24rats. Except the blank control group, the left two groups were molding by end to end or end adventitial suture after sharp transection of sciatic nerve of the right hind leg. The second day after molding, the electro-acupuncture treatment group choosed points "huantiao" and "zusanli", and were treated by electroacupuncture after conventional acupuncture, the positive pole connected to "huantiao" and the negative pole connected to "zusanli", using rarefaction-dense wave, the frequency of5Hz,current intensity was as set as the slight twitch of muscle, once a day with each time15minutes,7days per course of treatment, continuous three courses of treatment; There were not any treatment in model control group and blank control group. take8animals in each group for the SFI detection and tissue sampling and trimming from rats after the end of1to3course of treatment. Observing the influence of the electroacupuncture on Schwann and the nerve growth guidance factors:Slit1, slit2, netrin-1of the injured sciatic nerve tissue by immunohistochemistry, and observing the influence of the electroacupuncture on the nerve growth guidance factors: SlitlmRNA, slit2mRNA, netrin-1mRNA of the injured sciatic nerve and relative spinal cord tissue and by RT-PCR.
Results
1.Dynamic observation of result of SFI shows:after each treatment course, the SFI of model control group was obviously lower than blank control group(P<0.01);while the SFI of electroacupuncture treatment group was obviously higher than model control group(P<0.01).
2.The S100protein expression shows:in the injured sciatic nerve tissue, after the first course, the S100protein expression of model control group was higher than blank control group(P<0.01),after the second course, model group was lower than blank control group(P<0.01), after the third course, model control group was higher than blank control group(P>0.05). Electroacupuncture group was higher than model control group in the whole course, and after the first course, there were significant differences(P<0.05),even greater differences in the second and third course(P<0.01).
3.The expression results of Slitl and SlitlmRNA shows:the result of Slitland SlitlmRNA in injured sciatic nerve and its relative spinal cord tissue in model group reached the peak after the first course, and gradually decreased and higher than blank control group(P<0.01). Slit1and Slit1mRNA in injured sciatic nerve and its relative spinal cord tissue in electroacupuncture group also reached the peak after the first course, and gradually decreased while higher than model group(P<0.01,P<0.05).
4.The expression results of Slit2and Slit2mRNA shows:the result of Slit2and Slit2mRNA in injured sciatic nerve and its relative spinal cord tissue in model group reached the peak after the first course, and gradually decreased but higher than blank control group(P<0.01). Slit2and Slit2mRNA in injured sciatic nerve and its relative spinal cord tissue in electroacupuncture group also reached the peak after the first course, and gradually decreased while higher than model group(P<0.01).
5.The expression results of Netrin-land Netrin-1mRNA shows:the result of Netrin-land Netrin-1mRNA in injured sciatic nerve and its relative spinal cord tissue in model group reached the peak after the first course, and gradually decreased but higher than blank'control group(P<0.01,P<0.05). Netrin-land Netrin-1mRNA in injured sciatic nerve and its relative spinal cord tissue in electroacupuncture group also reached the peak after the first course, and gradually decreased while higher than model group(P<0.01).
Conclusion
1.Electroacupuncture can promote the recovery of nerve function of experimental peripheral injured rats.
2. Electroacupuncture can promote the expression of nerve growth guidance fact-ors (Slit1/Slit2/Netrin-1and Slit1mRNA/Slit2mRNA/Netrin-1mRNA) of the periphe-ral nerve injury tissue. And the expression level of each factor and its'mRNA are enhanced in sync.
3.Electroacupuncture promote the increasing of Scs in peripheral nerve tissue, and lay an important foundation for nerve regeneration and repair; Electroacupuncture may influence the expression of the nerve growth guidance factors and Swchann cells increasing to make a favorable microenvironment for nerve recovery, and carry out the promotion function on regeneration and repair of peripheral injury.
引文
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