针康法对局灶性脑缺血大鼠胶质细胞环境和细胞凋亡的影响
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摘要
目的
观察针康法对局灶性脑缺血大鼠缺血区BDNF、S100β、GFAP蛋白和BDNF、Caspase-3基因的表达,探讨该方法促进脑缺血大鼠神经功能恢复的可能机制。
方法
建立大鼠永久性局灶性脑缺血模型,将240只大鼠按随机数字法分为5组：假手术组、模型组、针刺组、康复组、针康组,每组各48只。每组再按3、7、14、21天分为4亚组,每组12只。假手术组、模型组不进行任何干预,针刺组采用头穴丛刺治疗,康复组采用跑台训练,针康组采用头穴丛刺结合跑台训练治疗。各时间点采用Bederson's神经功能评分观察大鼠神经功能的变化,采用免疫组化检测各组大鼠缺血区BDNF、S100β、 GFAP蛋白的表达变化,采用实时荧光定量PCR检测BDNFmRNA. Caspase-3mRNA的表达。
结果
1神经功能评分：与假手术组比较,各时间点,各组神经功能评分降低,有显著性差异(P<0.01)；术后7d,14d与模型组比较,针刺组,康复组,针康组神经功能评分降低,差异有统计学意义(P<0.05)；术后7d,14d与针刺组,康复组比较,针康组神经功能评分降低,差异有统计学意义(P<0.05)；各时间点,与针刺组比较,康复组神经功能评分趋势一致,差异无统计学意义(P>0.05)。
2缺血区皮层BDNF的表达：术后3d,7d,14d,21d与假手术组比较,各组BDNF阳性细胞表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组BDNF阳性细胞表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组BDNF阳性细胞表达增加(p<0.05)。
3缺血区皮层S100β的表达：术后3d,7d,14d,21d与假手术组比较,各组S100β阳性细胞表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组S100β阳性细胞表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组S100β阳性细胞表达增加(p<0.05)。
4缺血区GFAP蛋白的表达：术后3d,7d,14d,21d与假手术组比较,各组GFAP阳性细胞表达增加(p<0.01);术后7d,14d,与模型组相比,针刺组、康复组、针康组GFAP阳性细胞表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组GFAP阳性细胞表达增加(p<0.05)。
5脑缺血大鼠BDNFmRNA的表达：各时间点,与假手术比较,各组BDNFmRNA的表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组BDNFmRNA的表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组BDNFmRNA的表达增加(p<0.05)。
6脑缺血大鼠Caspase-3mRNA表达：各时间点,与假手术比较,模型组Caspase-3mRNA表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组Caspase-3mRNA表达减少(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组Caspase-3mRNA表达减少(p<0.05)；术后7d,14d,与针刺组比较,康复组Caspase-3mRNA的表达无明显差异,(P>0.05)。
结论
1.针康法能促进局灶性脑缺血后大鼠神经功能康复,且针康法优于单纯针刺和康复。
2.针康法能够促进局灶性脑缺血大鼠皮质缺血区周围GFAP,S100β的表达,促进胶质细胞活化,从而保护神经元,进而起到脑保护作用,且针康法优于单纯针刺和康复。
3.针康法能促进局灶脑缺血后BDNFmRNA的表达增加,保护神经元,与BDNF蛋白表达趋势一致,并且随时间递增其表达增加,且针康法优于单纯针刺和康复。
4.针康法能抑制局灶脑缺血后Caspase-3mRNA的表达,减少细胞凋亡,促进神经恢复,并且随时间递增其表达降低,且针康法优于单纯针刺和康复。
5.针康法能够调控脑缺血后胶质细胞环境,抑制神经细胞凋亡途径,这可能是针康法促进脑缺血神经恢复的机制之一。
Objective
To observe the effect of acupuncture in ischemic region of BDNF and S100β and GFAP protein and method on focal cerebral ischemia rats, and expression of BDNFmRNA, Caspase-3mRNA.
Method
Establishment of rat permanent focal cerebral ischemia model, In these standards in line with the selected240rats. Divided into5groups, Sham operation group, model group, acupuncture group, the rehabilitation group and acupuncture combined with rehabilitation group. Each group had48rats, Then each group at3,7,14.21days were divided into4groups,12rats in each group. Sham operation group, model group without any intervention, The acupuncture group were treated with head acupuncture therapy, The rehabilitation group was treated with treadmill training, Acupuncture combined with rehabilitation group was treated with head acupuncture treatment combined with treadmill training. Each time, Changes in the immunohistochemical detection of BDNF,S100β、GFAP in ischemic rats expression. point of the changes of nerve function test observation of neural function in rats, The detection of BDNF-mRNA,Caspase-3mRNA real-time fluorescent quantitative PCR
Result
1. The neurological function score, Each time point in each group, the neurological function score increased clearly (P<0.01); After7th days of treatment compared with the model group. The nerve function score of acupuncture group, rehabilitation group and comprehensive group decreased clearly (P<0.05). Compared with the acupuncture group and rehabilitation group, comprehensive group of neural function score was significantly lower (P<0.05). Acupuncture group and rehabilitation group was compared with similar.
2Expression of BDNF in the ischemic area:After the success of the model,3days,7days,14days, each group and sham operation were statistically significant(p<0.01).7days after making models, compared with the model group, expression of BDNF was significantly reduced in acupuncture group, rehabilitation group and comprehensive group (P<0.05).14days after making models, Comprehensive group compared with the model group were significantly different(p<0.01).
3The expression of S100β around ischemic cortex:on the3rd and7th day after the modeling, there were statistically difference between the sham operation group and other groups (P<0.05). On the3rd day after the modeling, to compared with the model group,S100β expression promoted in the CNSP combined with task-oriented training group and in the task-oriented training group (P<0.05). on the14th day after the modeling, there were statistically difference between the CNSP combined with task-oriented training group and model group (P<0.01). On the21St day after the modeling, there were statistically difference amongst in the CNSP combined with task-oriented training group, the task-oriented training group, the CNSP group and the model group(P<0.05).
4The expression of GFAP around ischemic cortex:On the7th and14th days after the modeling, to compared with the model group, the expression of GFAP in the CNSP combined with task-oriented training group, the task-oriented training group and the CNSP group enhanced (P<0.05), and they in the CNSP combined with task-oriented training groupwere superior to the task-oriented training group and the CNSP group (P<0.05) respective.There were statistically difference amongst in the CNSP combined with task-orientedtraining group, the task-oriented training group and the CNSP group (P<0.05). On the21St day after the modeling, the expression of GFAP in the CNSP combined with task-oriented training group were superior to the others (P<0.05).
5The expression of BDNFmRNA:Each time point in each group compared with the model group, BDNFmRNA expression was significantly increased (P<0.01). After7th days of treatment compared with the model group, expression of BDNFmRNA was significantly higher in acupuncture group, rehabilitation group and comprehensive group (P<0.05). Compared with the acupuncture group and rehabilitation group, Expression of BDNFmRNA was significantly higher in comprehensive group (P<0.05).Acupuncture group and rehabilitation group was compared with similar.
6. The expression of Caspase-3mRNA:Each time point in each group compared with the model group, Caspase-3mRNA expression was Increased significantly (P<0.01). After7th days of treatment compared with the model group, expression of Caspase-3mRNA was significantly reduced in acupuncture group, rehabilitation group and comprehensive group (P<0.05) Compared with the acupuncture group and rehabilitation group, Expression of Caspase-3mRNA was significantly reduced in comprehensive group (P<0.05).Acupuncture group and rehabilitation group was compared with similar.
Conclusion
1Acupuncture and rehabilitation can promote the recovery of neural function of rats after focal cerebral ischemia.
2Acupuncture and rehabilitation can promote the expression of proliferation in brain tissue after focal cerebral ischemia GFAP, S100β.
3Acupuncture and rehabilitation can promote the expression of proliferation in brain tissue after focal cerebral ischemia BDNF-mRNA.
4Acupuncture and rehabilitation promotes the expression of brain tissue after focal cerebral ischemia Caspase-3mRNA attenuation.
5Acupuncture combined with rehabilitation to glial cells after cerebral ischemia in environmental regulation, inhibition of neuronal apoptosis pathway. This may be one of the mechanisms of acupuncture combined with rehabilitation for cerebral ischemia nerve recovery.
观察针康法对局灶性脑缺血大鼠缺血区BDNF、S100β、GFAP蛋白和BDNF、Caspase-3基因的表达,探讨该方法促进脑缺血大鼠神经功能恢复的可能机制。
方法
建立大鼠永久性局灶性脑缺血模型,将240只大鼠按随机数字法分为5组：假手术组、模型组、针刺组、康复组、针康组,每组各48只。每组再按3、7、14、21天分为4亚组,每组12只。假手术组、模型组不进行任何干预,针刺组采用头穴丛刺治疗,康复组采用跑台训练,针康组采用头穴丛刺结合跑台训练治疗。各时间点采用Bederson's神经功能评分观察大鼠神经功能的变化,采用免疫组化检测各组大鼠缺血区BDNF、S100β、 GFAP蛋白的表达变化,采用实时荧光定量PCR检测BDNFmRNA. Caspase-3mRNA的表达。
结果
1神经功能评分：与假手术组比较,各时间点,各组神经功能评分降低,有显著性差异(P<0.01)；术后7d,14d与模型组比较,针刺组,康复组,针康组神经功能评分降低,差异有统计学意义(P<0.05)；术后7d,14d与针刺组,康复组比较,针康组神经功能评分降低,差异有统计学意义(P<0.05)；各时间点,与针刺组比较,康复组神经功能评分趋势一致,差异无统计学意义(P>0.05)。
2缺血区皮层BDNF的表达：术后3d,7d,14d,21d与假手术组比较,各组BDNF阳性细胞表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组BDNF阳性细胞表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组BDNF阳性细胞表达增加(p<0.05)。
3缺血区皮层S100β的表达：术后3d,7d,14d,21d与假手术组比较,各组S100β阳性细胞表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组S100β阳性细胞表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组S100β阳性细胞表达增加(p<0.05)。
4缺血区GFAP蛋白的表达：术后3d,7d,14d,21d与假手术组比较,各组GFAP阳性细胞表达增加(p<0.01);术后7d,14d,与模型组相比,针刺组、康复组、针康组GFAP阳性细胞表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组GFAP阳性细胞表达增加(p<0.05)。
5脑缺血大鼠BDNFmRNA的表达：各时间点,与假手术比较,各组BDNFmRNA的表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组BDNFmRNA的表达增加(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组BDNFmRNA的表达增加(p<0.05)。
6脑缺血大鼠Caspase-3mRNA表达：各时间点,与假手术比较,模型组Caspase-3mRNA表达增加(p<0.01)；术后7d,14d,与模型组相比,针刺组、康复组、针康组Caspase-3mRNA表达减少(p<0.05)；术后7d,14d,与针刺组、康复组比较,针康组Caspase-3mRNA表达减少(p<0.05)；术后7d,14d,与针刺组比较,康复组Caspase-3mRNA的表达无明显差异,(P>0.05)。
结论
1.针康法能促进局灶性脑缺血后大鼠神经功能康复,且针康法优于单纯针刺和康复。
2.针康法能够促进局灶性脑缺血大鼠皮质缺血区周围GFAP,S100β的表达,促进胶质细胞活化,从而保护神经元,进而起到脑保护作用,且针康法优于单纯针刺和康复。
3.针康法能促进局灶脑缺血后BDNFmRNA的表达增加,保护神经元,与BDNF蛋白表达趋势一致,并且随时间递增其表达增加,且针康法优于单纯针刺和康复。
4.针康法能抑制局灶脑缺血后Caspase-3mRNA的表达,减少细胞凋亡,促进神经恢复,并且随时间递增其表达降低,且针康法优于单纯针刺和康复。
5.针康法能够调控脑缺血后胶质细胞环境,抑制神经细胞凋亡途径,这可能是针康法促进脑缺血神经恢复的机制之一。
Objective
To observe the effect of acupuncture in ischemic region of BDNF and S100β and GFAP protein and method on focal cerebral ischemia rats, and expression of BDNFmRNA, Caspase-3mRNA.
Method
Establishment of rat permanent focal cerebral ischemia model, In these standards in line with the selected240rats. Divided into5groups, Sham operation group, model group, acupuncture group, the rehabilitation group and acupuncture combined with rehabilitation group. Each group had48rats, Then each group at3,7,14.21days were divided into4groups,12rats in each group. Sham operation group, model group without any intervention, The acupuncture group were treated with head acupuncture therapy, The rehabilitation group was treated with treadmill training, Acupuncture combined with rehabilitation group was treated with head acupuncture treatment combined with treadmill training. Each time, Changes in the immunohistochemical detection of BDNF,S100β、GFAP in ischemic rats expression. point of the changes of nerve function test observation of neural function in rats, The detection of BDNF-mRNA,Caspase-3mRNA real-time fluorescent quantitative PCR
Result
1. The neurological function score, Each time point in each group, the neurological function score increased clearly (P<0.01); After7th days of treatment compared with the model group. The nerve function score of acupuncture group, rehabilitation group and comprehensive group decreased clearly (P<0.05). Compared with the acupuncture group and rehabilitation group, comprehensive group of neural function score was significantly lower (P<0.05). Acupuncture group and rehabilitation group was compared with similar.
2Expression of BDNF in the ischemic area:After the success of the model,3days,7days,14days, each group and sham operation were statistically significant(p<0.01).7days after making models, compared with the model group, expression of BDNF was significantly reduced in acupuncture group, rehabilitation group and comprehensive group (P<0.05).14days after making models, Comprehensive group compared with the model group were significantly different(p<0.01).
3The expression of S100β around ischemic cortex:on the3rd and7th day after the modeling, there were statistically difference between the sham operation group and other groups (P<0.05). On the3rd day after the modeling, to compared with the model group,S100β expression promoted in the CNSP combined with task-oriented training group and in the task-oriented training group (P<0.05). on the14th day after the modeling, there were statistically difference between the CNSP combined with task-oriented training group and model group (P<0.01). On the21St day after the modeling, there were statistically difference amongst in the CNSP combined with task-oriented training group, the task-oriented training group, the CNSP group and the model group(P<0.05).
4The expression of GFAP around ischemic cortex:On the7th and14th days after the modeling, to compared with the model group, the expression of GFAP in the CNSP combined with task-oriented training group, the task-oriented training group and the CNSP group enhanced (P<0.05), and they in the CNSP combined with task-oriented training groupwere superior to the task-oriented training group and the CNSP group (P<0.05) respective.There were statistically difference amongst in the CNSP combined with task-orientedtraining group, the task-oriented training group and the CNSP group (P<0.05). On the21St day after the modeling, the expression of GFAP in the CNSP combined with task-oriented training group were superior to the others (P<0.05).
5The expression of BDNFmRNA:Each time point in each group compared with the model group, BDNFmRNA expression was significantly increased (P<0.01). After7th days of treatment compared with the model group, expression of BDNFmRNA was significantly higher in acupuncture group, rehabilitation group and comprehensive group (P<0.05). Compared with the acupuncture group and rehabilitation group, Expression of BDNFmRNA was significantly higher in comprehensive group (P<0.05).Acupuncture group and rehabilitation group was compared with similar.
6. The expression of Caspase-3mRNA:Each time point in each group compared with the model group, Caspase-3mRNA expression was Increased significantly (P<0.01). After7th days of treatment compared with the model group, expression of Caspase-3mRNA was significantly reduced in acupuncture group, rehabilitation group and comprehensive group (P<0.05) Compared with the acupuncture group and rehabilitation group, Expression of Caspase-3mRNA was significantly reduced in comprehensive group (P<0.05).Acupuncture group and rehabilitation group was compared with similar.
Conclusion
1Acupuncture and rehabilitation can promote the recovery of neural function of rats after focal cerebral ischemia.
2Acupuncture and rehabilitation can promote the expression of proliferation in brain tissue after focal cerebral ischemia GFAP, S100β.
3Acupuncture and rehabilitation can promote the expression of proliferation in brain tissue after focal cerebral ischemia BDNF-mRNA.
4Acupuncture and rehabilitation promotes the expression of brain tissue after focal cerebral ischemia Caspase-3mRNA attenuation.
5Acupuncture combined with rehabilitation to glial cells after cerebral ischemia in environmental regulation, inhibition of neuronal apoptosis pathway. This may be one of the mechanisms of acupuncture combined with rehabilitation for cerebral ischemia nerve recovery.
引文
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