【作者】 张哲荣；

【导师】 靳瑞；

【作者基本信息】 广州中医药大学 ， 针灸推拿学， 2011， 博士

【摘要】 背景新生儿缺氧缺血性脑病(hypoxic-ischemic brain damage, HIBD)是指由于围生期窒息、缺氧所导致的脑缺氧缺血性损害,临床出现一系列神经系统异常的表现。常见于严重窒息的足月新生儿,严重者可死于新生儿早期,幸存者多留有神经系统损伤后后遗症,如智能低下、脑瘫、癫痫、共济失调等。是围生期脑损伤的最重要原因。大量研究表明HIBD的发病原因及发病机制极为复杂,多方面综合因素有关,但存在共同的病理变化,即脑部缺氧缺血,引起脑区神经细胞凋亡,并且神经元细胞凋亡的发生与HIBD患儿的预后有着密切的联系。神经细胞凋亡受一系列基因表达的调控,表现在缺血损伤后许多基因转录翻译,这些基因编码合成的蛋白质产物直接或间接参与了脑缺血后神经元凋亡过程。在参与调控神经元凋亡的基因和蛋白质产物中,核因子κB (nuclear factor-κB, NF-κB)和caspase-3备受关注。针灸作为祖国医学的重要主成部分,在小儿脑瘫临床研究方面,取得了较好的成绩,明显改善了脑瘫患儿的神经功能缺损症状,但基础研究方面,开展较少,具体机制尚不清楚。本课题组,前期观察到了早期针灸干预缺血缺氧损伤的病理模型,可以对脑组织单胺类神经递质与氨基酸含量,以及细胞凋亡相关的Bax与Bcl-2造成影响。缺血缺氧损伤后的病理改变机制,甚为复杂,针灸干预可能是多方面的综合作用,借此,本研究在前期工作基础上,观察早期针灸干预对核因子-κB (nuclear factor-κB, NF-kB)和caspase-3基因表达的影响,试图从多方面探讨针灸对于新生儿缺血缺氧性脑病的作用机制。目的观察针刺结合艾灸督脉对脑瘫模型幼鼠运动功能、平衡功能、学习记忆功能及脑组织神经细胞凋亡相关NF-Kb、Caspase-3蛋白表达的影响,探讨早期针灸治疗小儿脑瘫凋亡相关的作用机制,为针灸疗法的临床应用提供客观的科学依据。方法(一)HIBD模型造模方法SPF级7日龄新生SD幼鼠,在室温26℃环境下,用皮筋在手术台上固定造模组幼鼠的四肢及头部,在乙醚吸入浅麻醉下,小心剪开颈部皮肤,分离迷走神经并暴露左侧颈总动脉,用细丝线双线结扎该动脉并中间离断,造成永久性断流。缝合好皮肤后放入37℃恒温水浴容器中恢复2h,然后密闭容器,整个手术时间控制在5min内,保证实验幼鼠皮肤红润。如果动物出血或者呼吸抑制,则弃去不用。以1.0L/min流速将含8%氧气的92%氮氧混合气持续通入缺氧箱中,缺氧箱置于水温37℃的恒温水浴锅内,缺氧2.5h,1h后作行为测定。(二)分组及治疗方法1.正常组：仅同方式抓取,不做造模及治疗干预。2.假手术组：麻醉后分离左颈总动脉,但不结扎,术后亦不作低氧处理3.模型组：进行造模,不做治疗干预。4.针刺组：所有需要针刺治疗的小鼠针四肢及头部穴位。以直径2mm,长20mm的针灸针,先在“内关”、“涌泉”速刺不留针,然后针四肢及头部穴位。四肢穴位“曲池”、“足三里”穴针1分深左右,头部穴位“百会”、“颞Ⅰ针”平刺,进针后觉针下沉紧即可,留针10min。5.针刺+艾灸组：针刺基础上结合艾灸督脉,温和灸“督脉”10分钟。(三)定位方法：穴位定位参照《实验针灸学》,结合人与动物骨度类比：“督脉”为尾根与肛门之间的凹陷中；顶骨正中定“百会”；外耳道口直上0.8cm处模拟“颞Ⅰ针”；桡骨近端的关节外侧,离腕关节约3mm左右的尺桡骨缝间取“内关”；“曲池”位于桡骨近端的关节外侧前方的凹陷中；“足三里”在膝关节后外侧,腓骨小头下约5mm处；后肢掌心前正中取“涌泉”。(四)治疗时间手术当天称重,术后1d休息,术后2d检测悬吊试验和斜坡试验,术后3d开始治疗6天,术后9d检测悬吊试验和斜坡试验,术后10再次治疗6天,术后16d检测悬吊试验和斜坡试验,术后18d称重。(五)指标观测1.行为运动能力及学习记忆能力测试：悬吊试验、斜坡试验、Y迷宫试验2.脑组织病理学形态：HE染色病理检查3.脑组织中NF-Kb、Caspase-3蛋白表达：免疫组化法测结果1.造模情况：死亡率18/54=33.3%,成功率33/54=61.11%2.体重变化治疗前各组幼鼠体重比较,差异无统计学意义(P>0.05),说明各组幼鼠之间体重均衡,组间具有可比性。治疗后,自身前后比较：五组的治疗后体重均高于治疗前,差异有统计学意义(P<0.05)；组间比较,差异有统计学意义(P<0.05)；进行两两比较后,模型组体重明显低于其他组,差异有统计学意义(P<0.05)；进行两次体重测量的差值进行比较,组间差异有统计学意义(P<0.05)；两两比较后,正常组、假手术组、针刺结合艾灸组比较差异无统计学意义(P>0.05)；模型组、单纯针刺组明显低于其他三组,差异有统计学意义(P<0.05)；单纯针刺组高于模型组,差异有统计学意义(P<0.05)；3.悬吊试验相同时间点,不同组间悬吊时间比较：治疗前,模型组、单纯针刺组、针刺结合艾灸组都低于正常组与假手术组(P<0.05)；第一阶段治疗后,模型组、单纯针刺组、针刺结合艾灸组仍全部低于正常组与假手术组(P<0.05)；但针刺结合艾灸组高于模型组及单纯针刺组(P<0.05)；第二阶段治疗后,模型组、单纯针刺组、针刺结合艾灸组仍全部低于正常组与假手术组(P<0.05)；但针刺结合艾灸组高于模型组及单纯针刺组(P<0.05)；单纯针刺组高于模型组(P<0.05)。同一组别,不同时间点悬吊时间比较：各组幼鼠治疗前、第一阶段治疗后、与第二阶段治疗后悬吊时间比较,差异有统计学意义(P<0.05)；分别进行两两比较,五组均存在：幼鼠悬吊时间第一阶段治疗后高于治疗前,第二阶段高于第一阶段,差异均有统计学意义(P<0.05)。4.斜坡试验相同时间点,不同组间斜坡时间比较：治疗前,模型组、单纯针刺组、针刺结合艾灸组都低于正常组与假手术组(P<0.05)；第一、二阶段治疗后,模型组、单纯针刺组、针刺结合艾灸组仍全部低于正常组与假手术组(P<0.05)；但针刺结合艾灸组高于模型组及单纯针刺组(P<0.05)；单纯针刺组高于模型组(P<0.05)。同一组别,不同时间点斜坡时间比较：各组幼鼠治疗前、第一阶段治疗后、与第二阶段治疗后悬吊时间比较,差异有统计学意义(P<0.05)；分别进行两两比较,五组均存在：幼鼠斜坡时间第一阶段治疗后高于治疗前,第二阶段高于第一阶段,差异均有统计学意义(P<0.05)。5.Y迷宫结果各组幼鼠Y迷宫正确次数不同,差异有统计学意义(P<0.05),进行两两比较,除正常组与假手术组比较,差异无统计学意义(P>0.05)外,其余组间两两比较,差异均有统计学意义(P<0.05)；由高到低依次为针刺+艾灸组、单纯针刺组、模型组。6.脑组织病理改变脑组织常规石蜡切片：HE染色,神经细胞胞浆呈淡紫色,核呈蓝黑色。正常组及假手术组神经细胞结构正常,胞浆丰富,胞核清晰可见,海马锥体细胞3-4层,相邻细胞间排列紧密。模型组皮层变薄,大部分神经元变性,体积变小,核固缩成不规则形,核仁消失,胞核与胞浆界限模糊。单纯针刺组有少量神经细胞变性,体积变小,核固缩少见。单纯针刺结合艾灸组神经细胞排列仍不整齐,少量神经元变性。针刺结合艾灸组神经细胞形态、排列基本正常,仅有少量的神经细胞变性,零星可见极少量损伤细胞,海马锥体细胞1-3层,相邻细胞排列比较紧密。7.各组大鼠脑皮质正常神经细胞数目的比较与正常组比较,模型组脑皮质正常神经细胞数目低于正常组及假手术组,差异具有统计学意义(P<0.05)；与模型组比较,针刺组、针灸组高于模型组正常神经细胞数目,差异具有统计学意义(P<0.05)；针刺组与针灸组比较,差异无统计学意义(P>0.05)。8.脑组织NF-Kb蛋白表达光密度值,与正常组比较,模型组脑组织NF-Kb蛋白表达高于正常组及假手术组,差异具有统计学意义(P<0.05)；与模型组比较,针刺组、针灸组低于模型组,差异具有统计学意义(P<0.05)；针刺组与针灸组比较,差异无统计学意义(P>0.05)。9.脑组织Caspase-3蛋白表达正常组及假手术组,仅在皮层个别区域有少量Caspase-3阳性细胞。模型组中,皮层出现明显的Caspase-3免疫阳性细胞,其平均光密度值与正常组比较,差异有统计学意义(P<0.05)；针刺及针灸治疗后Caspase-3蛋白表达较模型组明显减少,差异有统计学意义(P<0.05)；针刺组和针灸组间Caspase-3表达,差异无统计学意义(P>0.05)。结论1.缺血缺氧性损伤引起了幼鼠脑神经细胞凋亡,并且与细胞凋亡NF-κB、caspase-3基因表达密切相关。2.早期针灸干预对脑瘫幼鼠肌力(悬吊试验),平衡能力(斜坡试验)、学习记忆功能(Y迷宫)均有促进作用,并且针刺结合艾灸优于单纯针刺组。3.早期针灸干预对影响了脑瘫幼鼠脑组织病理形态学改变,治疗后,变性神经细胞数量显著减少,零星可见一些损伤细胞,海马锥体细胞层细胞形态、排列基本正常。4.早期针灸干预对脑瘫幼鼠脑组织神经细胞凋亡NF-κB、caspase-3基因表达具有良性调节作用,可能是针灸疗法治疗小儿脑瘫的作用机制。更多还原

【Abstract】 BackgroundNeonatal hypoxic-ischemic brain damage (HIBD) is due to asphyxia caused cerebral blood supply and gas exchange impairment caused by a brain injury, a serious disease endangering the health of newborn lives, have a higher morbidity and mortality, also one of the important reasons contributed to children’s mental retardation, cerebral palsy and other neurological disabilities. The causes and pathogenesis of HIBD are more complex; more and more evidence has showed that apoptosis is also involved and plays a unique role. The occurrence of neuronal apoptosis and prognosis in children is closely linked. At the molecular level, apoptosis is affected by many genes and the regulation of cytokines, nuclear factor-κB and caspase-3 are the research focus.Acupuncture and moxibustion are the important part of traditional Chinese medicine.They are very useful for children with cerebral palsy. We have did a lot of work in clinical research, but a little work in experiment research. We could not understand clearly the specific mechanism of acupuncture and moxibustion. For of all the reasons, our team investigated the effect of acupuncture and moxibustion under governor vessel treatment on movement function, balance function, study and memery function, and brain NF-Kb, Caspase-3 expressing in the HIBD rats. Attempts to explore the mechanism of acupuncture and moxibustion for hypoxic-ischemic brain damage from various aspects.ObjectiveTo investigate the effect of acupuncture and moxibustion under governor vessel treatment on movement function, balance function, study and memery function, and brain NF-Kb、Caspase-3 expressing in the CP rats. Meanwhile, to understand the mechanism of early acupuncture and moxibustion treatment for CP, and to provide the theoretical basis for the promotion of early acupuncture and moxibustion treatment in clinical use for children with cerebral palsy.Methods1. Established a simulation model of perinatal asphyxia HIBD2. The animals were randomly divided into five groups:normal group, sham group, model group, simple acupuncture group and acupuncture with moxibustion group. We developed a treatment timetable with acupoints reference from (Study of experimental acupuncture), combining human and animal bones analogy: acupoints taken for treatment include parietal bone center for’Baihui’ external ear canal, straight 0.8cm simulate take’Temporal 1 needle’; radial bone proximal lateral aspect from the wrist off 3cm or so take’Neiguan’ radius proximal lateral joint when elbow is flexed take’Quchi’; anterior aspect of lower leg under the fibular head and about 5mm take’Zusanli’ hind legs before the middle of the sole take’Yongquan’. After treatment for two stages, separately carry out tests for HIBD mouse model on behavior motor ability and on learning and memory ability, and then after mice were sacrificed, examine the model mouse brain tissue and related gene expression targets.3. Hanging test, Slope test and Y maze test were used for valuing neuroethology function in different groups.The method of immunohistochemistry was used to observe the HIDB mouse dynamic expression of apoptosis regulatory proteins NF-Kb and Caspase-3.Results1. Modeling resultsMortality rate:33.3%, Success rate:61.11%2. Change of weightMultiple comparison results showed that:the normal group, sham operation group, acupuncture group, acupuncture and moxibustion group were no significant difference between the four groups, model group and acupuncture group was no statistical difference neither. The first four groups compared with the other groups, P<0.01, both statistically significant weight gain can be considered the first four group weight gain more than two groups. Area group F=15.897, P>0.05, shows that factors between area group was not statistically significant.Before intervention, there were no significant difference among five groups. After intervention, the weight of rats were heavier than before. Model group was lighter than other groups. From heavy to light, the order was normal group, sham operation group, acupuncture and moxibustion group, acupuncture group and model group.3. Hanging testBefore intervention, model group, acupuncture group and acupuncture with moxibustion group were worse than normal group and sham operation group.After the first treatment, the situation was the same as before., but acupuncture with moxibustion group was better than acupuncture groupAfter the second treatment, model group, acupuncture group and acupuncture with moxibustion group were also worse than normal group and sham operation group. From better to worse,the order was acupuncture and moxibustion group, acupuncture group and model group.After intervention, the valu of Hanging test were better than before in five groups.4. Slope testBefore intervention, model group, acupuncture group and acupuncture with moxibustion group were worse than normal group and sham operation group.After the first treatment, the situation was the same as before., but acupuncture with moxibustion group was better than acupuncture groupAfter the second treatment, model group, acupuncture group and acupuncture with moxibustion group were also worse than normal group and sham operation group. From better to worse, the order was acupuncture and moxibustion group, acupuncture group and model group.After intervention, the valu of Slope test were better than before in five groups.5. Y maze testThe times of right movement, there were significant difference among five groups. From better to worse, the order was acupuncture and moxibustion group, acupuncture group, model group, normal group and sham operation group.6. Pathological changes in brain tissueHE staining:normal light microscope under 10X20 times for each section were observed within 5 View pathological changes after cerebral ischemia. Each group were pathological changes in rat brain:brain tissue of paraffin, HE staining, nerve cells of pale red, blue-black nuclear was. Normal control group and the sham operation group structure of the normal cortical brain cells, clear nucleus, cytoplasm-rich, interstitial edema-free performance, hippocampal pyramidal cells in 2 to 3 layers, closely arranged, round and large nucleus, clear nucleolus. Model group, cortical thinning, part of the neuronal degeneration and smaller, the nucleus and cytoplasm of ambiguous nuclear fast shrunk triangles or irregular, nucleoli disappear. Reduce hippocampal pyramidal cell layer, arranged in sparse, irregular, smaller cell volume, nuclear condensation phenomenon can be seen. Acupuncture group, acupuncture combined with moxibustion group, there are different degrees of nerve cell degeneration, but significantly reduced the number of nerve cell degeneration, sporadic seen a number of injured cells, hippocampal pyramidal cell layer of cell morphology, arrangement normal.7. Counting of normal nerve cells in brain cortexModel group was worse than normal group and sham operation group.Acupuncture with moxibustion group and acupuncture group were better than model group. There were no significant difference between acupuncture with moxibustion group and acupuncture group.8. Expressing of proteins NF-ΚbModel group was worse than normal group and sham operation group.Acupuncture with moxibustion group and acupuncture group were better than model group. There were no significant difference between acupuncture with moxibustion group and acupuncture group.9. Expressing of proteins Caspase-3Model group was worse than normal group and sham operation group.Acupuncture with moxibustion group and acupuncture group were better than model group. There were no significant difference between acupuncture with moxibustion group and acupuncture group.Conclusion1. Hypoxic-ischemic brain injury in rats caused neural cell apoptosis. There were clsed relationship between neural cell apoptosis and expressing of proteins NF-Κb and Caspase-3.2. The therapy of early combining acupuncture with moxibustion was useful for the improvement of neural function, and acupuncture with moxibustion group was better than acupuncture group.3. The therapy of early combining acupuncture could improve the pathological changes in brain tissue.4. The therapy of early combining acupuncture has positive regulation in the expressing of proteins NF-Kb and Caspase-3.更多还原