【作者】 李娜；

【导师】 邱幸凡； 张六通；

【作者基本信息】 湖北中医学院 ， 中医基础理论， 2009， 博士

【摘要】 目的：建立卫气虚大鼠模型，选择益气固表之玉屏风散制剂以治疗方式或预防方式作用于模型动物，观察动物造模及干预后在神经免疫、神经营养及神经递质方面的改变。根据实验结果，阐释卫气与神经传导之间的联系，明确卫气与神经传导相关的具体环节，从神经传导角度初步探讨卫气实现其生理功能的可能机制，为卫气的感传作用提供实验依据，为气脉理论作初步探索，丰富和充实中医的卫气学说和络脉学说内容。比较玉屏风散的治疗功效和预防功效，探讨益气固表之玉屏风散对卫气虚证的神经传导功能方面的影响，为玉屏风散临床运用范围的扩展提供依据。方法：理论研究以中医学卫气理论及络脉理论为基础，阐释卫气的内涵，并对神经传导的认识进行了分析。从卫气的生成、运行、功能三个方面与神经传导相比较，论证了卫气与神经传导的密切联系。实验研究：清洁级SD大鼠，200-250g，72只，随机分为6组：正常组、卫气虚模型组、低免疫对照组、模型预防组、模型治疗组及低免疫治疗组。卫气虚模型组、模型预防组、模型治疗组大鼠进行寒、热交替刺激实验：先将各组大鼠分次放入特制铁丝笼中，每笼6只，将笼子放入-20±1℃冰柜中20min，每隔10min观察一次。取出动物后，放常规饲养室饲养(室温25±1℃)，4h后进行温热刺激。即将各组大鼠分次放入40±1℃恒温箱中15min，每隔5min观察一次。取出后常规饲养。每天寒、热交替刺激一次，连续6d。低免疫对照组、低免疫治疗组采用低剂量腹腔注射环磷酰胺10mg／kg·d连续6d，造成免疫功能低下大鼠模型。正常组和卫气虚模型组、低免疫对照组：造模结束之日起灌服生理盐水，连续7天。模型预防组：造模之日起以玉屏风散冲剂灌胃1g／kg．d，连续7天。模型治疗组、低免疫治疗组：造模结束之日起以玉屏风散冲剂灌胃1g／kg．d，连续7天。灌胃结束之日摘眼球取大鼠血清，采取内固定法固定大鼠脑组织，取单侧坐骨神经及腹部皮肤。ELISA法测血清IL-2，血清及坐骨神经NGF含量；免疫组化染色检测皮肤及脑组织中P物质含量，脑组织的细胞凋亡。结果：卫气虚模型组血清IL-2含量较正常组升高，有非常显著性差异(P＜0．01)，与低免疫对照组比较无显著性差异(P＞0．05)，提示卫气虚模型具有免疫功能的变化；模型治疗组、模型预防组血清IL-2较卫气虚模型组降低，差异有非常显著性(P＜0．01)，模型预防组较模型治疗组血清IL-2降低，有非常显著性差异(P＜0．01)，提示玉屏风散对卫气虚有治疗作用，同时玉屏风散的预防作用有更好的疗效。卫气虚模型组血清NGF高于正常组，但无统计学意义；模型预防组血清NGF与卫气虚模型组、低免疫对照组、低免疫治疗组比较有非常显著性差异(P＜0．01)，与正常组比较有显著性差异(P＜0．05)；模型治疗组血清NGF与正常组、低免疫治疗组比较均有显著性差异(P＜0．05)，与卫气虚模型组、低免疫对照组比较有非常显著性差异(P＜0．01)；模型预防组血清NGF水平低于模型治疗组，但差异无统计学意义，说明玉屏风散的治疗和预防能够降低卫气虚模型的血清NGF水平。坐骨神经中的NGF水平变化与血清中的NGF水平变化并不同步，卫气虚模型组坐骨神经中的NGF含量较其它组低，与正常组比较有非常显著性差异(P＜0．01)，与模型预防组比较有显著性差异(P＜0．05)，提示卫气虚模型的周围神经中NGF含量下降，玉屏风散的预防作用可以防止此种变化。正常组、卫气虚模型组、模型治疗组、模型预防组、低免疫对照组及低免疫治疗组大鼠的腹部皮肤切片中皆可以见到棕黄色P物质阳性染色区域。卫气虚模型组与低免疫对照组皮肤组织中P物质阳性染色面积较正常组减少，有显著性差异(P＜0．05)，提示卫气虚损可引起皮肤P物质含量减少，此种变化可能与机体免疫机能降低有关。卫气虚模型脑组织中额叶皮质P物质阳性细胞计数低于正常组，有非常显著性差异(P＜0．01)，与模型预防组和模型治疗组比较有显著性差异(P＜0．05)，同时正常组与低免疫对照组比较有显著性差异(P＜0．05)，提示卫气虚模型可以降低大脑额叶皮质中P物质含量，玉屏风散的治疗和预防干预可以升高卫气虚模型脑组织中的P物质含量。低免疫对照组较正常组阳性细胞计数低，提示脑组织中的P物质改变与免疫状态相关。各组大脑额叶皮质中皆可见棕黄色TUNEL阳性细胞染色区域，其中卫气虚模型组TUNEL阳性细胞计数较正常组增加，有非常显著性差异(P＜0．01)，模型预防组TUNEL阳性细胞计数较卫虚组减少，有显著性差异(P＜0．05)，提示卫气虚变化可促进脑组织细胞的凋亡，而玉屏风散则可遏制此种变化。结论：通过寒热交替刺激制作的卫气虚模型具有免疫力低下的特征，符合卫气虚的微观辨证特点，同时发生了神经传导功能的改变，包括坐骨神经中NGF的降低，皮肤及脑组织中P物质的减少以及脑细胞凋亡的增加，说明卫气虚证候，不仅有着免疫功能的低下，也有神经系统功能的改变，证明了卫气除了共识的卫外免疫功能外还具有感应传导信息的功能，卫气与神经传导相关。益气固表的玉屏风散不仅能够改善低下的免疫力从而治疗卫气虚之证，还能通过影响血清及坐骨神经中NGF水平，影响皮肤及脑组织中P物质的含量以及脑组织中的细胞凋亡等途径来改善卫气虚所致的感应传导障碍。预防性地使用玉屏风散较玉屏风散的治疗作用更优。更多还原

【Abstract】 Objective: Establish the Weiqi-deficiency rat model, choosethe Yiqigubiao therapy _Yupingfengsan to the rats in treatment orprevent way. Obseve the change on the rats in neuro-immune,neurotrophic, neurotransmitter after making model andadministration. According to experiment results, interpret therelation between Weiqi and the nerve conduction and define theconcrete links, preliminarily investigate the perhaps mechanismthat Weiqi realize its physiological functions based on nerveconduction. Prove the conduction of Weiqi with experimentalapproach, preliminary exploration the theory of Qimai, Enrich theWeiqi theory and collaterals theory of traditional Chinese medicine.Compareing the treat effect and the prevention effect ofYupingfengsan, discuss the influence the nerve conduction onsyndrome of Weiqi-deficiency caused by Yupingfengsan. Provideevidence for extending the clinical application of Yupingfengsan.Methods: theoretical research is base oncollaterals and Weiqitheory of Chinese traditional medicine. Explained the connotationof Weiqi. Analyzed the theory of nerve conduction and comparedbetween Weiqi and nerve conduction from three aspects: generation,operation and function. Demonstrated the correlation of Weiqi andnerve conduction.Experimental research: 72 SD rats, weight range from 200g to250g, were divided into 6 groups by random: normal group, Weixugroup, lower immune group, model prevention group, model treating group and lower immune treating group. Took the cold and hotalternate stimulate on Weixu group, model prevention group andmodel treating group. First the rats were placed into specially madecages, 6 rats in one cage, then placed the cage into the refrigeratorunder the -20℃. 20 minutes later, took out the cage at normaltemperature. During this cold stimulate, observed the rats each10mins. After 4 hours, placed the rats into 40℃incubator for 15mins. Cold and hot alternate stimulate on rats once a day for 6 days.Rats of lower immune group and lower immune treating group wereintraperitoneal injected lower dose cyclophosphamide with 10mg/kgof once a day for 6 days to make lower immune model. Rats took thephysiological saline by gavage for 7 days after making model innormal group, Weixu model group and lower immune group. Rats ofmodel prevention group were taken 1g/kg.d Yupingfengsan for 7 daysby garage from the first day making model. Rats of the model treatinggroup and lower immune treating group were taken 1g/kg, dYupingfengsan for 7 days by gavage from the last day making model.Take blood serum, sciatic nerve, brain issue, ventral skin.Measured IL-2 content in serum and NGF content in serum and sciaticnerve by Enzyme-linked Immunosorbent Assay, measured SP content inskin and brain and apoptosis in brain by immunohistochemicalmethod.Results: The serum IL-2 content of Weixu group is higher thannormal group with extremely significant difference (P＜0.01)through the comparison, and without significant differencecomparing with lower immune group(P＞0.05).It suggests that Weixugroup have changed in immune. The serum IL-2 content of modeltreating group and model prevention group are lower than Weixu groupwith extremely significant difference (P＜0.01). The serum IL-2content of model prevention group is lower than model treating groupwith extremely significant difference (P＜0.01). It suggests thatYupingfengsan has the treat effect on Weixu model and the prevention effect is better than treatment. The NGF content in serum of Weixugroup is higher than normal group, but without significantdifference. The NGF content in serum of model prevention groupexists extremely significant difference (P＜0.01) comparing withWeixu group, lower immune group and lower immune treating group, andexists significant difference(P＜0.05)comparing with normal group.The NGF content in serum of model treating group exists extremelysignificant difference(P＜0.01)comparing with Weixu group and lowerimmune group, and exists significant difference (P＜0.05) comparingwith normal group and lower immune treating group. The NGF contentin serum of model prevention group is lower than model treatinggroup, but without significant difference (P＞0.05). It suggeststhat treatment or prevention with Yupingfengsan has the effect ofdecrease the NGF content in serum of Weixu model. The change on NGFcontent in sciatic nerve does not similar to it in serum. The NGFcontent in sciatic nerve of Weixu group is lower than normal groupwith extremely significant difference (P＜0.01),and existssignificant difference (P＜0.05) comparing with model preventiongroup. It suggests that NGF content in sciatic nerve have deceasedin Weixu group, and this change can be hindered by prevention effectof Yupingfengsan. The SP positive staining area can be seen in clipsof abdominal skin of all groups. But the area of SP positive stainingof Weixu group and lower immune group are smaller than normal groupwith significant difference (P＜0.05). It suggests that Weixu couldcause the decrease of SP in skin and this change may associate withthe low immune. The number of SP positive cell in frontal cortexof Weixu group is smaller than normal group with extremelysignificant difference (P＜0.01). The number of SP positive cellof model prevention group and model treating group has significantdifference (P＜0.05) comparing with Weixu group. It suggests thatWeixu could cause the decrease of SP in frontal cortex, and theprevention effect and treatment effect of Yupingfengsan could increase the contant of SP in skin of Weixu group. The number ofSP positive cell in frontal cortex of lower immune group is smallerthan normal group. It suggests that the change of SP in frontalcortex may associate with the low immune. Apoptosis in frontalcortex positive staining area can be seen in brain clips of allgroups. The number of TUNEL positive cell of Weixu group is biggerthan normal group with extremely significant difference (P＜0.01).And model prevention group is smaller than Weixu group withsignificant difference(P＜0.05). It suggests that making Weixu modelpromote the apoptosis in frontal cortex, and this change can behindered by prevention effect of Yupingfengsan.Conclusion: Weixu model made by the cold and hot alternatestimulate have the character of lower immune, which is accord withthe characteristics of syndrome differentiation in traditionalChinese medicine. Weixu model rats change on nerve conduction;include the decreasing NGF content in sciatic, decreasing SP of skinand frontal cortex and increasing apoptosis in frontal cortex. Itsuggests that the syndrome of Weixu has the characters not only oflower immune, but also on nerve conduction. It proved that Weiqihave the function not only in immune but also in informationconduction. Weiqi is related with nerve conduction.Yupingfengsan has the treat effect on syndrome of Weixu throughincreasing immune, and improve the conduction-hinder in syndromeof Weixu by influencing NGF content in serum, SP content in skinand frontal cortex and apoptosis in frontal cortex. And preventive-useYupingfengsan has better effect than treat use.更多还原