青鹏膏剂穴位贴敷对大鼠皮肤组织结构及肥大细胞释放的肝素等生物活性物质影响的研究
发布时间：2018-09-18 15:03
【摘要】：穴位贴敷疗法,最早见于《五十二病方》,是以中医整体观理论为指导,依据经络学说,将中药作用于腧穴上,继而通过经络作用于机体,从而达到疏通气血、调整阴阳的作用。其特点是操作简便、用药安全、经济廉价。青鹏膏剂为藏药经典方剂,是由选自于青藏高原的传统藏药亚大黄、铁棒锤、棘豆、诃子、毛诃子、人工麝香、安息香等9味组成,经特殊工艺加工而成的外用膏剂,具有清热解毒、活血化瘀、祛风除湿、消肿止痛等功效。青鹏膏剂穴位贴敷,是将药物、经络与穴位的有机融合,一方面发挥中药的功效,另一方面发挥经络腧穴对机体的影响,二者在临床治疗中应用广泛,但在实验研究方面欠缺。本实验通过组织形态学和分子生物学技术检验,观察青鹏膏剂穴位贴敷对皮肤组织的影响,进而研究对机体的影响,分析其可能的作用机制。目的在于为进一步研究青鹏膏剂提供实验依据,也为临床应用青鹏膏剂穴位贴敷治疗疾病寻求科学根据。目的:观察青鹏膏剂穴位贴敷对大鼠穴区内皮肤组织形态结构、血清中肝素等生物活性物质的含量以及皮下组织血管内皮细胞间粘附因子-1(ICAM-1)的表达的影响,探讨青鹏膏剂穴位贴敷可能的药理作用机制,为临床运用提供有价值的实验依据。方法:1实验动物分组及处理办法SPF级雄性Wistar大鼠40只,按随机数字表法,分为空白组(A组)、凡士林贴敷组(B组)、电针组(C组)和青鹏膏剂贴敷组(D组),每组10只。大鼠固定后,根据郭义主编的《实验针灸学》大鼠常用针灸穴位,选取大椎穴为中心、直径为1.5cm的区域,穴区局部剃毛。空白对照组大鼠相同时间和方法固定,但不给予涂药或针刺刺激。电针大椎穴组在大椎穴用毫针针刺,得气后针柄接电针治疗仪G6805-Ⅱ型,刺激参数为2/15Hz、1m A,2次/天,每次20min。凡士林贴敷大椎穴组大鼠在大椎穴区均匀涂抹医用凡士林(约25mg),青鹏膏剂贴敷大椎穴组大鼠每次于穴区局部均匀涂抹青鹏膏剂(约25mg),2次/天,每次20min。干预7天。2指标检测2.1改良的乙酰胆碱酯酶染色结合甲苯胺蓝复染方法观察大鼠穴区皮肤组织内肥大细胞(Mast cell,MC)的分布和形态特征以及与皮肤组织内其他结构的联系。2.2 ELISA法检测大鼠血清中肝素、缓激肽、前列腺素含量2.3原位杂交技术定位、定量检测大鼠皮下组织血管内皮细胞间粘附因子-1(ICAM-1)的表达以及观察肥大细胞在小血管周围的分布。结果:1青鹏膏剂穴位贴敷对大鼠穴区皮肤组织内MC的分布和形态特征以及对皮肤组织内其他结构的影响。1.1观察穴区内肥大细胞(MC)分布规律本实验的组织切片形态学观察显示:以青鹏膏剂组为例说明,各组大鼠“大椎”穴区真皮、皮下组织和肌肉组织中都分布着大小、数量不等的MC,多成条带状分布,以皮下组织层分布最为广泛;1.2各组“大椎”穴区MC总数及脱颗粒率的比较数据统计表明:与空白组、凡士林组相比,青鹏膏剂组和电针组两组穴区组织局部MC数量及脱颗粒显著升高,差异显著(P0.01);空白组与凡士林组、电针组与青鹏膏剂组分别做组间比较,差异无显著性(P0.05),各组穴区MC数量及脱颗粒率分析比较为:青鹏膏剂组电针组凡士林贴敷组空白对照组;1.3各组穴区内皮肤组织形态结构的变化MC多呈圆形、卵圆形,胞质内充满异染性蓝色颗粒,多沿神经或血管等分布。光学显微镜下,观察到青鹏膏剂组穴区内,有经过亚铁氰化铜法乙酰胆碱酯酶特殊染色后成蓝绿色的神经纤维束,以网状分布交织于血管,且周围分布有MC。MC与血管壁的乙酰胆碱酯酶阳性神经纤维发生直接接触。2青鹏膏剂穴位贴敷对大鼠血清中肝素、缓激肽、前列腺素含量的影响2.1青鹏膏剂穴位贴敷组和电针组大鼠血清肝素含量均明显高于空白对照组和凡士林贴敷组,差异显著(P0.01);青鹏膏剂穴位贴敷组大鼠血清肝素含量高于电针组,差异有统计学意义(P0.05),空白组与凡士林组间比较,差异不显著(P0.05);2.2青鹏膏剂穴位贴敷组血清缓激肽、前列腺素含量均明显低于其他三组,组间比较差异较显著(P0.05);电针组大鼠血清缓激肽、前列腺素含量均明显高于其他三组,组间比较差异有统计学意义(P0.01或P0.05);空白组与凡士林组间比较,差异不显著(P0.05)。3青鹏膏剂穴位贴敷对大鼠皮下组织血管内皮细胞间粘附因子-1(ICAM-1)的表达及肥大细胞分布的影响3.1组织切片形态学观察显示,青鹏膏剂贴敷组和电针组的穴区小血管内皮ICAM-1 mRNA阳性反应的显色程度和面积均明显强于其他两组。统计分析表明:青鹏膏剂穴位贴敷组和电针组大鼠血管内皮ICAM-1mRNA阳性反应物吸光度值明显高于其他两组,差异显著(P0.01);青鹏膏剂组与电针组,空白组与凡士林分别做组间比较,差异不显著(P0.05);3.2各组“大椎”穴区真皮、皮下组织和肌肉组织中都分布着大小、数量不等的MC,多成条带状分布,以皮下组织层分布最为广泛。青鹏膏剂组与电针组穴区组织内肥大细胞趋向中、小血管聚集,分布明显多于其他两组。数据统计表明:青鹏膏剂组与其他三组比较,穴区组织小血管周围MC数量显著升高,差异显著(P0.01);电针组MC数量统计高于凡士林组和空白组(P0.05);空白组与凡士林组做组间比较,差异无显著性(P0.05),各组小血管标记的穴区MC数量比较为青鹏膏剂组电针组凡士林贴敷组空白对照组。结论:1青鹏膏剂穴位贴敷可使大鼠皮肤组织内形成肥大细胞+血管+神经网络复合状条带结构,以及引起穴区内肥大细胞募集和脱颗粒,这与青鹏膏剂穴位贴敷显效密切相关;2青鹏膏剂穴位贴敷能够提高大鼠血清中肝素的含量,而降低血清中缓激肽、前列腺素含量,这可能与青鹏膏剂穴位贴敷的作用机理相关。3青鹏膏剂穴位贴敷可增强大鼠穴区组织血管内皮ICAM-1mRNA的表达,ICAM-1对青鹏膏剂促进肥大细胞向穴区迁移、募集有一定的趋化作用。
[Abstract]:Acupoint sticking therapy, first seen in <52 Disease Prescription>, is guided by the holistic view of traditional Chinese medicine, according to the theory of meridians and collaterals, the traditional Chinese medicine acts on acupoints, and then acts on the body through meridians and collaterals, so as to dredge blood and regulate Yin and yang. It is composed of 9 traditional Tibetan medicines selected from the Qinghai-Tibet Plateau, such as Yarhuba, Hammerhead, Oxytropis, Terminalia chebula, Musk, Benzoin, etc. It is processed by special technology and has the effects of clearing away heat and toxicity, activating blood circulation and removing stasis, dispelling wind and dampness, relieving swelling and pain. On the one hand, it exerts the effect of traditional Chinese medicine, on the other hand, it exerts the influence of meridians and acupoints on the body. Both of them are widely used in clinical treatment, but lack of experimental research. Objective: To provide experimental basis for further study of Qingpeng ointment and to provide scientific basis for clinical application of Qingpeng ointment in acupoint application for treating diseases. Methods: 1. 40 male Wistar rats of SPF grade were divided into blank group (group A) and vaseline group (group A) according to random number table method. Group B, group C and group D were treated with Qingpeng ointment. After fixation, according to the acupuncture points commonly used in rats edited by Guo Yi, Dazhui acupoint was selected as the center, and the area with diameter of 1.5 cm was shaved locally. In the electroacupuncture group of Dazhui point, the filiform needle was used for acupuncture at Dazhui point, and the needle handle was connected with the electroacupuncture therapeutic apparatus G6805-II. The stimulation parameters were 2/15 Hz, 1 mA, 2 times a day, 20 minutes each time. The distribution and morphological characteristics of mast cells (MC) in rat skin tissues were observed by modified acetylcholinesterase staining combined with toluidine blue re-staining method. The relationship between MC and other structures in rat serum was detected by ELISA. Heparin, bradykinin and prostaglandin were localized by in situ hybridization. The expression of ICAM-1 in rat subcutaneous tissue was quantitatively detected and the distribution of mast cells around small blood vessels was observed. Results: 1. The distribution and morphological characteristics of MC in rat skin and skin were observed by Acupoint Application of Qingpeng plaster. 1.1 Observation of the distribution regularity of mast cells (MC) in the acupoint area The morphological observation of the histological sections in this experiment showed that: Taking Qingpeng ointment group as an example, the size and quantity of MC were distributed in the dermis, subcutaneous tissue and muscle tissue of the "Dazhui" acupoint area of rats in each group. The total number of MC and degranulation rate in Dazhui acupoint area of each group were the most widely distributed. 1.2 Compared with the blank group and Vaseline group, the number and degranulation rate of local MC in Qingpeng ointment group and electroacupuncture group were significantly higher than those in the blank group and Vaseline group, electroacupuncture group and Qingpeng ointment group respectively (P 0.01). There was no significant difference between groups (P 0.05). The number and degranulation rate of MC in each group were as follows: the Qingpeng ointment group was treated with Vaseline as the blank control group; 1. Microscopically, the acupoints of Qingpeng ointment group were specially stained with cuprous ferrocyanide acetylcholinesterase to form blue-green nerve fiber bundles, which were interwoven with blood vessels in a reticular pattern, and MC. MC contacted directly with acetylcholinesterase positive nerve fibers of blood vessel wall. 2 Qingpeng ointment was applied to acupoints of rats. Effect of Qingzhong Heparin, Bradykinin, Prostaglandin Content 2.1 Qingpeng Plaster Acupoint Application Group and Electroacupuncture Group rats serum heparin content were significantly higher than the blank control group and Vaseline application group, the difference was significant (P 0.01); Qingpeng Plaster acupoint application group rats serum heparin content was higher than the electroacupuncture group, the difference was statistically significant (P 0.05), blank group and Electroacupuncture group Compared with the other three groups, the difference was not significant (P 0.05); 2.2 Qingpeng plaster acupoint application group serum bradykinin, prostaglandin content were significantly lower than the other three groups, the difference was significant (P 0.05); Electroacupuncture group rats serum bradykinin, prostaglandin content were significantly higher than the other three groups, the difference was statistically significant (P 0.01 or P 0.05). There was no significant difference between the blank group and the vaseline group (P Statistical analysis showed that the absorbance value of ICAM-1 mRNA positive reactant in the Qingpeng ointment acupoint application group and electroacupuncture group was significantly higher than that in the other two groups (P 0.01); Qingpeng ointment group and electroacupuncture group, blank group and vaseline group were compared, the difference was not significant. (P 0.05); 3.2 MCs of different sizes and quantities were distributed in the dermis, subcutaneous tissue and muscle tissue of Dazhui acupoint area of each group, and most widely distributed in subcutaneous tissue layer. Ming: Compared with the other three groups, the number of MC around the small vessels in Qingpeng ointment group increased significantly (P 0.01); the number of MC in electroacupuncture group was higher than that in vaseline group and blank group (P 0.05); there was no significant difference between the blank group and vaseline group (P 0.05); the number of MC around the small vessels in each group was higher than that in Qingpeng ointment group. Conclusion: Acupoint application of Qingpeng ointment can induce the formation of mast cell + blood vessel + neural network complex strip structure in rat skin tissue, and cause the collection and degranulation of mast cells in acupoint area, which is closely related to the obvious effect of Qingpeng ointment on acupoint application; 2 Qingpeng oint application can improve the effect of acupoint application. Higher serum heparin, lower serum bradykinin, prostaglandin content, which may be related to the Qingpeng ointment acupoint sticking mechanism. 3 Qingpeng ointment acupoint sticking can enhance the expression of ICAM-1 mRNA in vascular endothelium of rat acupoint tissue, ICAM-1 on Qingpeng ointment to promote the migration of mast cells to acupoint region, recruitment has a certain tendency. Chemical action.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
本文编号：2248288
[Abstract]:Acupoint sticking therapy, first seen in <52 Disease Prescription>, is guided by the holistic view of traditional Chinese medicine, according to the theory of meridians and collaterals, the traditional Chinese medicine acts on acupoints, and then acts on the body through meridians and collaterals, so as to dredge blood and regulate Yin and yang. It is composed of 9 traditional Tibetan medicines selected from the Qinghai-Tibet Plateau, such as Yarhuba, Hammerhead, Oxytropis, Terminalia chebula, Musk, Benzoin, etc. It is processed by special technology and has the effects of clearing away heat and toxicity, activating blood circulation and removing stasis, dispelling wind and dampness, relieving swelling and pain. On the one hand, it exerts the effect of traditional Chinese medicine, on the other hand, it exerts the influence of meridians and acupoints on the body. Both of them are widely used in clinical treatment, but lack of experimental research. Objective: To provide experimental basis for further study of Qingpeng ointment and to provide scientific basis for clinical application of Qingpeng ointment in acupoint application for treating diseases. Methods: 1. 40 male Wistar rats of SPF grade were divided into blank group (group A) and vaseline group (group A) according to random number table method. Group B, group C and group D were treated with Qingpeng ointment. After fixation, according to the acupuncture points commonly used in rats edited by Guo Yi, Dazhui acupoint was selected as the center, and the area with diameter of 1.5 cm was shaved locally. In the electroacupuncture group of Dazhui point, the filiform needle was used for acupuncture at Dazhui point, and the needle handle was connected with the electroacupuncture therapeutic apparatus G6805-II. The stimulation parameters were 2/15 Hz, 1 mA, 2 times a day, 20 minutes each time. The distribution and morphological characteristics of mast cells (MC) in rat skin tissues were observed by modified acetylcholinesterase staining combined with toluidine blue re-staining method. The relationship between MC and other structures in rat serum was detected by ELISA. Heparin, bradykinin and prostaglandin were localized by in situ hybridization. The expression of ICAM-1 in rat subcutaneous tissue was quantitatively detected and the distribution of mast cells around small blood vessels was observed. Results: 1. The distribution and morphological characteristics of MC in rat skin and skin were observed by Acupoint Application of Qingpeng plaster. 1.1 Observation of the distribution regularity of mast cells (MC) in the acupoint area The morphological observation of the histological sections in this experiment showed that: Taking Qingpeng ointment group as an example, the size and quantity of MC were distributed in the dermis, subcutaneous tissue and muscle tissue of the "Dazhui" acupoint area of rats in each group. The total number of MC and degranulation rate in Dazhui acupoint area of each group were the most widely distributed. 1.2 Compared with the blank group and Vaseline group, the number and degranulation rate of local MC in Qingpeng ointment group and electroacupuncture group were significantly higher than those in the blank group and Vaseline group, electroacupuncture group and Qingpeng ointment group respectively (P 0.01). There was no significant difference between groups (P 0.05). The number and degranulation rate of MC in each group were as follows: the Qingpeng ointment group was treated with Vaseline as the blank control group; 1. Microscopically, the acupoints of Qingpeng ointment group were specially stained with cuprous ferrocyanide acetylcholinesterase to form blue-green nerve fiber bundles, which were interwoven with blood vessels in a reticular pattern, and MC. MC contacted directly with acetylcholinesterase positive nerve fibers of blood vessel wall. 2 Qingpeng ointment was applied to acupoints of rats. Effect of Qingzhong Heparin, Bradykinin, Prostaglandin Content 2.1 Qingpeng Plaster Acupoint Application Group and Electroacupuncture Group rats serum heparin content were significantly higher than the blank control group and Vaseline application group, the difference was significant (P 0.01); Qingpeng Plaster acupoint application group rats serum heparin content was higher than the electroacupuncture group, the difference was statistically significant (P 0.05), blank group and Electroacupuncture group Compared with the other three groups, the difference was not significant (P 0.05); 2.2 Qingpeng plaster acupoint application group serum bradykinin, prostaglandin content were significantly lower than the other three groups, the difference was significant (P 0.05); Electroacupuncture group rats serum bradykinin, prostaglandin content were significantly higher than the other three groups, the difference was statistically significant (P 0.01 or P 0.05). There was no significant difference between the blank group and the vaseline group (P Statistical analysis showed that the absorbance value of ICAM-1 mRNA positive reactant in the Qingpeng ointment acupoint application group and electroacupuncture group was significantly higher than that in the other two groups (P 0.01); Qingpeng ointment group and electroacupuncture group, blank group and vaseline group were compared, the difference was not significant. (P 0.05); 3.2 MCs of different sizes and quantities were distributed in the dermis, subcutaneous tissue and muscle tissue of Dazhui acupoint area of each group, and most widely distributed in subcutaneous tissue layer. Ming: Compared with the other three groups, the number of MC around the small vessels in Qingpeng ointment group increased significantly (P 0.01); the number of MC in electroacupuncture group was higher than that in vaseline group and blank group (P 0.05); there was no significant difference between the blank group and vaseline group (P 0.05); the number of MC around the small vessels in each group was higher than that in Qingpeng ointment group. Conclusion: Acupoint application of Qingpeng ointment can induce the formation of mast cell + blood vessel + neural network complex strip structure in rat skin tissue, and cause the collection and degranulation of mast cells in acupoint area, which is closely related to the obvious effect of Qingpeng ointment on acupoint application; 2 Qingpeng oint application can improve the effect of acupoint application. Higher serum heparin, lower serum bradykinin, prostaglandin content, which may be related to the Qingpeng ointment acupoint sticking mechanism. 3 Qingpeng ointment acupoint sticking can enhance the expression of ICAM-1 mRNA in vascular endothelium of rat acupoint tissue, ICAM-1 on Qingpeng ointment to promote the migration of mast cells to acupoint region, recruitment has a certain tendency. Chemical action.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5
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