青鵬膏劑穴位貼敷對大鼠皮膚組織結(jié)構(gòu)及肥大細胞釋放的肝素等生物活性物質(zhì)影響的研究
發(fā)布時間：2018-09-18 15:03
【摘要】：穴位貼敷療法,最早見于《五十二病方》,是以中醫(yī)整體觀理論為指導(dǎo),依據(jù)經(jīng)絡(luò)學(xué)說,將中藥作用于腧穴上,繼而通過經(jīng)絡(luò)作用于機體,從而達到疏通氣血、調(diào)整陰陽的作用。其特點是操作簡便、用藥安全、經(jīng)濟廉價。青鵬膏劑為藏藥經(jīng)典方劑,是由選自于青藏高原的傳統(tǒng)藏藥亞大黃、鐵棒錘、棘豆、訶子、毛訶子、人工麝香、安息香等9味組成,經(jīng)特殊工藝加工而成的外用膏劑,具有清熱解毒、活血化瘀、祛風(fēng)除濕、消腫止痛等功效。青鵬膏劑穴位貼敷,是將藥物、經(jīng)絡(luò)與穴位的有機融合,一方面發(fā)揮中藥的功效,另一方面發(fā)揮經(jīng)絡(luò)腧穴對機體的影響,二者在臨床治療中應(yīng)用廣泛,但在實驗研究方面欠缺。本實驗通過組織形態(tài)學(xué)和分子生物學(xué)技術(shù)檢驗,觀察青鵬膏劑穴位貼敷對皮膚組織的影響,進而研究對機體的影響,分析其可能的作用機制。目的在于為進一步研究青鵬膏劑提供實驗依據(jù),也為臨床應(yīng)用青鵬膏劑穴位貼敷治療疾病尋求科學(xué)根據(jù)。目的:觀察青鵬膏劑穴位貼敷對大鼠穴區(qū)內(nèi)皮膚組織形態(tài)結(jié)構(gòu)、血清中肝素等生物活性物質(zhì)的含量以及皮下組織血管內(nèi)皮細胞間粘附因子-1(ICAM-1)的表達的影響,探討青鵬膏劑穴位貼敷可能的藥理作用機制,為臨床運用提供有價值的實驗依據(jù)。方法:1實驗動物分組及處理辦法SPF級雄性Wistar大鼠40只,按隨機數(shù)字表法,分為空白組(A組)、凡士林貼敷組(B組)、電針組(C組)和青鵬膏劑貼敷組(D組),每組10只。大鼠固定后,根據(jù)郭義主編的《實驗針灸學(xué)》大鼠常用針灸穴位,選取大椎穴為中心、直徑為1.5cm的區(qū)域,穴區(qū)局部剃毛。空白對照組大鼠相同時間和方法固定,但不給予涂藥或針刺刺激。電針大椎穴組在大椎穴用毫針針刺,得氣后針柄接電針治療儀G6805-Ⅱ型,刺激參數(shù)為2/15Hz、1m A,2次/天,每次20min。凡士林貼敷大椎穴組大鼠在大椎穴區(qū)均勻涂抹醫(yī)用凡士林(約25mg),青鵬膏劑貼敷大椎穴組大鼠每次于穴區(qū)局部均勻涂抹青鵬膏劑(約25mg),2次/天,每次20min。干預(yù)7天。2指標(biāo)檢測2.1改良的乙酰膽堿酯酶染色結(jié)合甲苯胺藍復(fù)染方法觀察大鼠穴區(qū)皮膚組織內(nèi)肥大細胞(Mast cell,MC)的分布和形態(tài)特征以及與皮膚組織內(nèi)其他結(jié)構(gòu)的聯(lián)系。2.2 ELISA法檢測大鼠血清中肝素、緩激肽、前列腺素含量2.3原位雜交技術(shù)定位、定量檢測大鼠皮下組織血管內(nèi)皮細胞間粘附因子-1(ICAM-1)的表達以及觀察肥大細胞在小血管周圍的分布。結(jié)果:1青鵬膏劑穴位貼敷對大鼠穴區(qū)皮膚組織內(nèi)MC的分布和形態(tài)特征以及對皮膚組織內(nèi)其他結(jié)構(gòu)的影響。1.1觀察穴區(qū)內(nèi)肥大細胞(MC)分布規(guī)律本實驗的組織切片形態(tài)學(xué)觀察顯示:以青鵬膏劑組為例說明,各組大鼠“大椎”穴區(qū)真皮、皮下組織和肌肉組織中都分布著大小、數(shù)量不等的MC,多成條帶狀分布,以皮下組織層分布最為廣泛;1.2各組“大椎”穴區(qū)MC總數(shù)及脫顆粒率的比較數(shù)據(jù)統(tǒng)計表明:與空白組、凡士林組相比,青鵬膏劑組和電針組兩組穴區(qū)組織局部MC數(shù)量及脫顆粒顯著升高,差異顯著(P0.01);空白組與凡士林組、電針組與青鵬膏劑組分別做組間比較,差異無顯著性(P0.05),各組穴區(qū)MC數(shù)量及脫顆粒率分析比較為:青鵬膏劑組電針組凡士林貼敷組空白對照組;1.3各組穴區(qū)內(nèi)皮膚組織形態(tài)結(jié)構(gòu)的變化MC多呈圓形、卵圓形,胞質(zhì)內(nèi)充滿異染性藍色顆粒,多沿神經(jīng)或血管等分布。光學(xué)顯微鏡下,觀察到青鵬膏劑組穴區(qū)內(nèi),有經(jīng)過亞鐵氰化銅法乙酰膽堿酯酶特殊染色后成藍綠色的神經(jīng)纖維束,以網(wǎng)狀分布交織于血管,且周圍分布有MC。MC與血管壁的乙酰膽堿酯酶陽性神經(jīng)纖維發(fā)生直接接觸。2青鵬膏劑穴位貼敷對大鼠血清中肝素、緩激肽、前列腺素含量的影響2.1青鵬膏劑穴位貼敷組和電針組大鼠血清肝素含量均明顯高于空白對照組和凡士林貼敷組,差異顯著(P0.01);青鵬膏劑穴位貼敷組大鼠血清肝素含量高于電針組,差異有統(tǒng)計學(xué)意義(P0.05),空白組與凡士林組間比較,差異不顯著(P0.05);2.2青鵬膏劑穴位貼敷組血清緩激肽、前列腺素含量均明顯低于其他三組,組間比較差異較顯著(P0.05);電針組大鼠血清緩激肽、前列腺素含量均明顯高于其他三組,組間比較差異有統(tǒng)計學(xué)意義(P0.01或P0.05);空白組與凡士林組間比較,差異不顯著(P0.05)。3青鵬膏劑穴位貼敷對大鼠皮下組織血管內(nèi)皮細胞間粘附因子-1(ICAM-1)的表達及肥大細胞分布的影響3.1組織切片形態(tài)學(xué)觀察顯示,青鵬膏劑貼敷組和電針組的穴區(qū)小血管內(nèi)皮ICAM-1 mRNA陽性反應(yīng)的顯色程度和面積均明顯強于其他兩組。統(tǒng)計分析表明:青鵬膏劑穴位貼敷組和電針組大鼠血管內(nèi)皮ICAM-1mRNA陽性反應(yīng)物吸光度值明顯高于其他兩組,差異顯著(P0.01);青鵬膏劑組與電針組,空白組與凡士林分別做組間比較,差異不顯著(P0.05);3.2各組“大椎”穴區(qū)真皮、皮下組織和肌肉組織中都分布著大小、數(shù)量不等的MC,多成條帶狀分布,以皮下組織層分布最為廣泛。青鵬膏劑組與電針組穴區(qū)組織內(nèi)肥大細胞趨向中、小血管聚集,分布明顯多于其他兩組。數(shù)據(jù)統(tǒng)計表明:青鵬膏劑組與其他三組比較,穴區(qū)組織小血管周圍MC數(shù)量顯著升高,差異顯著(P0.01);電針組MC數(shù)量統(tǒng)計高于凡士林組和空白組(P0.05);空白組與凡士林組做組間比較,差異無顯著性(P0.05),各組小血管標(biāo)記的穴區(qū)MC數(shù)量比較為青鵬膏劑組電針組凡士林貼敷組空白對照組。結(jié)論:1青鵬膏劑穴位貼敷可使大鼠皮膚組織內(nèi)形成肥大細胞+血管+神經(jīng)網(wǎng)絡(luò)復(fù)合狀條帶結(jié)構(gòu),以及引起穴區(qū)內(nèi)肥大細胞募集和脫顆粒,這與青鵬膏劑穴位貼敷顯效密切相關(guān);2青鵬膏劑穴位貼敷能夠提高大鼠血清中肝素的含量,而降低血清中緩激肽、前列腺素含量,這可能與青鵬膏劑穴位貼敷的作用機理相關(guān)。3青鵬膏劑穴位貼敷可增強大鼠穴區(qū)組織血管內(nèi)皮ICAM-1mRNA的表達,ICAM-1對青鵬膏劑促進肥大細胞向穴區(qū)遷移、募集有一定的趨化作用。
[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.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5
本文編號：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.
【學(xué)位授予單位】：承德醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R285.5
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