【摘要】：目的：本研究擬通過活體動(dòng)物實(shí)驗(yàn),觀察電針灸刺激眼周穴位及中藥潤目靈顆粒灌胃對(duì)干眼模型的療效,探索電針及中藥潤目靈與兔淚腺抗凋亡作用及與M型膽堿受體及其介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路上關(guān)鍵因子的相關(guān)性,推斷淚腺細(xì)胞主動(dòng)分泌的可能作用及機(jī)制。方法：健康新西蘭大白兔,實(shí)驗(yàn)分為以下六組：空白對(duì)照組、阿托品模型組、西藥干預(yù)組、中藥干預(yù)組、電針干預(yù)組、針?biāo)幐深A(yù)組,雌雄兼?zhèn)?每組各6只。模型組和各治療組均行1%硫酸阿托品滴眼液每日滴雙眼4次(上午8：00、12：00,16：00、20：00)制備干眼模型,3天后水液缺乏性干眼模型形成并穩(wěn)定后對(duì)各治療組分別行毛果蕓香堿滴眼液滴眼、給予潤目靈灌胃、電針干預(yù)及潤目靈灌胃結(jié)合電針干預(yù)治療。實(shí)驗(yàn)標(biāo)本采集和數(shù)據(jù)收集時(shí)間為：各組實(shí)驗(yàn)兔分別于實(shí)驗(yàn)前、實(shí)驗(yàn)第4天(模型穩(wěn)定)、實(shí)驗(yàn)第11天(治療第一周)、實(shí)驗(yàn)第18天(治療第二周)檢測(cè)淚流量、淚膜破裂時(shí)間、角膜染色分?jǐn)?shù)；并在實(shí)驗(yàn)第18天取淚腺組織行病理形態(tài)學(xué)觀察、Elisa法測(cè)乙酰膽堿含量、免疫組化及real-time PCR檢測(cè)。結(jié)果：Schirmer I、淚膜破裂時(shí)間及角膜染色均證實(shí)西藥、電針、中藥、針?biāo)幝?lián)合治療均可促進(jìn)淚液分泌、延長淚膜破裂時(shí)間、修復(fù)受損的角膜上皮,但針?biāo)幐深A(yù)組的效果最佳。實(shí)驗(yàn)18天,取各組的淚腺及角膜組織進(jìn)行相關(guān)實(shí)驗(yàn)室檢查,西藥干預(yù)組、中藥干預(yù)組及電針干預(yù)組的形態(tài)出現(xiàn)不同程度的改善,針?biāo)幐深A(yù)組的淚腺及角膜組織形態(tài)最接近于空白對(duì)照組。各組淚腺組織的乙酰膽堿含量較模型組有所上升,淚腺組織中的乙酰膽堿含量較模型組有統(tǒng)計(jì)學(xué)差異。針?biāo)幐深A(yù)組的凋亡因子Fas、FasL、Bax蛋白的半定量,與阿托品模型組比較有統(tǒng)計(jì)學(xué)差異。針?biāo)幐深A(yù)組中的淚腺組織中的M受體介導(dǎo)的的信號(hào)轉(zhuǎn)導(dǎo)通路上關(guān)鍵因子的蛋白和蛋白及mRNA檢測(cè)顯示M3AchR及MAPK,與模型組比較有統(tǒng)計(jì)學(xué)差異,但I(xiàn)P3與模型組比較,無統(tǒng)計(jì)學(xué)差異。結(jié)論：電針和中藥潤目靈均可以抑制淚腺細(xì)胞凋亡,增加淚液分泌。電針增加淚液分泌與神經(jīng)遞質(zhì)釋放以及淚腺組織上M受體介導(dǎo)的信號(hào)轉(zhuǎn)導(dǎo)通路關(guān)鍵因子也存在著一定的相關(guān)性。
[Abstract]:Objective: to observe the effect of electroacupuncture (EA) stimulation on acupoints around the eyes and Runmuling granule (RML) on dry eye model by living animal experiment. To explore the relationship between electroacupuncture and Runmuling in rabbit lacrimal gland and the key factors in M type choline receptor and its signal transduction pathway, the possible role and mechanism of active secretion of lacrimal gland cells were inferred. Methods: healthy New Zealand white rabbits were divided into the following six groups: blank control group, atropine model group, western medicine intervention group, traditional Chinese medicine intervention group, electroacupuncture intervention group, acupuncture and medicine intervention group, both sexes. The model group and the treatment group were treated with 1% atropine sulfate eye drops 4 times a day (8: 00: 12: 00: 16: 00 20: 00) to make dry eye model. After 3 days, the dry eye model of water deficiency was formed and stabilized. Each treatment group was treated with pilocarpine eye drops, treated with Runmuling, electroacupuncture and Runmuling combined with electroacupuncture intervention. The time of collecting samples and collecting data were: before experiment, 4 days (model stability), 11 days (the first week of treatment), 18 days (the second week of treatment) to detect the lacrimal flow and tear film rupture time. Corneal staining fraction; On the 18th day of the experiment, the lacrimal gland tissues were taken for histopathological observation, and the content of acetylcholine, immunohistochemistry and real-time PCR were measured by Elisa method. Results: Schirmer I, tear film rupture time and corneal staining confirmed that western medicine, electroacupuncture, Chinese medicine, acupuncture and medicine combined treatment can promote tear secretion, prolong tear film rupture time, repair damaged corneal epithelium, but acupuncture and medicine intervention group is the best. On the 18th day of the experiment, the lacrimal gland and corneal tissue of each group were taken for laboratory examination, and the morphology of the western medicine intervention group, the traditional Chinese medicine intervention group and the electroacupuncture intervention group were improved to some extent. The morphology of lacrimal gland and cornea in acupuncture and medicine intervention group was closest to that in blank control group. The content of acetylcholine in the lacrimal gland was higher than that in the model group, and the content of acetylcholine in the lacrimal gland was significantly higher than that in the model group. The semiquantitative expression of apoptotic factor Fas,FasL,Bax protein in acupuncture and drug intervention group was significantly different from that in atropine model group. The detection of protein, protein and mRNA of key factors in the signal transduction pathway mediated by M receptor in lacrimal gland tissue of acupuncture and drug intervention group showed that M3AchR and MAPK, were significantly different from model group, but IP3 had no statistical difference compared with model group. Conclusion: both electroacupuncture and Runmuling can inhibit the apoptosis of lacrimal gland cells and increase the secretion of tear. The increase of tear secretion by electroacupuncture is also related to the release of neurotransmitters and the key factors of signal transduction pathway mediated by M receptor in lacrimal gland.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R245

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