本文選題：哮喘氣道重塑 + 五虎湯　； 參考：《湖南中醫(yī)藥大學(xué)》2015年博士論文

【摘要】：目的：本文在建立病毒誘發(fā)幼年大鼠哮喘氣道重塑模型的基礎(chǔ)上,從體內(nèi)研究五虎湯對哮喘大鼠血清中NGF、肺組織TGF-β1、IL-13及氣道重塑的影響；體外研究五虎湯含藥血漿對NGF及Sema4D受體Plexin-B1和CD72表達(dá)的影響,以及Sema4D與NGF之間的相互關(guān)系,從調(diào)整NEI網(wǎng)絡(luò)紊亂達(dá)到改善氣道重塑,為進(jìn)一步闡明五虎湯防治病毒誘發(fā)兒童哮喘機(jī)理提供實(shí)驗(yàn)依據(jù)。 方法：1.建立模型：將30只幼年SD大鼠隨機(jī)分為正常對照組、哮喘模型組與地塞米松組,每組10只,于實(shí)驗(yàn)第1、2天哮喘模型組與地塞米松組以RSV滴鼻聯(lián)合10%OVA皮下注射,第9天以1%OVA霧化吸入,每次30min,隔日一次,持續(xù)2w；正常對照組以Hep-2細(xì)胞滴鼻聯(lián)合生理鹽水致敏、激發(fā)作為對照。通過測定氣道反應(yīng)性,留取BALF作細(xì)胞計(jì)數(shù)與分類,肺組織HE染色及Masson染色觀察其病理變化及膠原纖維沉積情況,驗(yàn)證模型建立成功與否。 2.體內(nèi)實(shí)驗(yàn)：將48只幼年SD大鼠隨機(jī)分為正常對照組、哮喘模型組、地塞米松組、五虎湯高、中、低劑量組,除正常對照組外,其余各組均建立哮喘氣道重塑模型,于實(shí)驗(yàn)第15天起,五虎湯高、中、低劑量組分別予4.752g/kg/d、2.376g/kg/d、1.188g/kg/d濃度的五虎湯,每日分2次灌胃,持續(xù)2w,正常對照組與哮喘模型組予生理鹽水灌胃,地塞米松組予地塞米松溶液(2.4mg/kg/d)灌胃,每日2次,持續(xù)2w。觀察各組大鼠反應(yīng)情況時(shí),病理形態(tài)學(xué)方法分析氣道重塑的相關(guān)指標(biāo),免疫組化法檢測肺組織α-SMA表達(dá),ELISA法測定血清中NGF水平,Western bl ot法檢測肺組織TGF-B1及IL-13蛋白相對含量,Real-time PCR法測定肺組織中NGF、TGF-β1及IL-13mRNA的表達(dá)。 3.體外實(shí)驗(yàn)：分離正常對照組和哮喘模型組大鼠氣道上皮細(xì)胞,進(jìn)行原代培養(yǎng)并傳至2-3代后,將細(xì)胞進(jìn)行分組：A組為正常氣道上皮細(xì)胞,不做任何處理；B組為哮喘氣道上皮細(xì)胞,不做任何處理;C組為哮喘氣道上皮細(xì)胞+Sema4D:D組為哮喘氣道上皮細(xì)胞+加入Sema4D、抗Sema4D;E1組、E2組分別為哮喘氣道上皮細(xì)胞+五虎湯高、中劑量含藥血漿;F1組、F2組分別為哮喘氣道上皮細(xì)胞+Sema4D與五虎湯高、中劑量含藥血漿,均處理24h后,應(yīng)用MTT法和Transwell小室法分別檢測氣道上皮細(xì)胞增殖量及遷移數(shù)量,RT-PCR法及Western Blot法分別測定Sema4D受體Plexin-B1和CD72mRNA及蛋白表達(dá)水平,ELISA法檢測氣道上皮細(xì)胞中NGF表達(dá)。 結(jié)果：1.建立模型：與正常對照組比較,哮喘模型組大鼠出現(xiàn)明顯的喘息、煩躁、豎毛聳肩、口唇發(fā)紺、體重增長緩慢及精神差等,HE染色及Masson染色觀察發(fā)現(xiàn)大量炎性細(xì)胞浸潤,以嗜酸性粒細(xì)胞為主,量化氣道學(xué)參數(shù)明顯升高,膠原纖維沉積大量增多等,氣道阻力顯著增高(P0.01)；地塞米松組較哮喘模型組哮喘癥狀及相應(yīng)病理變化均有所減輕。 2.體內(nèi)實(shí)驗(yàn)：(1)與正常組比,模型組大鼠出現(xiàn)明顯喘息癥狀,五虎湯高、中劑量組喘息等癥狀較模型組明顯減輕,地塞米松組大鼠癥狀亦有所緩解,五虎湯低劑量組大鼠喘息緩解不明顯；(2)與正常組比較,模型組病理切片鏡下可見大量炎性細(xì)胞、管腔狹窄、粘液分泌增多、氣道平滑肌顯著增厚且排列紊亂、粘膜下可見較多膠原樣物質(zhì)沉積,氣道重塑量化指標(biāo)、肺組織α-SMA表達(dá)IOD值均顯著增高(P0.01);與模型組比較,五虎湯高、中劑量組及地塞米松組鏡下病理改變程度明顯減輕,且氣道形態(tài)學(xué)參數(shù)各項(xiàng)指標(biāo)、α-SMA日性表達(dá)IOD值明顯降低(P0.01),五虎湯低劑量組與其差異不明顯(P0.05);(3)與正常組比較,模型組大鼠血清中NGF表達(dá)明顯增高(P0.01),各治療組均能降低NGF水平,以五虎湯高、中劑量組較明顯(P0.01),且下降程度較低劑量組、地塞米松組差異具有統(tǒng)計(jì)學(xué)意義(P0.01),五虎湯各劑量組降低NGF表達(dá)程度均優(yōu)于地塞米松組(P0.01,P0.05)；(4)與正常組比較,模型組肺組織TGF-β1、IL-13蛋白的表達(dá)均明顯升高(P0.01),予藥物干預(yù)后,五虎湯高、中劑量及地塞米松組TGF-β1及IL-13蛋白表達(dá)顯著下降(P0.01),且三組間無差異(P0.05),五虎湯低劑量組TGF-β1及IL-13蛋白減弱較模型組差異無統(tǒng)計(jì)學(xué)意義(P0.05)；(5)與正常組比,其余各組肺組織NGFmRNA相對表達(dá)量均升高,與模型組比,各治療組NGFmRNA的表達(dá)均顯著下降(P0.01),五虎湯高、中劑量組較低劑量組、地塞米松組下降有明顯統(tǒng)計(jì)學(xué)意義(P0.01),此二組之間無差異(P0.05)；(6)與正常組比較,模型組大鼠TGF-β1mRNA、IL-13mRNA的表達(dá)明顯升高(P0.01),與模型組比,五虎湯高、中劑量組TGF-β1mRNA表達(dá)水平顯著降低(P均0.01),且二組之間無明顯差異(P0.05),但仍高于正常組(P0.01),低劑量組及地塞米松組與模型組比較無顯著差異(P均0.05)；五虎湯高、中劑量組及地塞米松組IL-13mRNA表達(dá)水平較模型組均明顯降低(P0.01),五虎湯高、中劑量組之間差異無統(tǒng)計(jì)學(xué)意義(P0.05),較地塞米松組IL-13mRNA表達(dá)差異具有顯著性(P0.01)；五虎湯低劑量組較模型組差異無統(tǒng)計(jì)學(xué)意義(P0.05)。 3.體外實(shí)驗(yàn)：(1)與A組比較,B組氣道上皮細(xì)胞增殖量OD值明顯升高(P0.01)：與B組比較,C組OD值亦明顯升高(P0.01)；D組、F1組、F2組加入OD值有所下降(P0.01);E1、E2組OD值有所降低(P0.01)。以上各組與A組比較,均具有顯著性差異(P均0.01)。(2)Transwell小室法測定A組、B組、C組、D組、E1組、E2組、F1組與F2組的細(xì)胞遷移數(shù)分別為70,102,155,140,102,93,126,137。(3)與A組比較,B組Sema4D及其受體Plexin-B1表達(dá)與NGF含量均有升高,受體CD72表達(dá)降低；與B組比較,C組在Sema4D作用下,其OD值及遷移細(xì)胞數(shù)明顯增多(P0.01),且Sema4D受體Plexin-B1mRNA及蛋白、氣道上皮細(xì)胞中NGF表達(dá)水平均明顯升高(P0.01),CD72受體的表達(dá)顯著降低(P0.01)與C組比較,E1組、E2組、F1組與F2組Plexin-B1mRNA及蛋白表達(dá)水平亦明顯下降(P0.01),同時(shí)NGF的表達(dá)隨之顯著降低(P0.01)；CD72mRNA及蛋白表達(dá)相應(yīng)地明顯升高(P0.01)。與D組比較,E1、E2組PlexinB1mRNA及蛋白與NGF表達(dá)水平亦明顯下降(P0.01),CD72mRNA及蛋白表達(dá)水平亦明顯升高(P0.01)。F1、F2組與D組各指標(biāo)的表達(dá)結(jié)果一致,差異無統(tǒng)計(jì)學(xué)意義(P0.05)相對與C組而言,F1、F2組細(xì)胞OD值及遷移數(shù)量的下降程度,較E1、E2組下降程度不明顯(P0.05),其Plexin-BlmRNA及蛋白與NGF表達(dá)水平的下降程度和CD72mRNA及蛋白的升高程度均不及E1、E2組對應(yīng)指標(biāo)的改變程度(P0.05)。 結(jié)論：1.應(yīng)用RSV聯(lián)合OVA致敏激發(fā)方法能夠成功制備幼年SD大鼠哮喘氣道重塑模型。 2.五虎湯高、中劑量通過改善RSV氣道重塑模型大鼠支氣管壁、平滑肌增厚及上皮下膠原沉積,并下調(diào)a-SMA的表達(dá),從而減慢氣道重塑的進(jìn)程。 3.五虎湯高、中劑量通過降低RSV氣道重塑模型大鼠肺組織TGF-β1及IL-13的表達(dá)而下調(diào)NGF表達(dá)實(shí)現(xiàn)對哮喘中NEI網(wǎng)絡(luò)紊亂的調(diào)整,從而延緩氣道重塑及氣道炎癥進(jìn)程。 4.Sema4D參與哮喘氣道上皮細(xì)胞的增殖與遷移,并通過與NGF相互促進(jìn)作用,增強(qiáng)其與受體PlexinB1的結(jié)合能力,同時(shí)抑制與其受體CD72結(jié)合力,使氣道上皮細(xì)胞增殖與遷移過程加速,從而促進(jìn)氣道重塑的發(fā)生與發(fā)展。 5.五虎湯高、中劑量的含藥血漿能夠發(fā)揮抗Sema4D作用,通過抑制RSV氣道重塑模型大鼠Sema4D與NGF結(jié)合,降低Sema4D與其受體PlexinB1的結(jié)合能力,增強(qiáng)與其受體CD72的結(jié)合力,調(diào)整哮喘NEI網(wǎng)絡(luò)紊亂,緩解哮喘氣道重塑。
[Abstract]:Objective: To study the effect of five tiger soup on NGF, TGF- beta 1, IL-13 and airway remodeling in the serum of asthmatic rats, and the effect of five tiger soup containing plasma on the expression of NGF and Sema4D receptor Plexin-B1 and CD72 in vitro, and between Sema4D and NGF in vitro. The relationship can help improve airway remodeling by adjusting NEI network disorder, and provide experimental evidence for further elucidate the mechanism of Wuhu Decoction in preventing and treating asthma in children.
Methods: 1. a model was established: 30 young SD rats were randomly divided into normal control group, asthma model group and dexamethasone group, 10 rats in each group. On day 1,2, the asthma model group and dexamethasone group were subcutaneously injected with RSV dripping nose combined with 10%OVA, 1%OVA atomization inhalation, each time 30min each time, continuous 2W, and normal control group with Hep-2. The cells were sensitized with saline and stimulated as control. By measuring airway responsiveness, taking BALF as a cell count and classification, HE staining and Masson staining of lung tissue were used to observe the pathological changes and the deposition of collagen fibers, and the success of the model was verified.
2. in vivo experiment: 48 young SD rats were randomly divided into normal control group, asthma model group, dexamethasone group, five tiger soup high, middle, low dose group, except the normal control group, the other groups were established asthma airway remodeling model, from the fifteenth day of the experiment, the five tiger soup high, middle, low dose group were given 4.752g/kg/d, 2.376g/kg/d, 1.188g/kg/d concentration, respectively. The five tiger soup was intragastric 2 times a day and lasted 2W. The normal control group and the asthma model group were given the physiological saline for gastric perfusion. The dexamethasone group was given the dexamethasone solution (2.4mg/kg/d) to gavage, 2 times a day, and 2W. was used to observe the response of each group. The pathological morphological method was used to analyze the correlation index of the airway remodeling and the immunohistochemical method was used to detect the pulmonary tissue alpha -SMA. The expression of NGF in serum was measured by ELISA, and the relative content of TGF-B1 and IL-13 in lung tissue was detected by Western BL ot. Real-time PCR method was used to determine NGF, TGF- beta 1 and IL-13mRNA.
3. in vitro experiment: the airway epithelial cells in the normal control group and the asthmatic model group were isolated and cultured and passed to 2-3 generations. The cells were divided into groups: the A group was normal airway epithelial cells and did not do any treatment; the B group was asthma airway epithelial cells and did not do any treatment; group C was asthma airway epithelial cell +Sema4D:D as asthma. Airway epithelial cells + Sema4D, anti Sema4D, group E1, group E2 were asthma airway epithelial cells + five tiger soup high, medium dose of drug plasma, F1 group, F2 group of asthma airway epithelial cells +Sema4D and five tiger soup high, medium dose of plasma, 24h, MTT method and Transwell cell method were used to detect the proliferation of airway epithelial cell proliferation, respectively. The expression of Sema4D receptor Plexin-B1 and CD72mRNA and protein expression were measured by RT-PCR method and Western Blot method respectively. NGF expression in airway epithelial cells was detected by ELISA method.
Results: 1. the model was established: compared with the normal control group, the rats in the model group of asthma had obvious wheezing, irritability, hair shrug, lip cyanosis, slow weight growth and poor spirit. HE staining and Masson staining found a large number of inflammatory cells infiltrating, mainly eosinophilic cells, quantified airway parameters and collagen fibrous sedimentation Airway resistance increased significantly (P0.01), and asthma symptoms and corresponding pathological changes were relieved in the dexamethasone group compared with asthma model group.
2. in vivo experiment: (1) compared with the normal group, the rats in the model group had obvious wheezing symptoms, the five tiger soup high and the middle dose group were obviously relieved than the model group, and the symptoms of the rats in the dexamethasone group were also relieved. (2) compared with the normal group, the model group showed a large number of inflammation under the pathological section microscope. In sex cells, narrowing of the lumen, mucus secretion increased, the airway smooth muscle was thickened and arranged in disorder, more gel samples were deposited under the mucous membrane, the quantitative index of airway remodeling and the IOD value of the expression of alpha -SMA in the lung tissue were significantly increased (P0.01). Compared with the model group, the five tiger soup was higher, the medium dose group and the dexamethasone group were significantly reduced in the pathological changes. The IOD value of the expression of alpha -SMA was significantly lower (P0.01), and the difference in the low dose group of five tiger soup was not obvious (P0.05). (3) compared with the normal group, the expression of NGF in the serum of the model group was significantly higher (P0.01), and all the treatment groups could reduce the level of NGF, with the five tiger soup high, the middle dose group more obvious (P0.01), and decreased. In the lower dose group, the difference in dexamethasone group was statistically significant (P0.01). The level of NGF expression in each dose group of Wuhu decoction was better than that of dexamethasone group (P0.01, P0.05). (4) compared with the normal group, the expression of TGF- beta 1 and IL-13 protein in the lung tissue of the model group was significantly increased (P0.01), the prognosis of the drug drying, the high level of five tiger soup, the medium dose and dexamethasone. The expression of TGF- beta 1 and IL-13 protein in the pine group was significantly decreased (P0.01), and there was no difference between the three groups (P0.05). The decrease of TGF- beta 1 and IL-13 protein in the low dose group of the five tiger soup was not statistically significant (P0.05). (5) the relative amount of NGFmRNA in the lung tissues of the other groups increased, compared with the normal group, and the expression of NGFmRNA in each group was significantly higher than that of the model group. Decrease (P0.01), five tiger soup, low dose group, lower dose group, dexamethasone group decreased significantly (P0.01), there was no difference between the two groups (P0.05); (6) compared with the normal group, the expression of TGF- beta 1mRNA, IL-13mRNA increased significantly (P0.01) in the model group, compared with the model group, the five tiger soup was higher, and the medium dose group TGF- beta 1mRNA expression level was significant. There was no significant difference (P 0.01), and there was no significant difference between the two groups (P0.05), but it was still higher than the normal group (P0.01). There was no significant difference between the low dose group and the dexamethasone group (P 0.05). The IL-13mRNA expression level in the medium dose group and the dexamethasone group was significantly lower than that in the model group (P0.01), the five tiger soup and the middle dose group were different. There was no statistical significance (P0.05), and there was significant difference in IL-13mRNA expression compared with dexamethasone group (P0.01), and there was no significant difference between the low dose group and the model group (P0.05).
3. in vitro experiment: (1) compared with group A, the proliferation of airway epithelial cell proliferation in B group increased significantly (P0.01): compared with group B, the OD value of group C was also significantly increased (P0.01); D group, F1 group, F2 group decreased (P0.01) and E1, E2 group O value decreased (0.01). (2) The cell migration number of A group, group B, group C, group D, group E1, E2 group, F1 group and F2 group was 70102155140102,93126137. (3) compared with A group. The expression level of NGF in the airway epithelial cells increased significantly (P0.01), and the expression of CD72 receptor decreased significantly (P0.01), and the expression level of Plexin-B1mRNA and protein in E1, E2, F1 and F2 groups also decreased significantly (P0.01), and the expression of CD72 was significantly decreased (P0.01), and the expression of CD72 was significantly decreased. Compared with group D, the expression level of PlexinB1mRNA and protein and NGF in E1, E2 group also decreased (P0.01), the expression level of CD72mRNA and protein was also significantly increased (P0.01).F1, F2 group was in accordance with the expression results of each index in D group, and the difference was not statistically significant. The decrease degree of E1, E2 group was not obvious (P0.05). The decrease of Plexin-BlmRNA and protein and NGF expression level and the increase of CD72mRNA and protein were not as good as that of E1 and E2 group (P0.05).
Conclusion: 1.. RSV combined with OVA sensitization stimulation method can successfully prepare airway remodeling model of asthmatic SD rats.
2. wutiger soup was high, medium dose was used to improve the bronchial wall of RSV airway remodeling model rats, smooth muscle thickening and collagen deposition, and down regulation of a-SMA expression, thus slowing the process of airway remodeling.
3. five tiger soup was high, and the medium dose reduced the expression of TGF- beta 1 and IL-13 in the lung tissue of the RSV airway remodeling model and downregulated the expression of NGF to adjust the NEI network disorder in asthma, thus postponing the airway remodeling and airway inflammation.
4.Sema4D participates in the proliferation and migration of airway epithelial cells in asthmatic airway, and enhances the binding ability of NGF to receptor PlexinB1, and inhibits the binding force of its receptor with its receptor, accelerating the proliferation and migration of airway epithelial cells, thus promoting the development and development of airway remodeling.
5. five tiger soup, medium dose of drug containing plasma can play the anti Sema4D effect, by inhibiting the RSV airway remodeling model rats Sema4D and NGF binding, reducing the binding capacity of Sema4D and its receptor PlexinB1, enhancing the binding force of the receptor CD72, adjusting the disorder of the asthma NEI network and alleviating the airway remodeling of asthma.
【學(xué)位授予單位】：湖南中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R285.5;R-332

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