本文選題：香煙提取物 + 肺間質(zhì)纖維化　； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：香煙煙霧中含有多種有害化學(xué)物質(zhì)，是多種疾病的主要誘因之一，而且被公認為是引起肺間質(zhì)纖維化的重要危險因素。香煙煙霧中的氧化劑和自由基可通過多種途徑產(chǎn)生氧化應(yīng)激，大量證據(jù)表明氧化應(yīng)激在肺間質(zhì)纖維化的發(fā)病機制中扮演著重要的角色，無論是在急性發(fā)作期還是緩解期，病人均存在氧化／抗氧化失衡，然而其分子機制尚不清楚。在臨床上肺間質(zhì)纖維化尚無特效的治療方法，近來抗氧化治療引起許多學(xué)者關(guān)注，研究顯示還原性谷胱甘肽（GSH）是機體內(nèi)重要的還原劑，具有抗氧化作用，可清除自由基及其他活性氧簇，保護肺組織免受氧化損傷。因此本實驗就以肺泡Ⅱ型上皮細胞來源的A549細胞為模型，用不同濃度香煙提取物(cigarette smoke extract，CSE)刺激體外培養(yǎng)的A549細胞，，觀察細胞生長的形態(tài)學(xué)改變及增殖抑制情況。通過檢測細胞上清液中各項氧化指標及反應(yīng)纖維化程度的指標，來探討吸煙對肺間質(zhì)纖維化影響的可能機制以及還原型谷胱甘肽對吸煙引起的肺間質(zhì)纖維化是否有逆轉(zhuǎn)作用。 方法：體外培養(yǎng)肺泡Ⅱ型上皮細胞來源的A549細胞，取對數(shù)生長期的細胞進行試驗。參照Carp and Janoff[1]的方法，通過特殊裝置制備香煙提取物。抽吸2支去過濾煙嘴的香煙，把煙霧融入不含血清1640培養(yǎng)基中，調(diào)整PH值，并除去細菌和大顆粒，然后稀釋至不同的濃度，30min中內(nèi)用于試驗。本實驗首先嘗試應(yīng)用0%、2.5%、5%、7.5%、10%、12.5%濃度的香煙提取物培養(yǎng)液培養(yǎng)細胞，采用MTT法測定細胞活力，通過相差顯微鏡觀察CSE對細胞增殖形態(tài)的影響。依據(jù)其結(jié)果選定合適的濃度進行實驗分組。實驗分為空白對照組、2.5%CSE組、5%CSE組、7.5%CSE組，7.5%CSE+GSH組(GSH1mmol/L).分別收集空白對照組和各干預(yù)組細胞培養(yǎng)24h小時后的上清液，應(yīng)用比色分析法測細胞上清液超氧化物歧化酶(SOD)活性和丙二醛（MDA）含量，ELISA法檢測細胞上清液中轉(zhuǎn)化生長因子1(TGF-β1)的水平。 結(jié)果：1倒置顯微鏡下觀察A549細胞在不同濃度CSE作用下形態(tài)學(xué)變化 倒置顯微鏡拍攝結(jié)果顯示CSE作用后A549細胞生長密度減低，細胞之間空隙加大，漂浮細胞增多，貼壁細胞也呈回縮變圓的態(tài)勢，出現(xiàn)萎縮，與對照組的細胞形態(tài)區(qū)別明顯。2不同濃度CSE對A549細胞的生長抑制作用 通過MTT比色法檢測，CSE對體外培養(yǎng)的A549細胞有較強的增殖抑制作用，抑制作用呈劑量和時間依賴性。相同濃度香煙提取物不同時間（12、24、36h）作用后，隨作用時間的延長其抑制率增大，差異有統(tǒng)計學(xué)意義(P0.05)。相同時間不同濃度（0%、2.5%、5%、7.5%、10%、12.5%）香煙提取物作用后，隨著濃度的增高其抑制率增大，差異有統(tǒng)計學(xué)意義(P0.05)。在10%CSE組作用24h時抑制率達70.30±2.21%，在12.5%CSE組作用24h時抑制率高達90.10±1.01%。3不同濃度CSE對A549細胞上清液TGF-β1、MDA、SOD水平的影響 不同濃度CSE組TGF-β1、MDA顯著高于對照組，差異有統(tǒng)計學(xué)意義（P0.05），且CSE濃度越高以上各因子含量越高，呈濃度依賴性，差異有統(tǒng)計學(xué)意義（P0.05）。不同濃度CSE組SOD活性低于于對照組，差異有統(tǒng)計學(xué)意義（P0.05），且CSE濃度越高SOD活性越低，差異有統(tǒng)計學(xué)意義（P0.05）。4GSH作用后A549細胞上清液TGF-β1、MDA、SOD水平的變化 7.5%CSE+GSH組TGF-β1、MDA含量顯著低于7.5%CSE組，差異有統(tǒng)計學(xué)意義（P0.05），而SOD活性高于7.5%CSE組，差異有統(tǒng)計學(xué)意義（P0.05）。 結(jié)論： 1香煙提取物可抑制肺泡上皮細胞的生長，抑制作用呈劑量和時間依賴性。 2香煙提取物可誘導(dǎo)肺泡上皮細胞分泌TGF-β1、MDA，使SOD活性下降，從而引起其氧化損傷及損傷后細胞修復(fù)變化。 3還原型谷胱甘肽可以使CSE刺激后的A549細胞產(chǎn)生的MDA、TGF-β1減少，SOD增加，對吸煙引起的氧化損傷有一定逆轉(zhuǎn)作用。
[Abstract]:Objective: cigarette smoke contains a variety of harmful chemicals, which is one of the main causes of various diseases and is recognized as an important risk factor for pulmonary fibrosis. Oxidizing agents and free radicals in cigarette smoke can produce oxidative stress through a variety of pathways, and a large number of evidence indicate that oxidative stress occurs in the pathogenesis of pulmonary fibrosis. The mechanism plays an important role, in both acute and remission phase, the patient has oxidation / antioxidant imbalance, but its molecular mechanism is not clear. There is no special therapeutic method for pulmonary interstitial fibrosis in clinical. Recently, many researchers pay more attention to antioxidant therapy. The research shows that reduced glutathione (GSH) is a machine. An important reductant in the body has antioxidant effects, which can remove free radicals and other active oxygen clusters and protect lung tissue from oxidative damage. Therefore, this experiment uses A549 cells derived from alveolar type II epithelial cells as a model to stimulate A549 cells cultured in vitro by different concentrations of cigarette smoke extract (CSE) and observe the cells. The possible mechanism of smoking on pulmonary fibrosis and the reverse effect of glutathione on pulmonary interstitial fibrosis caused by smoking were examined by detecting the indexes of oxidation and the degree of fibrosis in the supernatant of the cell.
Methods: A549 cells derived from alveolar type II epithelial cells were cultured in vitro, and the cells in the logarithmic growth period were tested. Cigarette extracts were prepared through special devices with reference to the method of Carp and Janoff[1]. 2 cigarettes were drawn to filter the cigarette mouth, and the smoke was incorporated into the 1640 culture medium without serum, and the pH value was adjusted and the bacteria and large particles were removed. Then diluted to different concentrations, 30min was used in the experiment. First of all, we tried to use 0%, 2.5%, 5%, 7.5%, 10%, 12.5% concentration of cigarette extract culture medium to determine cell viability by MTT method, and observe the effect of CSE on the cell proliferation by phase contrast microscope. The experiment was divided into blank control group, 2.5%CSE group, 5%CSE group, 7.5%CSE group and 7.5%CSE+GSH group (GSH1mmol/L). The supernatant of cell supernatant superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in cell supernatant were measured by colorimetric analysis and ELISA method was used to detect the transformation of cell supernatant. The level of growth factor 1 (TGF- beta 1).
Results: 1 the morphological changes of A549 cells under different concentrations of CSE were observed under inverted microscope.
The results of inverted microscope showed that the growth density of A549 cells decreased after CSE, the gap between cells increased, floating cells increased, and the adherent cells also appeared to shrink and round and atrophy. The difference between the cells of the control group and the cell morphology of the control group was obviously inhibited by the different concentrations of.2 and CSE on the growth of A549 cells.
The MTT colorimetric assay showed that CSE had a strong inhibitory effect on the proliferation of A549 cells in vitro, and the inhibitory effect was dosed and time dependent. After the effect of the same concentration of cigarette extracts at different time (12,24,36h), the inhibition rate increased with the time of action (P0.05). The same time (0%, 2.5%, 5%, 7). .5%, 10%, 12.5%) after the effect of cigarette extract, the inhibitory rate increased with the increase of concentration (P0.05). The inhibition rate was 70.30 + 2.21% in 10%CSE group 24h, and the inhibition rate of CSE in 12.5%CSE group was up to 90.10 + 1.01%.3 and CSE on A549 cell supernatant TGF- beta 1, MDA, SOD level
Different concentrations of CSE group TGF- beta 1, MDA was significantly higher than the control group, the difference was statistically significant (P0.05), and the higher the concentration of CSE, the higher the content of each factor, the concentration dependence, the difference was statistically significant (P0.05). The activity of SOD in the CSE group of different concentrations was lower than that of the control group, the difference was statistically significant (P0.05), and the higher the CSE concentration was, the lower the SOD activity was, the difference was the difference. The change of TGF- TGF- 1, MDA and SOD levels in A549 cell supernatant after P0.05.4GSH treatment was statistically significant.
The contents of TGF- beta 1 and MDA in group 7.5%CSE+GSH were significantly lower than those in group 7.5%CSE (P0.05), while SOD activity was higher than that in 7.5%CSE group (P0.05).
Conclusion:
1 cigarette extract inhibited the growth of alveolar epithelial cells in a dose and time dependent manner.
2 cigarette extract can induce alveolar epithelial cells to secrete TGF- beta 1 and MDA, resulting in a decrease in SOD activity, resulting in oxidative damage and cell repair after injury.
3 reduced glutathione can reduce the production of MDA and TGF- beta 1 from A549 cells stimulated by CSE, and increase SOD, which may reverse the oxidative injury caused by smoking.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R563

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