【摘要】：目的：探討人氣道上皮細(xì)胞三磷酸腺苷(ATP)釋放量的改變?cè)跈C(jī)械通氣所導(dǎo)致的氣道黏液高分泌中所起到的作用。 方法： (1)人氣道上皮細(xì)胞(HBE16)行體外培養(yǎng)，在培養(yǎng)板傾斜30°（即1/3細(xì)胞無(wú)培養(yǎng)液覆蓋并暴露于空氣中）0、10、15、30、45s時(shí)分別行四甲基偶氮唑鹽光吸收法(MTT法)測(cè)定細(xì)胞活性，并確定最佳傾斜時(shí)間； （2）通過(guò)有節(jié)律的傾斜細(xì)胞培養(yǎng)板，利用液體表面張力、大氣壓及液體重力所產(chǎn)生的牽張力及壓力(可分解為剪應(yīng)力與壓應(yīng)力)給予牽張刺激,并利用向密閉盒中注入醫(yī)用氧氣增加壓力以模擬機(jī)械通氣； （3）各組細(xì)胞依施加條件不同而分為8組：①對(duì)照組、②單純傾斜組、③傾斜+胞內(nèi)鈣離子（Ca~(2+)）螯合劑（BAPTA-AM）、④傾斜+BAPTA-AM+胞外Ca~(2+)螯合劑（EGTA）、⑤加壓+傾斜、⑥加壓+傾斜+RB-2、⑦加壓+傾斜+BAPTA-AM、⑧加壓+傾斜+BAPTA-AM+EGTA； （4）分別采用MTT法測(cè)定細(xì)胞活性、逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)各組黏蛋白(MUC)5ACmRNA表達(dá)水平、酶聯(lián)免疫反應(yīng)(ELISA)法檢測(cè)細(xì)胞培養(yǎng)上清液中MUC5AC含量、高效液相色譜(HPLC)法檢測(cè)培養(yǎng)液中ATP釋放量、倒置熒光顯微鏡觀察細(xì)胞內(nèi)Ca~(2+)濃度變化。 結(jié)果： （1）加壓+傾斜組細(xì)胞MUC5ACmRNA相對(duì)含量、培養(yǎng)上清液中ATP釋放量和MUC5AC蛋白分泌量分別為：（11.8±0.01）、（7.41±0.45）μmol/g、(0.77±0.26)，，顯著高于單純傾斜組的(6.6±0.01)、(2.76±0.47)μmol/g、(0.25±0.01)及對(duì)照組的(3.4±0.01)、(0.00±0.01）μmol/g、（0.02±0.01）（均P0.05）。 （2）RB-2、EGTA+BAPTA-AM處理后的加壓+傾斜組細(xì)胞的MUC5ACmRNA相對(duì)含量分別為：（10.5±0.03)、（11.9±0.11），與加壓+傾斜組(11.80±0.01)相比抑制作用不顯著（P0.05），但顯著高于對(duì)照組的（3.40±0.01）（P0.05）。 （3）RB-2、EGTA+BAPTA-AM處理后的加壓+傾斜組細(xì)胞培養(yǎng)上清液中MUC5AC蛋白含量分別為(0.07±0.05),(0.08±0.05),較加壓+傾斜組(0.74±0.27)顯著降低（均P0.05）。同時(shí)，與對(duì)照組(0.02±0.01)相比，加壓+傾斜組能顯著增加人氣道上皮細(xì)胞培養(yǎng)上清液中MUC5AC蛋白的含量(P0.05)； （4）RB-2、EGTA+BAPTA-AM處理后的加壓+傾斜組細(xì)胞培養(yǎng)上清液中ATP釋放量分別為：（0.05±0.02）μmol/g、（0.08±0.02）μmol/g，較加壓+傾斜組[(7.41±0.45)μmol/g]顯著降低（均P0.05）。同時(shí)，與對(duì)照組[(0.00±0.01)μmol/g]相比，加壓+傾斜能明顯提升細(xì)胞培養(yǎng)上清中ATP的含量(P0.05)； （5）培養(yǎng)板傾斜角度最大時(shí)，胞內(nèi)Ca~(2+)濃度達(dá)高峰，傾斜組胞內(nèi)Ca~(2+)濃度峰明顯高于對(duì)照組，加壓+傾斜組內(nèi)Ca~(2+)濃度峰明顯高于傾斜組；給予BAPTA-AM及RB-2預(yù)處理后胞內(nèi)Ca~(2+)濃度峰明顯降低，而EGTA預(yù)處理后Ca~(2+)濃度峰無(wú)明顯變化。結(jié)論：（1）機(jī)械通氣可以顯著提高氣道黏膜上皮MUC5AC的分泌與表達(dá)；（2）氣道上皮細(xì)胞依賴Ca~(2+)的ATP過(guò)量釋放與氣道黏膜上皮MUC5AC的高分泌密切相關(guān)，但與MUC5ACmRNA表達(dá)的關(guān)系不密切；（3）該機(jī)制中的Ca~(2+)幾乎全部由胞內(nèi)存儲(chǔ)鈣提供。
[Abstract]:Aim: to investigate the role of adenosine triphosphate (ATP) release in airway mucus hypersecretion induced by mechanical ventilation. Methods: (1) Human duct epithelial cells (HBE16) were cultured in vitro. When the culture plate tilted 30 擄(that is, 1 / 3 cells were not covered by culture medium and exposed to air), the cell activity was determined by tetramethylazolium photoabsorption assay (MTT) for 45 s, and the optimum tilting time was determined. (2) through a rhythmically inclined cell culture plate, tension is stimulated by the tension and pressure generated by liquid surface tension, atmospheric pressure and liquid gravity (which can be decomposed into shear stress and compressive stress). In order to simulate mechanical ventilation, medical oxygen is injected into the closed box to increase pressure. (3) the cells in each group were divided into 8 groups according to different applied conditions: 1 control group, 2 simple tilt group, 3 tilted intracellular calcium ion (Ca~ (2) chelating agent (BAPTA-AM), 4 tilted BAPTA-AM extracellular Ca~ (2) chelator (EGTA), 5 pressurized tilt, 6 pressurized tilt RB-2,7 pressurized tilt BAPTA-AM,8 pressurized tilt BAPTA-AM EGTA; (4) the cell activity was measured by MTT assay, the expression of mucin (MUC) 5ACmRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR), and the content of MUC5AC in the supernatant of cell culture was detected by enzyme-linked immunosorbent assay (ELISA). High performance liquid chromatography (HPLC) (HPLC) method was used to detect the amount of ATP released in the culture medium, and the concentration of Ca~ (2) was observed by inverted fluorescence microscope. Results: (1) the relative content of MUC5ACmRNA, the release of ATP and the secretion of MUC5AC protein in the culture supernatant were (11.8 鹵0.01), (7.41 鹵0.45) 渭 mol/g, (0.77 鹵0.26), respectively. It was significantly higher than that in tilting group (6.6 鹵0.01), (2.76 鹵0.47) 渭 mol/g, (0.25 鹵0.01) and control group (3.4 鹵0.01), (0.00 鹵0.01) 渭 mol/g, (0.02 鹵0.01) (P0.05). (2) the relative content of MUC5ACmRNA was (10.5 鹵0.03), (11.9 鹵0.11) in the pressure-inclined group treated with RB-2,EGTA BAPTA-AM. Compared with the pressure tilt group (11.80 鹵0.01), the inhibitory effect was not significant (P0.05), but significantly higher than the control group (3.40 鹵0.01) (P0.05). (3) the content of MUC5AC protein in the supernatant of the cell culture supernatant treated with RB-2,EGTA BAPTA-AM was (0.07 鹵0.05), (0.08 鹵0.05). Compared with the pressure tilt group (0.74 鹵0.27), significantly decreased (P0.05). At the same time, compared with the control group (0.02 鹵0.01), the pressure tilt group significantly increased the content of MUC5AC protein in the supernatant of human duct epithelial cells (P0.05). (4) the amount of ATP released from the supernatant of the cell culture supernatant treated with RB-2,EGTA BAPTA-AM was (0.05 鹵0.02) 渭 mol/g, (0.08 鹵0.02) 渭 mol/g, respectively. Compared with the pressure tilt group [(7.41 鹵0.45) 渭 mol/g], it was significantly lower (P0.05). At the same time, compared with the control group [(0.00 鹵0. 01) 渭 mol/g], the ATP content in the supernatant was significantly increased (P0.05). (5) when the tilting angle of culture plate was the largest, the concentration of intracellular Ca~ (2) reached the peak, the concentration of intracellular Ca~ (2) in tilted group was significantly higher than that in control group, and the concentration peak of Ca~ (2) in tilted group was higher than that in tilted group. After pretreatment with BAPTA-AM and RB-2, the concentration peak of Ca~ (2) decreased significantly, but the concentration peak of Ca~ (2) did not change after EGTA pretreatment. Conclusion: (1) Mechanical ventilation can significantly increase the secretion and expression of MUC5AC in airway mucosal epithelium; (2) the excessive release of ATP dependent on Ca~ (2) in airway epithelial cells was closely related to the high secretion of MUC5AC in airway mucosal epithelium, but not to the expression of MUC5ACmRNA. (3) almost all of the Ca~ (2) in this mechanism was provided by intracellular storage calcium.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R562

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相關(guān)期刊論文 前1條

1 周向東,童瑾,蘭箭;慢性阻塞性肺疾病患者氣道粘蛋白分子表型的研究[J];中華結(jié)核和呼吸雜志;2002年07期

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