【摘要】：目的：經(jīng)全氟化碳（perfluorocarbon，PFC）治療脂多糖（lipopolysaccharide，LPS）致急性肺損傷（acute lung injury, ALI）A549細(xì)胞模型，探討PFC對(duì)ALI人肺泡上皮細(xì)胞表面活性蛋白的影響。 方法：實(shí)驗(yàn)共分為四組：（1）、空白對(duì)照組（C組）：含有A549細(xì)胞（106/ml）的培養(yǎng)液；（2）、脂多糖組（L組）：含有A549細(xì)胞（106/ml）的培養(yǎng)液加入10ug/ml脂多糖溶液；（3）、全氟化碳組（P組）：含有A549細(xì)胞（106/ml）的培養(yǎng)液加入30%（V/V%）全氟化碳溶液；（4）、脂多糖+全氟化碳組（L+P組）：含有A549細(xì)胞（106/ml）的培養(yǎng)液加入10ug/ml脂多糖及30%（V/V%）全氟化碳溶液。各組實(shí)驗(yàn)標(biāo)本均設(shè)定6個(gè)觀察時(shí)間點(diǎn)：1h、2h、3h、4h、5h、6h，每個(gè)時(shí)間點(diǎn)配制為總體積1ml混合溶液裝入離心管（1.5ml），經(jīng)超聲振蕩三次，每次振蕩5秒鐘，間隔20秒，，分別于每個(gè)時(shí)間點(diǎn)提取A549細(xì)胞（離心5000rpm×5min），－20℃保存?zhèn)鋵?shí)驗(yàn)用。 第一部分：（1）、應(yīng)用實(shí)時(shí)熒光定量PCR（Real-time Quantitative PCR,q-PCR）檢測(cè)技術(shù)分別檢測(cè)C組與L組炎性因子IL-1β、IL-6、TNF-α mRNA表達(dá)情況，確定ALI細(xì)胞損傷模型的建立；（2）、將P組按PFC濃度分別為10%、30%、50%、70%分成四個(gè)亞組，與C組對(duì)照進(jìn)行實(shí)驗(yàn)并應(yīng)用q-PCR技術(shù)檢測(cè)表面活性蛋白SP-D和相關(guān)蛋白TTF-1mRNA表達(dá)情況，確定PFC的作用濃度。 第二部分：分別應(yīng)用q-PCR、Western blot和ELISA技術(shù)檢測(cè)各實(shí)驗(yàn)分組細(xì)胞裂解液中表面活性蛋白SP-A、SP-B、SP-C、SP-D的表達(dá)情況。 第三部分：應(yīng)用q-PCR技術(shù)檢測(cè)各實(shí)驗(yàn)分組細(xì)胞裂解液中表面活性蛋白相關(guān)的蛋白ABCA3、TTF-1的mRNA表達(dá)情況。 結(jié)果：（1）、IL-1β、IL-6、TNF-α的mRNA表達(dá)在2h至6h期間各時(shí)間點(diǎn)L組較C組明顯增強(qiáng)。30%PFC亞組與50%PFC亞組較C組SP-D mRNA、TTF-1mRNA表達(dá)明顯增強(qiáng)，70%PFC亞組SP-D mRNA、TTF-1mRNA表達(dá)較其他各組明顯降低。（2）、與L組比較，L+P組SP-A在2h時(shí)間點(diǎn)，SP-B、SP-C、SP-D在3h時(shí)間點(diǎn)的mRNA表達(dá)明顯增強(qiáng)，細(xì)胞裂解液中蛋白含量增加。（3）、ABCA3mRNA在2h、3h時(shí)間點(diǎn)L+P組較L組表達(dá)增強(qiáng)；TTF-1mRNA在3h時(shí)間點(diǎn)L+P組較L組表達(dá)增強(qiáng)。 結(jié)論：（1）、PFC作用于LPS致ALI-A549細(xì)胞模型，使該模型中SP-A、SP-B、SP-C、SP-D四種表面活性蛋白的mRNA和蛋白表達(dá)水平均有升高。（2）、應(yīng)用PFC作用于LPS致ALI-A549細(xì)胞模型，促進(jìn)了該模型中ABCA3、TTF-1的mRNA表達(dá)；上述兩種蛋白mRNA表達(dá)與其各自分別對(duì)應(yīng)的SP mRNA表達(dá)結(jié)果一致，同時(shí)證實(shí)了前期有關(guān)SP實(shí)驗(yàn)部分的結(jié)果的準(zhǔn)確性。（3）、以上結(jié)果排除了PFC本身作用的因素，考慮為PFC改善肺損傷的作用結(jié)果，其作用機(jī)制尚有待進(jìn)一步研究。
[Abstract]:AIM: To investigate the effect of perfluorocarbon (PFC) on the expression of surfactant protein (SAP) in human alveolar epithelial cells of acute lung injury (ALI) induced by lipopolysaccharide (LPS).
Methods: The experiment was divided into four groups: (1) blank control group (group C): culture medium containing A549 cells (106/ml); (2) lipopolysaccharide group (L group): culture medium containing A549 cells (106/ml) added 10 ug/ml lipopolysaccharide solution; (3) perfluorocarbon group (P group): culture medium containing A549 cells (106/ml) added 30% (V/V%) perfluorocarbon solution; (4), lipopolysaccharide group (L group). Polysaccharide + perfluorocarbon group (L + P group): The culture medium containing A549 cells (106 / ml) was added with 10 UG / ml lipopolysaccharide and 30% (V / V%) perfluorocarbon solution. The A549 cells were extracted at 20 seconds interval (5 000 rpm
The first part: (1) Real-time quantitative PCR (q-PCR) was used to detect the expression of inflammatory factors IL-1 beta, IL-6 and TNF-alpha mRNA in group C and group L, respectively, to establish the model of ALI cell injury; (2) P group was divided into four subgroups according to the PFC concentration of 10%, 30%, 50%, 70%, respectively, and the experiment was carried out with the control group C. The expression of SP-D and TTF-1 mRNA was detected by q-PCR to determine the concentration of PFC.
The second part: The expression of SP-A, SP-B, SP-C and SP-D in cell lysates of different experimental groups were detected by q-PCR, Western blot and ELISA respectively.
The third part: The expression of ABCA3 and TTF-1 mRNA in cell lysates was detected by q-PCR.
Results: (1) The expression of IL-1 beta, IL-6 and TNF-alpha mRNA in L group was significantly higher than that in C group at 2 h to 6 h. The expression of SP-A, SP-B, SP-C and SP-D in 30% PFC subgroup and 50% PFC subgroup were significantly higher than that in C group, while the expression of SP-D mRNA and TTF-1 mRNA in 70% PFC subgroup were significantly lower than that in other groups. (2) Compared with L group, SP-A in L + P group was significantly higher at 2 h, SP-B, SP-C, SP-D, SP-C, SP-D-D at 3 h. The expression of ABCA3 mRNA in L + P group was higher than that in L group at 2 h and 3 h, and the expression of TTF-1 mRNA in L + P group was higher than that in L group at 3 h.
CONCLUSIONS: (1) PFC increased the expression of mRNA and protein of SP-A, SP-B, SP-C and SP-D in ALI-A549 cell model induced by LPS. (2) PFC promoted the expression of ABCA3 and TTF-1 mRNA in ALI-A549 cell model induced by LPS, and the expression of these two protein mRNAs was increased respectively. The expression of SP mRNA was consistent with that of SP mRNA, which confirmed the accuracy of the previous experimental results of SP. (3) The above results excluded the factors of PFC itself. Considering the effect of PFC on improving lung injury, its mechanism needs further study.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R563.8

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