全氟化碳对脂多糖诱导急性肺损伤A549细胞表面活性蛋白的影响

发布时间：2018-08-13 11:34

【摘要】：目的：经全氟化碳（perfluorocarbon，PFC）治疗脂多糖（lipopolysaccharide，LPS）致急性肺损伤（acute lung injury, ALI）A549细胞模型，探讨PFC对ALI人肺泡上皮细胞表面活性蛋白的影响。方法：实验共分为四组：（1）、空白对照组（C组）：含有A549细胞（106/ml）的培养液；（2）、脂多糖组（L组）：含有A549细胞（106/ml）的培养液加入10ug/ml脂多糖溶液；（3）、全氟化碳组（P组）：含有A549细胞（106/ml）的培养液加入30%（V/V%）全氟化碳溶液；（4）、脂多糖+全氟化碳组（L+P组）：含有A549细胞（106/ml）的培养液加入10ug/ml脂多糖及30%（V/V%）全氟化碳溶液。各组实验标本均设定6个观察时间点：1h、2h、3h、4h、5h、6h，每个时间点配制为总体积1ml混合溶液装入离心管（1.5ml），经超声振荡三次，每次振荡5秒钟，间隔20秒，，分别于每个时间点提取A549细胞（离心5000rpm×5min），－20℃保存备实验用。第一部分：（1）、应用实时荧光定量PCR（Real-time Quantitative PCR,q-PCR）检测技术分别检测C组与L组炎性因子IL-1β、IL-6、TNF-α mRNA表达情况，确定ALI细胞损伤模型的建立；（2）、将P组按PFC浓度分别为10%、30%、50%、70%分成四个亚组，与C组对照进行实验并应用q-PCR技术检测表面活性蛋白SP-D和相关蛋白TTF-1mRNA表达情况，确定PFC的作用浓度。第二部分：分别应用q-PCR、Western blot和ELISA技术检测各实验分组细胞裂解液中表面活性蛋白SP-A、SP-B、SP-C、SP-D的表达情况。第三部分：应用q-PCR技术检测各实验分组细胞裂解液中表面活性蛋白相关的蛋白ABCA3、TTF-1的mRNA表达情况。结果：（1）、IL-1β、IL-6、TNF-α的mRNA表达在2h至6h期间各时间点L组较C组明显增强。30%PFC亚组与50%PFC亚组较C组SP-D mRNA、TTF-1mRNA表达明显增强，70%PFC亚组SP-D mRNA、TTF-1mRNA表达较其他各组明显降低。（2）、与L组比较，L+P组SP-A在2h时间点，SP-B、SP-C、SP-D在3h时间点的mRNA表达明显增强，细胞裂解液中蛋白含量增加。（3）、ABCA3mRNA在2h、3h时间点L+P组较L组表达增强；TTF-1mRNA在3h时间点L+P组较L组表达增强。结论：（1）、PFC作用于LPS致ALI-A549细胞模型，使该模型中SP-A、SP-B、SP-C、SP-D四种表面活性蛋白的mRNA和蛋白表达水平均有升高。（2）、应用PFC作用于LPS致ALI-A549细胞模型，促进了该模型中ABCA3、TTF-1的mRNA表达；上述两种蛋白mRNA表达与其各自分别对应的SP mRNA表达结果一致，同时证实了前期有关SP实验部分的结果的准确性。（3）、以上结果排除了PFC本身作用的因素，考虑为PFC改善肺损伤的作用结果，其作用机制尚有待进一步研究。
[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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R563.8

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