谷氨酰胺对人肺泡Ⅱ型上皮细胞热休克蛋白70表达的影响

Effect of glutamine on heat shock protein 70 expression in human pulmonary alveolar type Ⅱ epithelial cells

目的评价谷氨酰胺对人肺泡Ⅱ型上皮细胞系A549细胞热休克蛋白（HSP）70表达的影响。方法取人肺泡Ⅱ型上皮细胞系A549细胞,在不含谷氨酰胺的DMEM培养液中孵育24h作为空白对照组（C组）,43℃孵育1h、37℃恢复4h作为阳性对照组（PC组）,不同浓度（2、4、8、12和16 mmol/L）谷氨酰胺的DMEM培养液中孵育24h作为不同浓度谷氨酰胺诱导组（Gln2组、Gln4组、Gln8组、Gln（12）组和Gln（16）组）,8mmol/L谷氨酰胺的DMEM培养液中孵育不同时间（1、2、6、12、24和48 h）作为不同时间谷氨酰胺诱导组（T1组、T2组、T3组、T4组、T5组和T6组）。分别采用RT-PCR和Western blot法检测HSP70 mRNA和蛋白的表达。结果与C组比较,PC组和不同浓度谷氨酰胺诱导组人肺泡Ⅱ型上皮细胞系A549细胞HSPTO mRNA和蛋白的表达均升高,Gln8组HSP70 mRNA和蛋白表达水平高于Gln2组、Gln4组、Gln（12）组和Gln（16）组（P＜0．01）,而与PC组相比差异无统计学意义（P＞0．05）。与C组比较,PC组和不同时间谷氨酰胺诱导组HSP70 mRNA和蛋白的表达均升高,T5组HSP70 mRNA和蛋白表达水平高于T1组、T2组、T3组、T4组和T5组（P＜0．01）,而与PC组相比差异无统计学意义（P＞0．05）。结论谷氨酰胺可明显上调体外培养人肺泡Ⅱ型上皮细胞系A549细胞HSP70 mRNA和蛋白的表达,并呈浓度和时间依赖性。

Objective To investigate the effect of glutamine on heat shock protein （HSP） 70 expression in the human pulmonary alveolar type Ⅱ epithelial cells. Methods Human pulmonary alveolar type Ⅱ epithelial cell line A549 was purchased from Shandong Provincial Academy of Medical Sciences and cultured in Dulbecco＇ s modified Eagle medium （DMEM） with different concentrations of glutamine （2, 4, 8. 12 and 16 mmol/L） for 24 h （glutamine group 1） （GI）. In another glutamine group （G2） the cells were incubated in DMEM with 8 mmol/L glutamine for 1, 2, 6, 12, 24 and 48 h respectively. In control group （C） the cells were incubated in DMEM without glutamine for 24 h. In positive control group （PC） the cells were incubated at 43℃ for 1 h and harvested 4 h later. RT-PCR and Western blotting were used to determine the expression of HSP70 mRNA and protein. Results （1） In PC group HSP70 mRNA and protein expression in the A549 cells was significantly increased after being exposed to heat （43℃ × 1 h） and harvested 4 h later. （2） In group G1 after being incubated with different concentrations of glutamine for 24 h the HSP70 mRNA and protein expression in A549 cells were significantly increased as compared with group C and reached the peak at 8 mmol/L glutamine. （3） In group G2 after being incubated with 8 mmol/L glutamine for different time, the HSP70 mRNA and protein expression increased significantly with time as compared with group C and reached the peak at 24 h. Conclusion Glutamine can significantly uprogulate the expression of HSP70 mRNA and protein in human pulmonary alveolar type Ⅱ epithelial cells in a time and concentration dependent manner.