低氘水对人肺癌细胞A549增殖和分化的影响及机制

Effects of deuterium-depleted water on proliferation and differentiation of lung cancer A549 cells

目的研究低氘水(DDW)对人肺癌A549细胞增殖和分化的影响,并探讨其作用机制。方法取A549细胞分为观察组和对照组,分别用氘含量为50 ppm的DDW配制的培养基、正常水配制的培养基培养30 d,倒置显微镜下观察两组细胞形态,用CCK-8法测定48 h内细胞生长情况。观察组和对照组细胞先分别在无血清的DDW培养基和正常水培养基中培养48 h使细胞生长同步化,然后分别加入含10%血清的DDW培养基和正常水培养基继续培养,用碘化丙啶染色和流式细胞分析仪测定两组36、48 h时处于细胞周期G_1、S和G_2/M期的细胞比例;取两组细胞裂解液,抽提细胞总蛋白,Western blot法检测丝裂原活化的细胞外信号调节激酶(MEK1)、表皮生长因子受体(EGFR)表达,MEK1第217和第298位丝氨酸磷酸化水平,EGFR第1016和第1110位酪氨酸磷酸化水平;比较两组肺泡Ⅰ型上皮细胞特异性蛋白标志物AQP5、T1a和肺泡Ⅱ型上皮细胞特异性蛋白标志物SPB、SPC1、SPC2、CFTR的表达。结果对照组细胞呈立方形、细胞间紧密接触和堆积生长,观察组细胞呈纺锤形、细胞伸长、细胞间接触较松散。观察组36、48 h时的OD值均较对照组降低(P均<0.05)。血清饥饿后再刺激36 h,观察组处于G1期的细胞比例为71.75%,高于对照组的57.01%;观察组处于S期的细胞比例为22.43%,低于对照组的35.88%(P均<0.01);48 h时两组细胞周期比例比较差异无统计学意义。两组MEK1、EGFR表达及EGFR第1016位和第1110位酪氨酸磷酸化水平比较差异无统计学意义,观察组MEK1第217位和第298位丝氨酸磷酸化的水平降低(P均<0.01)。观察组SPC2水平较对照组升高(P<0.01)。结论 A549细胞用DDW长期培养,可显著抑制其细胞增殖和分化,其机制可能与降低MEK1的磷酸化水平、阻滞细胞周期G_1/S期转变以及诱导A549细胞由Ⅱ型肺泡上皮细胞向Ⅰ型肺泡上皮细胞转化、提高细胞分化程度有关。

Objective To study the effects of deuterium-depleted water（DDW） on human lung cancer cell line A549 and to explore the underlying molecular mechanisms.Methods A549 cells were randomly divided into two groups：the observation group and the control group,which were seperately cultured in the medium prepared with 50 ppm DDW and in the medium prepared with normal water for 30 d.The morphology of cells was observed under an inverted microscope.Cell growth was measured using the colorimetric cell counting kit-8（CCK-8） reagent to compare the growth rate of cells between the two groups.The cells in the two groups were synchronized by serum starvation and then were separately stimulated with medium containing 10% serum of DDW and medium containing normal water.At 36 and 48 h after the stimulation,cells from each group were trypsinized,stained with propidium iodide（PI） and then were analyzed by fluorescenceactivated cell sorting（FACS） to determine the percentages of cells distributed in G1,S and G2/M phases during cell cycle.Total proteins were extracted from cells cultured in normal medium or DDW medium.Western blotting was conducted to detect the expression levels of MEK1 and EGFR,phosphorylation of serine 217 and serine 298 on MEK1,and phosphorylation of tyrosine 1016 and tyrosine 1110.The expression levels of several alveolus-specific protein markers（type Ⅰalveolus markers：AQP5 and T1 a,type Ⅱ alveolus markers SPB,SPC1,SPC2 and CFTR） in cells of the two groups were also compared.Results In the control group,the cells were cuboidal and in close contact with accumulation of cells.In the observation group,the cells were spindle,and intercellular contact was loose with cell elongation.The OD of the observation group was lower than that of the control group（all P0.05）.After the serum starvation,cells were stimulated for36 h,the percentage of cells in G1 phase of the observation group was 71.75%,which was higher than that of the control group（57.01%）,the percentage of cells in S phase was 22.43%,which was lower than that of the control group（35.88%）,（all P0.05）.There was no significant difference in cell cycle ratio between the two groups at 48 h.No significant difference was found in the expression of MEK1,EGFR and the phosphorylation of EGFR on tyrosine 1016 and1110 between these two groups.In the observation group,the phosphorylation of MEK1 on both serine 217 and serine 298 was significantly decreased（all P0.01）.The expression level of SPC2 in the observation group was significantly increased as compared with that of the control group（P0.01）.Conclusions Long-term cultivation of A549 lung cancer cells with DDW medium significantly inhibits the cell proliferation and differentiation,which is related to decreasing phosphorylation level of MEK1,arresting the G_1/S phase transformation,inducing A549 cells from type Ⅱ alveolar epithelial cells to type I alveolar epithelial cells and improving the differentiation of A549 cells.