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目的研究益气除痰方对缺氧条件下A549细胞上皮间质转化的影响并初步探讨其与脯氨酸4-羟化酶β亚基(P4HB)、波形蛋白(Vimentin)表达的相关性。方法将A549细胞分为3组培养:常氧对照组(常规培养),缺氧模型组(加入CoCl2培养模拟缺氧微环境诱导A549细胞上皮间质转化),益气除痰方含药血清组(在CoCl2培养基础上加入益气除痰方含药血清培养)。相差显微镜下观察3组A549细胞形态的变化;RT-PCR检测3组549细胞P4HB、Vimentin mRNA的表达含量。结果相差显微镜下常规培养的A549细胞呈不典型上皮细胞形态,经CoCl2刺激后,大部分细胞形态明显拉长,呈现明显的间质细胞形态。益气除痰方含药血清组与常氧对照组比较间质细胞形态的细胞增多,较缺氧模型组间质细胞形态的细胞明显减少。缺氧模型组A549细胞P4HB、Vimentin mRNA的表达量较常氧对照组明显上调,差异有统计学意义(P<0.01);益气除痰方含药血清组P4HB、Vimentin mRNA的表达量较缺氧模型组明显下调,差异有统计学意义(P<0.01,P<0.05)。结论益气除痰方可抑制缺氧诱导的上皮间质转化,可能与其下调P4HB mRNA表达相关。
Objective To study the effect of Yiqi Chu Tan Recipe (MDC) on epithelial-mesenchymal transition in A549 cells under hypoxia condition and to explore its relationship with proline 4-hydroxylase β subunit (P4HB) and vimentin expression. Methods A549 cells were divided into three groups: normoxia control group (conventional culture), hypoxia model group (CoCl2 culture induced hypoxia microenvironment to induce epithelial-mesenchymal transition of A549 cells), Yiqi Chu Tan Decoction-containing serum group (CoCl2 culture on the basis of adding Qi Qi and Phlegm Fang containing serum culture). The morphological changes of A549 cells were observed under phase contrast microscope. The expressions of P4HB and Vimentin mRNA in 3 groups of 549 cells were detected by RT-PCR. Results A549 cells cultured under phase contrast microscope showed typical epithelial cell morphology. After stimulation with CoCl2, the morphology of most of the cells was obviously elongated, showing obvious stromal cell morphology. Compared with the normoxia control group, the number of cells in interstitial cells increased, and the number of cells in interstitial cells in hypoxic model group decreased obviously. The expressions of P4HB and Vimentin mRNA in A549 cells were significantly upregulated compared with normoxia control group (P <0.01), and the expressions of P4HB and Vimentin mRNA in A549 cells in hypoxia model group were more than those in normoxic control group Oxygen model group was significantly decreased, the difference was statistically significant (P <0.01, P <0.05). Conclusion Yiqi Shutan can inhibit hypoxia-induced epithelial-mesenchymal transition, which may be related to its down-regulation of P4HB mRNA expression.