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为研究1-磷酸鞘氨醇受体1(S1P1)通路在模拟缺血的OGD(氧气和糖剥夺)条件下麦冬多糖MDG-1的细胞保护作用中的角色,以阐明MDG-1细胞保护作用机制。利用RT-PCR和Western blotting实验,研究MDG-1对S1P1通路的调节作用,并利用化学合成siRNA阻断S1P1表达,同时构建S1P1-全长cDNA序列的真核表达载体作为阳性对照,在OGD条件下,用LDH法检测细胞死亡率。结果表明MDG-1具有显著的细胞保护作用,可保护细胞减少OGD诱导的细胞死亡,上调S1P1蛋白表达,而转染siRNA能阻断S1P1表达,并使MDG-1的细胞保护作用受到明显抑制。限制性酶切消化、PCR、测序结果证实S1P1-pcDNA3.1b载体构建成功,转染入S1P1-pcDNA3.1b载体的HEK293细胞S1P1表达增强,并可减少OGD诱导的细胞死亡。这些结果表明MDG-1可能是通过激活S1P1通路,减少OGD诱导的细胞死亡,起到细胞保护作用。
To investigate the role of the Sphingosine-1-phosphate receptor 1 (S1P1) pathway in mimicking the cytoprotective effect of Ophiopogon japonicus polysaccharide MDG-1 under conditions of ischemic OGD (oxygen and glucose deprivation) to elucidate MDG-1 cell protection Mechanism. RT-PCR and Western blotting experiments were used to investigate the regulatory effect of MDG-1 on the S1P1 pathway, and the use of chemically synthesized siRNA to block S1P1 expression. At the same time, the eukaryotic expression vector of S1P1-full-length cDNA sequence was constructed as a positive control under OGD conditions. Next, the cell death rate was measured by the LDH assay. The results showed that MDG-1 had a significant cytoprotective effect, which could protect cells from reducing OGD-induced cell death and up-regulate the expression of S1P1 protein, while siRNA transfection could block the expression of S1P1 and significantly inhibited the cytoprotection of MDG-1. Restriction digestion, PCR and sequencing confirmed the successful construction of S1P1-pcDNA3.1b vector. The expression of S1P1 in HEK293 cells transfected with S1P1-pcDNA3.1b vector was enhanced and OGD-induced cell death was reduced. These results suggest that MDG-1 may play a cytoprotective role by activating the S1P1 pathway, reducing OGD-induced cell death.