硫酸铍诱发人胚肺细胞凋亡机制及枸杞多糖保护作用的初步研究
发布时间：2018-08-18 12:29
【摘要】：目的：用体外实验的研究方法，观察硫酸铍（BeSO_4）致人胚肺成纤维细胞（MRC-5）活性氧（ROS）的变化、相关凋亡基因c-fos、bcl-2、bax、Caspase-3的表达水平及细胞内钙离子浓度及三磷酸肌醇受体（IP3R）表达的改变，探讨硫酸铍是否通过影响活性氧和钙离子导致人胚肺细胞损伤，同时观察枸杞多糖（LBP）是否对硫酸铍致人胚肺细胞损伤具有保护作用。 方法：体外培养人胚肺成纤维细胞，共分为空白对照组、硫酸铍低中高剂量组、枸杞多糖保护组及枸杞多糖对照组等6组。硫酸铍给予剂量分别为0、1、10、100μmol/L，枸杞多糖剂量为10μmol/L。细胞生长至对数生长期，根据实验需求，达到细胞密度后，加入药物培养24h收集细胞，分别进行以下实验：1.流式细胞术检测细胞内活性氧（ROS）；2.免疫组化（SP）法检测c-fos、bcl-2、bax、Caspase-3相关的表达3.激光共聚焦法检测细胞内[Ca2+]的水平；4.RT-PCR测定三磷酸肌醇受体(IP3R)的表达。 结果： 1．BeSO_4中、高剂量组ROS含量与空白对照组相比明显增高，表达细胞数增多，具有统计学意义（P0.05）。 2.细胞加药培养24h后，免疫组化结果显示： BeSO_4低、中、高剂量组c-fos、bcl-2表达均高于空白对照组，，差异有统计学意义（P0.05），且随着药物剂量增加表达水平不断增高。BeSO_4中、高剂量组bax表达均低于空白对照组、BeSO_4低、中、高剂量组Caspase-3表达均低于空白对照组，差异有统计学意义（P0.05）且随着药物剂量增加表达水平不断降低。 3.硫酸铍高剂量组细胞内Ca2+浓度明显高于空白对照组，差异有统计学意义（P0.05）。 4.激光共聚焦显微镜下，100μmol/L BeSO_4组中，细胞核呈现出明亮的绿色荧光，细胞分界清楚呈梭型，BeSO_4+LBP组细胞呈圆形，胞体淡绿色，细胞周围可见绿色荧光小点。 5. BeSO_4低、中、高剂量组Ⅲ型IP3R表达与空白对照组比较差异均有统计学意义(P0.01）； 6. LBP对BeSO_4损伤细胞的保护作用：BeSO_4组ROS含量高于空白对照组（P0.01）、LBP组ROS含量低于空白对照组（P0.01），二者具有交互作用（P0.05）。对于bcl-2、Caspase-3的表达BeSO_4和LBP具有交互效应（P0.05或P0.01）。Ca2+和IP3R检测结果显示BeSO_4和LBP具有交互作用（P0.05或P0.01）。 结论： 1. BeSO_4可致MRC-5细胞内ROS含量升高，LBP对此有一定保护作用。 2. BeSO_4可致MRC-5细胞内的c-fos、bcl-2基因表达量升高；bax、Caspase-3基因表达量减少，LBP对此有一定的调节作用。 3. BeSO_4可致MRC-5细胞内钙离子浓度升高，LBP对其有一定保护作用。 4. BeSO_4可能通过IP3R受体通道，造成MRC-5细胞钙超载，LBP对IP3R受体的表达有一定的调节作用。
[Abstract]:Aim: to observe the changes of reactive oxygen (ROS) (Ros) in human embryonic lung fibroblasts (MRC-5) induced by beryllium sulfate (BeSO_4) in vitro, the expression of c-fosbcl-2nbax-caspase-3, the intracellular calcium concentration and the expression of inositol triphosphate receptor (IP3R) in human embryonic lung fibroblasts (MRC-5). To investigate whether beryllium sulfate (beryllium sulfate) can damage human embryonic lung cells by affecting reactive oxygen species (Ros) and calcium ions, and to observe whether Lycium barbarum polysaccharide (LBP) has protective effect on human embryonic lung cell injury induced by beryllium sulfate. Methods: human embryonic lung fibroblasts were cultured in vitro and divided into six groups: blank control group, beryllium sulfate low and high dose group, Lycium barbarum polysaccharide protection group and Lycium barbarum polysaccharide control group. The doses of beryllium sulfate and Lycium barbarum polysaccharides were 10 渭 mol / L and 10 渭 mol / L, respectively. The cells grew to logarithmic growth stage. According to the experimental demand, the cell density was reached, then the cells were collected after 24 hours of drug culture, and the following experiments were carried out: 1. Intracellular reactive oxygen species (ROS) were detected by flow cytometry. The expression of Caspase-3 was detected by immunohistochemical (SP) method. The level of [Ca2] was detected by laser confocal method. 4. RT-PCR was used to detect the expression of inositol triphosphate receptor (IP3R). Results: in 1.BeSO_4, the content of ROS in the high dose group was significantly higher than that in the blank control group, and the number of expressed cells was increased, which was statistically significant (P0.05). After 24 hours of cell culture, the immunohistochemical results showed that the expression of c-fossil-bcl-2 in the low, medium and high dose groups of BeSO_4 was significantly higher than that in the blank control group (P0.05), and the expression level of c-fossil-bcl-2 was increased with the increase of drug dosage. The expression of bax in the high dose group was lower than that in the blank control group, and the expression of Caspase-3 in the high dose group was lower than that in the blank control group. The difference was statistically significant (P0.05) and the expression level decreased with the increase of drug dose. The intracellular Ca2 concentration in the high dose group of beryllium sulfate was significantly higher than that in the blank control group (P0.05). In the laser confocal microscope (LSCM) group, the nucleus showed bright green fluorescence, and the cell line was clear and round, the cell body was light green, and the green fluorescence dots were observed around the cells. The expression of type 鈪
本文编号：2189474
[Abstract]:Aim: to observe the changes of reactive oxygen (ROS) (Ros) in human embryonic lung fibroblasts (MRC-5) induced by beryllium sulfate (BeSO_4) in vitro, the expression of c-fosbcl-2nbax-caspase-3, the intracellular calcium concentration and the expression of inositol triphosphate receptor (IP3R) in human embryonic lung fibroblasts (MRC-5). To investigate whether beryllium sulfate (beryllium sulfate) can damage human embryonic lung cells by affecting reactive oxygen species (Ros) and calcium ions, and to observe whether Lycium barbarum polysaccharide (LBP) has protective effect on human embryonic lung cell injury induced by beryllium sulfate. Methods: human embryonic lung fibroblasts were cultured in vitro and divided into six groups: blank control group, beryllium sulfate low and high dose group, Lycium barbarum polysaccharide protection group and Lycium barbarum polysaccharide control group. The doses of beryllium sulfate and Lycium barbarum polysaccharides were 10 渭 mol / L and 10 渭 mol / L, respectively. The cells grew to logarithmic growth stage. According to the experimental demand, the cell density was reached, then the cells were collected after 24 hours of drug culture, and the following experiments were carried out: 1. Intracellular reactive oxygen species (ROS) were detected by flow cytometry. The expression of Caspase-3 was detected by immunohistochemical (SP) method. The level of [Ca2] was detected by laser confocal method. 4. RT-PCR was used to detect the expression of inositol triphosphate receptor (IP3R). Results: in 1.BeSO_4, the content of ROS in the high dose group was significantly higher than that in the blank control group, and the number of expressed cells was increased, which was statistically significant (P0.05). After 24 hours of cell culture, the immunohistochemical results showed that the expression of c-fossil-bcl-2 in the low, medium and high dose groups of BeSO_4 was significantly higher than that in the blank control group (P0.05), and the expression level of c-fossil-bcl-2 was increased with the increase of drug dosage. The expression of bax in the high dose group was lower than that in the blank control group, and the expression of Caspase-3 in the high dose group was lower than that in the blank control group. The difference was statistically significant (P0.05) and the expression level decreased with the increase of drug dose. The intracellular Ca2 concentration in the high dose group of beryllium sulfate was significantly higher than that in the blank control group (P0.05). In the laser confocal microscope (LSCM) group, the nucleus showed bright green fluorescence, and the cell line was clear and round, the cell body was light green, and the green fluorescence dots were observed around the cells. The expression of type 鈪
本文编号：2189474
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