【摘要】：目的：用體外實(shí)驗(yàn)的研究方法，觀察硫酸鈹（BeSO_4）致人胚肺成纖維細(xì)胞（MRC-5）活性氧（ROS）的變化、相關(guān)凋亡基因c-fos、bcl-2、bax、Caspase-3的表達(dá)水平及細(xì)胞內(nèi)鈣離子濃度及三磷酸肌醇受體（IP3R）表達(dá)的改變，探討硫酸鈹是否通過(guò)影響活性氧和鈣離子導(dǎo)致人胚肺細(xì)胞損傷，同時(shí)觀察枸杞多糖（LBP）是否對(duì)硫酸鈹致人胚肺細(xì)胞損傷具有保護(hù)作用。 方法：體外培養(yǎng)人胚肺成纖維細(xì)胞，共分為空白對(duì)照組、硫酸鈹?shù)椭懈邉┝拷M、枸杞多糖保護(hù)組及枸杞多糖對(duì)照組等6組。硫酸鈹給予劑量分別為0、1、10、100μmol/L，枸杞多糖劑量為10μmol/L。細(xì)胞生長(zhǎng)至對(duì)數(shù)生長(zhǎng)期，根據(jù)實(shí)驗(yàn)需求，達(dá)到細(xì)胞密度后，加入藥物培養(yǎng)24h收集細(xì)胞，分別進(jìn)行以下實(shí)驗(yàn)：1.流式細(xì)胞術(shù)檢測(cè)細(xì)胞內(nèi)活性氧（ROS）；2.免疫組化（SP）法檢測(cè)c-fos、bcl-2、bax、Caspase-3相關(guān)的表達(dá)3.激光共聚焦法檢測(cè)細(xì)胞內(nèi)[Ca2+]的水平；4.RT-PCR測(cè)定三磷酸肌醇受體(IP3R)的表達(dá)。 結(jié)果： 1．BeSO_4中、高劑量組ROS含量與空白對(duì)照組相比明顯增高，表達(dá)細(xì)胞數(shù)增多，具有統(tǒng)計(jì)學(xué)意義（P0.05）。 2.細(xì)胞加藥培養(yǎng)24h后，免疫組化結(jié)果顯示： BeSO_4低、中、高劑量組c-fos、bcl-2表達(dá)均高于空白對(duì)照組，，差異有統(tǒng)計(jì)學(xué)意義（P0.05），且隨著藥物劑量增加表達(dá)水平不斷增高。BeSO_4中、高劑量組bax表達(dá)均低于空白對(duì)照組、BeSO_4低、中、高劑量組Caspase-3表達(dá)均低于空白對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）且隨著藥物劑量增加表達(dá)水平不斷降低。 3.硫酸鈹高劑量組細(xì)胞內(nèi)Ca2+濃度明顯高于空白對(duì)照組，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 4.激光共聚焦顯微鏡下，100μmol/L BeSO_4組中，細(xì)胞核呈現(xiàn)出明亮的綠色熒光，細(xì)胞分界清楚呈梭型，BeSO_4+LBP組細(xì)胞呈圓形，胞體淡綠色，細(xì)胞周圍可見(jiàn)綠色熒光小點(diǎn)。 5. BeSO_4低、中、高劑量組Ⅲ型IP3R表達(dá)與空白對(duì)照組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.01）； 6. LBP對(duì)BeSO_4損傷細(xì)胞的保護(hù)作用：BeSO_4組ROS含量高于空白對(duì)照組（P0.01）、LBP組ROS含量低于空白對(duì)照組（P0.01），二者具有交互作用（P0.05）。對(duì)于bcl-2、Caspase-3的表達(dá)BeSO_4和LBP具有交互效應(yīng)（P0.05或P0.01）。Ca2+和IP3R檢測(cè)結(jié)果顯示BeSO_4和LBP具有交互作用（P0.05或P0.01）。 結(jié)論： 1. BeSO_4可致MRC-5細(xì)胞內(nèi)ROS含量升高，LBP對(duì)此有一定保護(hù)作用。 2. BeSO_4可致MRC-5細(xì)胞內(nèi)的c-fos、bcl-2基因表達(dá)量升高；bax、Caspase-3基因表達(dá)量減少，LBP對(duì)此有一定的調(diào)節(jié)作用。 3. BeSO_4可致MRC-5細(xì)胞內(nèi)鈣離子濃度升高，LBP對(duì)其有一定保護(hù)作用。 4. BeSO_4可能通過(guò)IP3R受體通道，造成MRC-5細(xì)胞鈣超載，LBP對(duì)IP3R受體的表達(dá)有一定的調(diào)節(jié)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

本文編號(hào)：2189474