BPDE诱导Swan 71滋养层细胞线粒体损伤的机制

发布时间：2018-11-03 09:32

【摘要】：目的苯并[α]芘(Benzo(a)pyrene,B(a)P)是已知的致畸、致癌、致突变及内分泌干扰物,大量的研究显示,B(a)P可以引起子痫、子宫内生长受限、流产等滋养层相关疾病,然而潜在机制尚不明确。本研究拟通过B(a)P在体内的代谢终致突变物7,8-二羟基-9,10-环氧苯并[α]芘(BPDE)对滋养层细胞系Swan 71进行染毒,观察其细胞和线粒体功能的改变及其具体分子机制。探索线粒体损伤在滋养层细胞功能紊乱中的作用,为滋养层相关疾病的治疗提供新的思路。方法1.MTT法检测BPDE对Swan 71细胞活力的影响,根据结果确立染毒浓度和时间。采用Transwell的方法检测各组细胞的侵袭能力;采用ELISA和Western blot的方法测定各组细胞HCG分泌能力;流式细胞仪检测细胞凋亡;酶标仪检测细胞内ROS、MDA和SOD的含量;RT-qPCR检测促炎因子(TNF-а、IL-6)mRNA的改变;荧光显微镜观察线粒体膜电位的改变;透射电镜观察线粒体形态和结构的改变;实时荧光定量PCR检测线粒体DNA拷贝数的改变。2.采用RT-qPCR、Western blot技术检测线粒体各组分裂融合基因和蛋白(Mfn1、Mfn2、OPA1、Drp1和Fis1)的表达情况。采用Western blot的方法对凋亡相关蛋白(P53、Bcl-2、Bak和Bax)、线粒体和胞浆中的Cyt c以及Caspase3前体和活化产物进行检测。结果1.BPDE对Swan 71细胞功能的影响与对照组相比,0.1μmol/L、0.2μmol/L、0.4μmol/L和0.8μmol/L组Swan 71细胞的侵袭能力逐渐减弱,且差异均具有统计学意义(P0.001);与对照组相比,0.5μmol/L、1μmol/L和2μmol/L组Swan 71细胞上清中HCG的含量和细胞绒毛膜促性腺激素β蛋白的表达量逐渐降低,差异具有统计学意义(P0.05);BPDE可以促进Swan 71细胞凋亡,且0.5μmol/L、1μmol/L和2μmol/L组与对照组相比差异具有统计学意义(P0.001)2.BPDE对Swan 71细胞氧化损伤的影响与对照组相比,0.25μmol/L、0.5μmol/L、1μmol/L和2μmol/L组细胞内ROS和MDA的水平逐渐增加,SOD的水平逐渐下降,差异均具有统计学意义(P0.05);与对照组相比,1μmol/L和2μmol/L组促炎因子TNF-а和IL-6 mRNA表达逐步上调,且差异具有统计学意义(P0.001)。3.BPDE对Swan 71细胞线粒体结构和功能的影响随着BPDE浓度的增加,线粒体膜电位逐渐降低;透射电镜观察,0.5μmol/L组线粒体线粒体肿胀扭曲,线粒体嵴紊乱,膜片段化,内质网肿胀。1μmol/L组线粒体显示出更严重的空泡化、破裂和溶解,线粒体嵴溶解,内质网呈泡状结构。线粒体DNA拷贝数呈现先上升后下降的趋势,0.25μmol/L、0.5μmol/L和1μmol/L组与对照组相比差异具有统计学意义(P0.05)。4.BPDE对Swan 71细胞凋亡相关蛋白的影响随着BPDE浓度的增加,P53、Bak1和Bax的蛋白表达量逐渐增加,Bcl-2的蛋白表达量逐渐降低。且除P53 0.25μmol/L组外各染毒组与对照组相比差异均具有统计学意义(P0.05)。5.BPDE对Swan 71细胞分裂融合基因的影响与对照组相比,0.5μmol/L、1μmol/L和2μmol/L组线粒体融合基因OPA1、Mfn1和Mfn2表达逐渐下降,差异均具有统计学意义(P0.05);0.5μmol/L、1μmol/L和2μmol/L组线粒体分裂基因Fis1表达逐渐上调,1μmol/L和2μmol/L组Drp1表达逐渐上调,差异均具有统计学意义(P0.05)。与对照组相比,0.5μmol/L、1μmol/L和2μmol/L组线粒体融合蛋白OPA1表达逐渐下降,各染毒组中Mfn1和Mfn2表达逐渐下降,差异均具有统计学意义(P0.05);各染毒组中Drp1和Fis1表达逐渐上调,差异具有统计学意义(P0.001)。6.BPDE对Swan 71细胞Cyt c释放和Caspase 3的影响随着BPDE浓度的增加,胞浆中的Cyt c蛋白的含量逐渐增加,而线粒体中的Cyt c蛋白的含量逐渐下降,Caspase 3前体逐渐减少,而活化的Caspase 3逐渐增多,且除0.25μmol/L组外差异均具有统计学意义(P0.05)。结论BPDE可诱导Swan 71细胞产生氧化损伤引起线粒体分裂增加而融合下降,造成线粒体膜片段化释放Cyt c,引发线粒体途径的凋亡,导致Swan 71细胞侵袭和HCG分泌功能障碍,为BPDE引起的滋养层相关疾病治疗提供实验依据。
[Abstract]:Benzo (a) ptyrene, B (a) P) is a known carcinogens, carcinogenic, mutagenic and endocrine disruptors, and a large number of studies show that B (a) P can induce trophoblast-related diseases such as eclampsia, intrauterine growth restriction, abortion, etc. However, the underlying mechanism is not yet clear. In this study, the change of cell and mitochondrial function and its specific molecular mechanism were observed by B (a) P metabolism in the body. The changes of cell and mitochondrial function and their specific molecular mechanism were observed in the trophoblast cell line Swan 71. To explore the role of mitochondrial damage in the functional disturbance of trophoblast cells and to provide a new idea for the treatment of trophoblast-related diseases. Methods 1. MTT assay was used to detect the effect of BPDE on the viability of Swan 71 cells, and the concentration and time of exposure were established according to the results. The invasion ability of all groups of cells was detected by Transwell's method, and the secretion ability of the cells was determined by ELISA and Western blot. The cell apoptosis was detected by flow cytometry. The contents of ROS, MDA and SOD in cells were detected by ELISA. The changes of the expression of TNF-CoV and IL-6 mRNA were detected by RT-qPCR. The changes of mitochondrial DNA number were observed by fluorescence microscope; the changes of mitochondrial morphology and structure were observed by transmission electron microscope; the change of mitochondrial DNA copy number was detected by real-time fluorescence quantitative PCR. RT-qPCR and Western blot were used to detect the expression of fusion genes and proteins (Mfn1, Mfn2, OPA1, Drp1, and CD1). Apoptosis-related proteins (P53, Bcl-2, Bk and Bax), Cyt c in mitochondria and cytoplasm, and Caspase3 precursor and activation products were detected by Western blot. Results 1. The effect of BPDE on the function of Swan 71 cells was 0. 1. mol/ L, 0.2. mol/ L, 0.4. mol/ L and 0.9. mol/ L, and the invasive ability of Swan 71 cells was decreased gradually, and the difference was statistically significant (P0.05). In 1. mol/ L and 2. m mol/ L group of Swan 71 cells, there was a statistically significant difference (P0.05). The BPDE could promote the apoptosis of Swan 71 cells and 0.5mumol/ L. Compared with the control group, the effect of BPDE on the oxidative damage of Swan 71 cells was 0. 25 ug/ L, 0.5. mol/ L, 1. m mol/ L and 2. m mol/ L, the levels of ROS and MDA increased gradually, and the level of SOD decreased gradually. The difference was statistically significant (P0.05). Compared with the control group, the expression of TNF-and IL-6 mRNA was up-regulated in 1. m u.mol/ L and 2. m mol/ L group, and the difference was statistically significant (P0.001). The effect of BPDE on the mitochondrial structure and function of Swan 71 cell decreased with the increase of BPDE concentration. Mitochondrial DNA was gradually decreased; transmission electron microscope observation, 0. 5 ug/ L group of mitochondrial swelling distortion, mitochondrial membrane disturbance, membrane fragmentation, reticulum swelling. 1. mu mol/ L group of mitochondria showed more serious vacuolization, rupture and dissolution, mitochondria were dissolved, and the Endoplasmic reticulum was in a bubble structure. Compared with the control group, the effect of BPDE on the apoptosis-related protein of Swan 71 cells increased with the increase of BPDE concentration, and the expression of P53, Bak1 and Bax increased gradually. The expression of Bcl-2 protein decreased gradually. Compared with the control group, the expression of OPA1, Mfn1 and Mfn2 in the mitochondrial fusion gene OPA1, Mfn1 and Mfn2 decreased gradually. The differences were statistically significant (P 0.05); 0. 5. m u.mol/ L, 1. m mol/ L and 2. m u.mol/ L, the expression of DS1 was gradually increased, and the expression of Drp1 in 1. m u.mol/ L and 2. m u.mol/ L groups was gradually increased, and the difference was statistically significant (P0.05). Compared with the control group, the expression of OPA1 in 0.5. mol/ L, 1. m mol/ L and 2. m mol/ L group decreased gradually, and the expression of Mfn1 and Mfn2 in each group was gradually decreased, and the difference was statistically significant (P0.05). The difference was statistically significant (P0.001). The effect of BPDE on the release of Cyt c and Caspase 3 in Swan 71 cells increased with the increase of BPDE concentration, the content of Cyt c protein in cytoplasm gradually increased, and the content of Cyt c protein in mitochondria gradually decreased, Caspase 3 precursor decreased gradually, and the activated Caspase 3 increased gradually. All the differences were statistically significant except for 0. 25 ug/ L group (P0.05). Conclusion BPDE can induce the increase of mitochondrial fragmentation caused by oxidative damage of Swan 71 cells, which causes mitochondrial membrane fragmentation to release Cyt c and induce apoptosis of mitochondrial pathway, which leads to the invasion of Swan 71 cells and the dysfunction of urinary tract secretion. provides experimental basis for the treatment of trophoblast related diseases caused by BPDE.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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