纳米镍对大鼠睾丸生精—支持共培养细胞的凋亡作用及机制研究
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摘要
目的:研究纳米镍对大鼠睾丸生精-支持共培养细胞的作用及其相关机制。方法:以体外共培养的大鼠睾丸生精-支持共培养细胞为实验对象,分别加入不同浓度的纳米镍(0、25、50、100、200、400和800μg/mL)染毒24 h后,采用MTT法检测细胞存活率,Hoechst33258染色法检测细胞凋亡形态及Annexin V/PI双染法检测细胞凋亡率;lncRNA基因芯片检测细胞凋亡相关lncRNA的差异表达。结果:与对照组相比,不同浓度纳米镍染毒后,细胞存活率均降低(P<0.01),并呈剂量效应关系(P<0.01)。Hoechst33258染色结果显示,与对照组比较,纳米镍染毒组细胞核或细胞质内显示出浓染致密的颗粒块状荧光及明显核形态变化。Annexin V/PI双染法流式检测结果显示,染毒浓度为25、50、100、200、400μg/mL时,细胞凋亡率分别为8.80%±0.50%、11.00%±0.70%、12.53%±0.71%、15.95%±0.54%和17.80%±0.76%,各浓度组细胞凋亡率与对照组比较均明显增加(P<0.01),且呈浓度效应关系(P<0.01)。LncRNA基因芯片检测结果显示,与凋亡相关的lncRNA共169个,其中,100μg/mL纳米镍染毒组与对照组比较,上调的有117个,下调的有52个。结论:纳米镍能引起大鼠睾丸生精-支持共培养细胞凋亡,lncRNA可能在其中起重要作用。
OBJECTIVE:Study the effects of nickel nanoparticles(Ni NPs) on apoptosis in SD rat Sertoli-germ cell culture and research on related mechanisms. METHODS:We established a rat Sertoli-germ cell co-culture system in vitro and the cells were treated with Ni NPs. Survive rates were determined by using the MTT assay. Apoptosis morphology were determined by Hoechst33258. Apoptosis index of Sertoli cells and germ cells were determined by Annexin V/PI staining;lncRNA expression profiles were detected to screen out the apoptosis-related genes. RESULTS:The survival rates in the Ni NPs exposure groups(25,50,100,200,400 and 800 μg/mL) were significantly lower than that in the control group(0 μg/mL) after 24 h(P<0.01). The survival rates decreased as the concentrations of Ni NPs increased. As the Hochest33258 shows, the treatment groups showed the dense granular luminescence and obvious nuclear morphological changes in the nucleus or cytoplasm. The apoptosis indexes were 8.80%±0.50%,11.00%±0.70%,12.53%±0.71%,15.95%±0.54% and 17.80%±0.76% as the doses were 25,50,100,200,400 μg/mL. Apoptosis indexes increased as the concentrations of Ni NPs increased. The apoptosis indexes in the Ni NPs exposure groups were significantly higher than that in the control group(P<0.01). Compared with the control group,there were 169 lncRNA(117 up and 52 down) associated with apoptosis in the exposure group of 100 μg/mL. CONCLUSION:The study shows that Ni NPs can cause apoptosis of SD rat Sertoli-germ cell co-culture with significant cytotoxicity. Lnc RNA were closely related to the apoptosis of SD rat co-culture Sertoli-germ cell.
OBJECTIVE:Study the effects of nickel nanoparticles(Ni NPs) on apoptosis in SD rat Sertoli-germ cell culture and research on related mechanisms. METHODS:We established a rat Sertoli-germ cell co-culture system in vitro and the cells were treated with Ni NPs. Survive rates were determined by using the MTT assay. Apoptosis morphology were determined by Hoechst33258. Apoptosis index of Sertoli cells and germ cells were determined by Annexin V/PI staining;lncRNA expression profiles were detected to screen out the apoptosis-related genes. RESULTS:The survival rates in the Ni NPs exposure groups(25,50,100,200,400 and 800 μg/mL) were significantly lower than that in the control group(0 μg/mL) after 24 h(P<0.01). The survival rates decreased as the concentrations of Ni NPs increased. As the Hochest33258 shows, the treatment groups showed the dense granular luminescence and obvious nuclear morphological changes in the nucleus or cytoplasm. The apoptosis indexes were 8.80%±0.50%,11.00%±0.70%,12.53%±0.71%,15.95%±0.54% and 17.80%±0.76% as the doses were 25,50,100,200,400 μg/mL. Apoptosis indexes increased as the concentrations of Ni NPs increased. The apoptosis indexes in the Ni NPs exposure groups were significantly higher than that in the control group(P<0.01). Compared with the control group,there were 169 lncRNA(117 up and 52 down) associated with apoptosis in the exposure group of 100 μg/mL. CONCLUSION:The study shows that Ni NPs can cause apoptosis of SD rat Sertoli-germ cell co-culture with significant cytotoxicity. Lnc RNA were closely related to the apoptosis of SD rat co-culture Sertoli-germ cell.
引文
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[14]季佳佳.二硫化碳对共培养大鼠睾丸支持—生精细胞凋亡和凋亡相关蛋白表达的影响[D].武汉:华中科技大学,2008.
[15]GALLO A,BONI R,BUTTINO I,TOSTI E.Spermiotoxicity of nickel nanoparticles in the marine invertebrate Ciona intestinalis(ascidians)[J].Nanotoxicology,2016,10(8):1096-1104.
[2]MIRI A H,SHAKIB E S,EBRAHIMI O,et al.Impacts of nickel nanoparticles on grow characteristics,photosynthetic pigment content and antioxidant activity of coriandrum sativum L[J].Orient J Chem,2017,33(3):1297-1303.
[3]WAN R,MO Y,CHIEN S,et al.The role of hypoxia inducible factor-1αin the increased MMP-2 and MMP-9production by human monocytes exposed to nickel nanoparticles[J].Nanotoxicology,2011,5(4):568-582.
[4]ZHANG W,LI Y,NIU J,et al.Photogeneration of reactive oxygen species on uncoated silver,gold,nickel,and silicon nanoparticles and their antibacterial effects[J].Langmuir,2013,29(15):4647-4651.
[5]PETERS K,UNGER R E,GATTI A M,et al.Metallic nanoparticles exhibit paradoxical effects on oxidative stress and pro-inflammatory response in endothelial cells in vitro[J].Int J Immunopathol Pharmacol,2007,20(4):685-695.
[6]AHAMED M.Toxic response of nickel nanoparticles in human lung epithelial A549 cells[J].Toxicol In Vitro,2011,25(4):930-936.
[7]ALARIFI S,ALI D,ALAKHTANI S,et al.Reactive oxygen species-mediated DNA damage and apoptosis in human skin epidermal cells after exposure to nickel nanoparticles[J].Biol Trace Elem Res,2014,157(1):84-93.
[8]ISPAS C,ANDREESCU D,PATEL A,et al.Toxicity and developmental defects of different sizes and shape nickel nanoparticles in zebrafish[J].Environ Sci Technol,2009,43(16):6349-6356.
[9]KONG L,TANG M,ZHANG T,WANG D,HU K,LU W,et al.nickel nanoparticles exposure and reproductive toxicity in healthy adult rats[J].Int J Mol Sci,2014,15(11):21253-21269.
[10]KONG L,GAO X,ZHU J,et al.Reproductive toxicity induced by nickel nanoparticles in Caenorhabditis elegans[J].Environ Toxicol,2017,32(5):1530-1538.
[11]KINO T,HURT D E,ICHIJO T,et al.Noncoding RNA gas5is a growth arrest-and starvation-associated repressor of the glucocorticoid receptor[J].Sci Signal,2010,3(107):8.
[12]HUARTE M,GUTTMAN M,FELDSER D,et al.A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response[J].Cell,2010,142(3):409-419.
[13]HUNG T,WANG Y,LIN M F,et al.Extensive and coordinated transcription of noncoding RNAs within cell-cycle promoters[J].Nat Genet,2011,43(7):621-629.
[14]季佳佳.二硫化碳对共培养大鼠睾丸支持—生精细胞凋亡和凋亡相关蛋白表达的影响[D].武汉:华中科技大学,2008.
[15]GALLO A,BONI R,BUTTINO I,TOSTI E.Spermiotoxicity of nickel nanoparticles in the marine invertebrate Ciona intestinalis(ascidians)[J].Nanotoxicology,2016,10(8):1096-1104.