Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus ...Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.展开更多
背景与目的目前,云南个旧锡矿有大量矿工从事开采工作,这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关,大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡Ⅱ型上皮细胞(immortalized rat alveolar cells ty...背景与目的目前,云南个旧锡矿有大量矿工从事开采工作,这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关,大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡Ⅱ型上皮细胞(immortalized rat alveolar cells type Ⅱ,RLE-6TN)恶性转化过程中,瘦素(leptin)及其介导的细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)信号通路所起的作用。方法采用200μg/mL的云锡矿粉隔代毒染RLE-6TN至第9代,建立毒染细胞模型,命名为R_(200)细胞,正常培养组命名为R细胞,通过Western blot法检测两种细胞leptin受体的表达情况。通过MTT法筛选出leptin及丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)抑制剂(U0126)对R_(200)细胞的最佳作用浓度。自第20代起,将R组、R_(200)组细胞分别与leptin及MEK抑制剂U0126共培养,对各组细胞的形态改变进行观察,并利用苏木素-伊红(hematoxylin-eosin,HE)染色技术鉴别第40代细胞的形态学差异,通过刀豆凝集素A(concanavalin A,ConA)及锚着独立性生长实验法检测细胞恶性转化情况。通过Western blot法检测leptin作用后上皮细胞ERK信号通路的变化。结果R组和R_(200)组细胞均表达leptin受体(OB-R)。与R_(200)组比较,当leptin浓度达100 ng/mL时,其促增殖效应最为显著,30μmol/L U0126可抑制毒染细胞R_(200)增殖,与对照组相比具有统计学差异(P<0.05)。自第25代起,leptin诱导的R_(200)组(R_(200)L组)细胞形态发生变化,至第30代出现恶性转化,至第40代时恶性转化特征明显;而R_(200)组细胞及U0126诱导的R_(200)组(R_(200)LU组)细胞则在第40代时才出现恶性转化特征。R_(200)L组细胞凝集速度较R_(200)LU组快,其余各组细胞P30出现凝集,且随ConA浓度增加,细胞凝集速度加快。R_(200)L组细胞自P40可见克隆形成,克隆形成率为2.25‰±0.5‰,R_(200)LU组及R_(200)组未见克隆集落。R_(200)L组细胞pERK表达增强;加入U0126阻断后,R_(200)L组细胞pERK磷酸化水平降低。结论Leptin可以促进云锡矿粉毒染肺上皮细胞的恶性转化,ERK信号通路可能是其促进云锡矿粉引发的肺泡Ⅱ型上皮细胞转化的重要途径。展开更多
Tin miners in Gejiu, Yunnan Province, China are at high risk of developing lung cancer with significant occupational characteristics. Tissue samples from these miners presented pathological characteristics, such as fi...Tin miners in Gejiu, Yunnan Province, China are at high risk of developing lung cancer with significant occupational characteristics. Tissue samples from these miners presented pathological characteristics, such as fibroplasia in carcinomas, peri-cancerous tissue in lung cancers, and hyperplasia and dysplasia of epithelial cells in peri-cancerous tissue. Carcinomas induced by Yunnan tin mine dust in the animal experiment underwent inflammation, fibroplasia, hyperplasia, dysplasia, and carcinogenesis of epithelial ceils. A correlated and synergistic relationship was observed between bronchial epithelial cell transformation and fibroblast activation in vitro induced by mine dust. Fibroblast hyperplasia and activation are important factors that promote the transformation and carcinogenesis of epithelial cells. Our findings suggested that pulmonary fibrosis may increase the risk and promote the occurrence of lung cancer, which can lead to lun~ fiber hyperplasia.展开更多
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.31902052 and 31972450)the National Key Research and Development Program of China(Grant No.2016YFC0500300)+1 种基金the Natural Science Foundation of Heilongjiang Province of China(Grant No.C2018021)the‘Academic backbone’Project of Northeast Agricultural University of China(Grant No.18XG08).
文摘Dianthus spiculifolius is a perennial herbaceous flower with strong environmental adaptability and is an important ornamental ground cover plant.In this study,seeds of D.spiculifolius were used as explants for callus induction,adventitious bud differentiation,and rooting by adding different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D),6-benzyl aminopurine(6-BA),and naphthaleneacetic acid(NAA)to Murashige and Skoog medium.The calli generated were co-cultured with Agrobacterium tumefaciens EHA105 containing pBI121-GUS or pBI121-GFP plasmids for 30 min,and transgenic regenerated plants were obtained by kanamycin(30mg·L^−1)screening.RT-PCR confirmed the stable expression of the exogenous GUS and GFP genes in the D.spiculifolius.Theβ-glucuronidase(GUS)histochemical staining confirmed GUS gene expression in transgenic calli,adventitious buds,and regenerated plants of D.spiculifolius.The green fluorescent protein(GFP)visual analysis showed GFP gene expression in transgenic calli.Furthermore,subcellular localization analysis showed that the three organelle marker proteins were not only successfully expressed but also accurately localized to their corresponding organelles in D.spiculifolius callus cells.These results indicated a successful establishment of a reliable and efficient A.tumefaciens-mediated genetic transformation system,which will contribute to functional gene research and genetic improvement of D.spiculifolius.
文摘背景与目的目前,云南个旧锡矿有大量矿工从事开采工作,这种职业环境与接触粉尘颗粒、重金属、多环芳烃和放射性氡有关,大大增加了患肺癌的风险。本研究旨在探讨在云锡矿粉诱导大鼠肺泡Ⅱ型上皮细胞(immortalized rat alveolar cells type Ⅱ,RLE-6TN)恶性转化过程中,瘦素(leptin)及其介导的细胞外调节蛋白激酶(extracellular regulated protein kinase,ERK)信号通路所起的作用。方法采用200μg/mL的云锡矿粉隔代毒染RLE-6TN至第9代,建立毒染细胞模型,命名为R_(200)细胞,正常培养组命名为R细胞,通过Western blot法检测两种细胞leptin受体的表达情况。通过MTT法筛选出leptin及丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinase,MEK)抑制剂(U0126)对R_(200)细胞的最佳作用浓度。自第20代起,将R组、R_(200)组细胞分别与leptin及MEK抑制剂U0126共培养,对各组细胞的形态改变进行观察,并利用苏木素-伊红(hematoxylin-eosin,HE)染色技术鉴别第40代细胞的形态学差异,通过刀豆凝集素A(concanavalin A,ConA)及锚着独立性生长实验法检测细胞恶性转化情况。通过Western blot法检测leptin作用后上皮细胞ERK信号通路的变化。结果R组和R_(200)组细胞均表达leptin受体(OB-R)。与R_(200)组比较,当leptin浓度达100 ng/mL时,其促增殖效应最为显著,30μmol/L U0126可抑制毒染细胞R_(200)增殖,与对照组相比具有统计学差异(P<0.05)。自第25代起,leptin诱导的R_(200)组(R_(200)L组)细胞形态发生变化,至第30代出现恶性转化,至第40代时恶性转化特征明显;而R_(200)组细胞及U0126诱导的R_(200)组(R_(200)LU组)细胞则在第40代时才出现恶性转化特征。R_(200)L组细胞凝集速度较R_(200)LU组快,其余各组细胞P30出现凝集,且随ConA浓度增加,细胞凝集速度加快。R_(200)L组细胞自P40可见克隆形成,克隆形成率为2.25‰±0.5‰,R_(200)LU组及R_(200)组未见克隆集落。R_(200)L组细胞pERK表达增强;加入U0126阻断后,R_(200)L组细胞pERK磷酸化水平降低。结论Leptin可以促进云锡矿粉毒染肺上皮细胞的恶性转化,ERK信号通路可能是其促进云锡矿粉引发的肺泡Ⅱ型上皮细胞转化的重要途径。
基金Acknowledgements We thank Dn Xianbo Zhou for his assistance with the final manuscript. This project was supported by two grants from the National Natural Science Foundation of China （81460355 and 81160276） and two grants from the Department of Science and Technology of Yunnan Province, Kunming Medical University Collaborative Special Projects （2014FB020 and 2011FBI81）.
文摘Tin miners in Gejiu, Yunnan Province, China are at high risk of developing lung cancer with significant occupational characteristics. Tissue samples from these miners presented pathological characteristics, such as fibroplasia in carcinomas, peri-cancerous tissue in lung cancers, and hyperplasia and dysplasia of epithelial cells in peri-cancerous tissue. Carcinomas induced by Yunnan tin mine dust in the animal experiment underwent inflammation, fibroplasia, hyperplasia, dysplasia, and carcinogenesis of epithelial ceils. A correlated and synergistic relationship was observed between bronchial epithelial cell transformation and fibroblast activation in vitro induced by mine dust. Fibroblast hyperplasia and activation are important factors that promote the transformation and carcinogenesis of epithelial cells. Our findings suggested that pulmonary fibrosis may increase the risk and promote the occurrence of lung cancer, which can lead to lun~ fiber hyperplasia.
