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ESTABLISHMENT OF CELL CLONE OF LYMPHOMA AND A CELL LINE INFECTED WITH LEUKEMIA VIRUS AND STUDY ON ITS INDUCTED DIFFERENTIATION
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作者 程立 孔宪寿 +3 位作者 刘小沅 许菡 邓平 殷莲华 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 1992年第3期64-68,共5页
Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS... Since 1960, the tumor strains of L6565 viral leukemia, SRS lymphoma and L783 transplantable leukemia were established successively in our laboratory. Recently, derived from the strains of threse leukemia/lymphoma, SRS-82 cell line, SACIIB2, SACIIC3 cell clones and a cell line infected with SRS leukemia virus (SRSV/3T3) were obtained at vitro. The main results of study on the biology, virology and Its Induction of differentiation are summarily reported. 展开更多
关键词 cell line cell clone lymphoma leukemia nude mice cell differentiation.
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ELECTRON MICROSCOPIC OBSERVATION OF DIFFERENTIATION OF LEUKEMIC CELL CLONE AFTER CFU-MIX CULTURE
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作者 陈燕 王辨明 +3 位作者 李崇渔 喻东姣 阮幼冰 郑清平 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 1992年第1期45-50,共6页
By scanning and transmission electron microscopy, leukemlc celb were obwrved after CFU-Mix culture. Even though granulocytlc growth factor, erythropoietin and lymphocytlc growth factor were added at vitor, acute leuke... By scanning and transmission electron microscopy, leukemlc celb were obwrved after CFU-Mix culture. Even though granulocytlc growth factor, erythropoietin and lymphocytlc growth factor were added at vitor, acute leukemlc celb still showed defects In differentiation and maturation. These were characterized by abnormal colony which consisted of smooth cells, bizarre shape, nuclear-cytoplasmic asynchrony In development, and appearance of nuclear bleb. However, chronic myelogenous leukemlc celb were more nature than the acute ones, manifesting in normal colony with finger- like projections and ruffled membrane. Macrophages and eosinophils could be observed. It b suggested that there b a difference In differentiation between acute and chronic leukemia cells. 展开更多
关键词 In cell ELECTRON MICROSCOPIC OBSERVATION OF DIFFERENTIATION OF LEUKEMIC cell clone AFTER CFU-MIX CULTURE CML CFU AML
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Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97 被引量:111
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作者 Yan Li Zhao-You Tang Sheng-Long Ye Yin-Kun Liu Jie Chen Qiong Xue Jun Chen Dong-Mei Gao Wei-Hua Bao Liver Cancer Institute and Zhongshan Hospital of Fudan University (Former Liver Cancer Institute of Shanghai Medical University),Shanghai 200032,China 《World Journal of Gastroenterology》 SCIE CAS CSCD 2001年第5期630-636,共7页
ALM To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97. and... ALM To establish clone cells with different metastatic potential for the study of metastasis-related mechanisms. METHODS Cloning procedure was performed on parental hepatocellular carcinoma (HCC) cell line MHCC97. andbiological characteristics of the target clones selected by in vivo screening were studied.``RESULTS Two clones with high MHCC97-H and IowMHCC9--L1 metastatic potential were isolated from theparent cell line. Compared with MHCC97-L. MHCC97-H hadsmaller cell size average cell diameter 43 um vs 50 μmand faster in vitro and in vivo growth rate tumor celldoubling time was 34.2 h vs 60.0 h. The main ranges ofchromosomes were 5.5 58 in MHCC97-H and 57 62 inMHCC97-L. Boyden chamber in vitro invasion assay demonstrated that the number of penetrating cells through the artificial basement membrane was 137.5 - 11 .0) cellsfield for MHC_C99--H vs 17.7 - 6.3) field for MHCC97-L.The proportions of cells in GO Gl phase. S phase, and G_ M phase for MHCC97-H MHCC97-L were 0.56 6.65.0.28 0.25 and 0.l6 0.10, respectively, as measured by flow cytometry. The serum AFP levels in nude mice 5 wk after orthotopic implantation of tumor tissue were ( 24666 μg. L for MHCC97-H and (91- 66) μg' L 1 for MHCC97L. The pulmonary metastatic rate was 100% (10-10) vs40% 4- 10).``CONCLUSION Two clones of the same genetic background but with different biological behaviors were established, which could be valuable models for investigation on HCC metastasis. 展开更多
关键词 HEPATOcellULAR carcinoma clone cells: METASTASIS
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Immunophenotypic analysis of abnormal plasma cell clones in bone marrow of primary systemic light chain amyloidosis patients 被引量:3
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作者 Hu Yang Wang Mangju +6 位作者 Chen Yan Chen Xue Fang Fang Liu Shiqin Zhang Ying Wu Xueqiang Zhu Ping 《Chinese Medical Journal》 SCIE CAS CSCD 2014年第15期2765-2770,共6页
Background Primary systemic light chain amyloidosis (AL) is a rare plasma cell disease,our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients,and explore wh... Background Primary systemic light chain amyloidosis (AL) is a rare plasma cell disease,our purpose was to analyze the immunophenotypic characteristics of the plasma cells in bone marrow in AL patients,and explore whether the detection of abnormal plasma cell clones in bone marrow by flow cytometry (FCM) could be used as an important indicator of AL diagnosis.Methods Fresh bone marrow samples were collected from 51 AL,21 multiple myeloma (MM),and 5 Waldenstr(o)m's macroglobulinemia (WM) patients.The immunophenotype of bone marrow cells were analyzed and compared by FCM using a panel of antibodies including CD45,CD38,CD138,CD117,CD56,and CD19.Results In AL,light chain restriction could be identified in 31 cases (60.9%),in which the λ light chain restriction was found in 24 cases (77.4%).In MM,κ light chain restriction was found in 13 cases (61.9%),and λ light chain restriction in eight cases.CD45 on abnormal plasma cells was negative to weakly positive in both AL and MM,but was positive to strongly positive in WM.In the bone marrow plasma cells of the 51 AL,78.4% were CD56+,68.6% were CD117+,and 88.2% were CD19-.While in the 21 MM cases,66.7% were CD56+,38.1% were CD117+,and 90.4% were CD19-.The plasmacytoid lymphocytes in the five WM patients were CD19+ and CD56-,CD117-.Conclusion Detection of abnormal plasma cell clones in bone marrow by FCM is valuable for the diagnosis of AL. 展开更多
关键词 primary systemic light chain amyloidosis plasma cell clone light chain restriction IMMUNOPHENOTYPE flow cytometry
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Research on Isolation and Clone of Embryonic Stem Cell-Like in Bovine 被引量:2
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作者 AN Li-long, YANG Qi, XIAO Mei, FENG Xiu-Liang, YANG Chun-rong, LEI An-min, GAO Zhi-min, DOU Zhong-ying and QIU Huai( Agriculture College of Zhanjiang Ocean University, Zhanjiang 524008 , P. R . China Key laboratory of AnimalReproductive Endocrinology and Embryo Biotechnology of Agricultural Ministry of China , Northwestern Scienceand Technology University of Agriculture and Forestry, yangling 712100 , P.R. China) 《Agricultural Sciences in China》 CAS CSCD 2002年第4期450-458,共9页
Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture ... Bovine embryonic stem cell would be invaluable for researching the aspect of animal cloning, production transgenic animal and discussion of gene function in vitro. With the object of establishing an effective culture system for isolation and clone of bovine pluripotent stem cell, we cultured bovine embryos and mouse embryos including morula blastula and hatached blastula and obtained animal ICM on Primary murine embryonic fibroblast (Primary murine embryonic fibroblast, PMEF) feeder layer with tissue medium(DMEM supplemented with 15ml/100ml NBS,0.1μmol/L Na2SeO3, 0.1mmol/L p-mercaptoethanol, 1000ng/ml LIF, 10 ng/ml IGF, 1mmol/L necessary amino acid and 1mmol/L L-glutamine),then,we obtained mouse ICM and bovine ICM. Moreover, we isolated and cloned the 6 passage bovine ES like cells(12 cell lines) and 9 passage murine ES like cells (52 cell lines) deriving from bovine ICM and murine ICM respectively on the feeder layer of PMEF by disaggregating ICM and ES cell clones of bovine and murine into smaller clumps through digesting with 0.125g/100ml trypsin and 0.02g/100ml EDTA and scattering with a glass needle. The pluripotency of both murine and bovine ES like cells was identified with morphological character, histochemistry identification , karyotype analysis and differentiation of ES cells in vitro or in vivo. This result showed that bovine embryonic stem cell and murine embryonic stem cell had developmental pluripotency. 展开更多
关键词 BOVINE MURINE Embryonic stem cell Inner cell mass ISOLATION clone
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Hepatoprotective Effect of Guava (<i>Psidium guajava</i>L.) Leaf Extracts on Ethanol-Induced Injury on Clone 9 Rat Liver Cells 被引量:1
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作者 Hung-Hui Chen Po-Hua Wu +2 位作者 Diana Lo Yun-Chieh Pan Ming-Chang Wu 《Food and Nutrition Sciences》 2011年第9期983-988,共6页
Guava (Psidium guajava L.), a tropical fruit, belongs to Myrtaceae family. Leaves and fruits of guava have been reported to have an anti-diarrheal, hypoglycemic, lipid lowering, anti-bacterial in addition to antioxida... Guava (Psidium guajava L.), a tropical fruit, belongs to Myrtaceae family. Leaves and fruits of guava have been reported to have an anti-diarrheal, hypoglycemic, lipid lowering, anti-bacterial in addition to antioxidant activities. The aim of this study was to investigate several guava leaf extract cytotoxic effects on healthy clone 9 liver cells and its hepatoprotective effects on ethanol-induced heap-toxicity. It was discovered that when the clone 9 liver cells were treated with guava (Psidium guajava Linn.) extracts for 24 hours, there was no retardation of growth as well as when ethanol and acetone extracts at low concentrations 100 μg/mL and 50 μg/mL were administered however cytototoxic effects were detected at higher concentrations. Water and hot water extracts in concentrations lower than or equal to 500 μg/mL revealed no cytotoxic effects. Injury induction to healthy clone 9 liver cells using 5% alcohol concentration for 30 minutes revealed the hepatoprotective properties of guava (Psidium guajava Linn.) extracts. This was significant in concentrations of 100 μg/mL or lower for ethanol and all concentrations for hot water extracts. Hot water extracts showed higher hepatoprotective and lower cytotoxic properties than other extracts. 展开更多
关键词 GUAVA (Psidium Guajava Linn.) Alcohol-Injured cell Hepatoprotective Properties clone 9 cell Cytotoxicity
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lncRNA NEAT1降表达人喉鳞状细胞癌细胞增殖凋亡变化及与miR-214靶向关系
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作者 罗德艳 冯俊 +2 位作者 彭涛 唐一萍 杨久梅 《山东医药》 CAS 2024年第18期26-30,共5页
目的观察长链非编码RNA(lncRNA)核富集转录体1(NEAT1)降表达的人喉鳞状细胞癌(LSCC)细胞增殖凋亡变化,探讨其与微小RNA-214(miR-214)的靶向关系。方法采用实时荧光定量PCR(RT-qPCR)法检测人永生化表皮细胞Hacat和人LSCC细胞系(FD-LSC-1... 目的观察长链非编码RNA(lncRNA)核富集转录体1(NEAT1)降表达的人喉鳞状细胞癌(LSCC)细胞增殖凋亡变化,探讨其与微小RNA-214(miR-214)的靶向关系。方法采用实时荧光定量PCR(RT-qPCR)法检测人永生化表皮细胞Hacat和人LSCC细胞系(FD-LSC-1、Hep-2、TU177)中lncRNA NEAT1、miR-214,选择TU177细胞为受试细胞。取对数生长期TU177细胞,分为甲、乙组,分别转染si-lncRNA NEAT1(敲低lncRNA NEAT1表达)、si-NC(乱序无意义序列),培养至24 h时采用CCK8法观察两组细胞增殖情况、采用平板克隆形成实验观察两组细胞克隆形成情况、采用流式细胞术观察两组细胞周期和细胞凋亡情况、采用RT-qPCR法检测两组细胞lncRNA NEAT1及miR-214。另取对数生长期TU177细胞,分为一二三四组,一组顺序转染WT-lncRNA NEAT1、miR-214 mimic,二组顺序转染WT-lncRNA NEAT1、miR-NC,三组顺序转染MUT-lncRNA NEAT1、miR-214 mimics,四组顺序转染MUT-lncRNA NEAT1、miR-NC。采用双荧光素酶报告基因实验验证lncRNA NEAT1及miR-214的靶向关系。结果与乙组相比,培养24、48、72 h时甲组TU177细胞OD值低,培养14 d时甲组TU177细胞克隆形成数少,甲组TU177细胞G0/G1期细胞比例高、S期细胞比例低,细胞凋亡率高(P均<0.05);与乙组相比,转染24 h时甲组TU177细胞lncRNA NEAT1相对表达量低、miR-214相对表达量高(P均<0.05)。培养48 h时一、二、三、四组TU177细胞细胞荧光素酶活性分别为0.63±0.08、0.99±0.01、1.02±0.02、0.98±0.03,与二组相比,一组细胞荧光素酶活性低(P<0.05)。结论敲低lncRNA NEAT1表达可抑制TU177细胞的增殖和克隆,阻滞细胞周期于G0/G1期,并促进细胞的凋亡。TU177细胞中lncRNA NEAT1与miR-214靶向相关。lncRNA NEAT1可能通过与miR-214靶向结合,抑制TU177细胞的增殖克隆,阻滞细胞周期于G0/G1期,促进细胞的凋亡。 展开更多
关键词 长链非编码RNA 长链非编码RNA核富集转录体1 微小RNA-214 喉鳞状细胞癌 细胞增殖 细胞凋亡 细胞克隆 细胞周期
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七彩神仙鱼催乳素基因的克隆、定位及表达分析
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作者 成果 温彬 +1 位作者 高建忠 陈再忠 《广东农业科学》 CAS 2024年第4期35-44,共10页
【目的】七彩神仙鱼(Symphysodon aequifasciatus)具有特殊的亲代抚育行为,克隆并定位七彩神仙鱼催乳素基因,分析其在亲代抚育中的表达模式,为理解催乳素在七彩神仙鱼亲代抚育中的作用提供依据。【方法】通过BLASTP对七彩神仙鱼全基因... 【目的】七彩神仙鱼(Symphysodon aequifasciatus)具有特殊的亲代抚育行为,克隆并定位七彩神仙鱼催乳素基因,分析其在亲代抚育中的表达模式,为理解催乳素在七彩神仙鱼亲代抚育中的作用提供依据。【方法】通过BLASTP对七彩神仙鱼全基因组进行分析,鉴定出1个催乳素基因,命名为dfprl。基于dfprl基因的CDS区,设计克隆和PCR引物,通过RACE技术克隆获得dfprl基因全长,并利用生物信息学对dfprl基因进行结构分析,对其氨基酸序列进行理化性质和进化分析。对不同抚育阶段七彩神仙鱼的脑、性腺和皮肤进行转录组分析,探究dfprl基因在亲代抚育中的表达特征,并利用原位杂交技术对dfprl基因在七彩神仙鱼皮肤中的表达进行定位。【结果】dfprl全长为1282 bp,cDNA序列开放阅读框长度为639 bp,共编码212个氨基酸,5'-UTR为309 bp,3'-UTR为334 bp。dfprl蛋白存在PRL家族的典型结构域Hormone_1,dfprl蛋白与慈鲷科其他鱼类PRL蛋白相似性较高,与尼加拉瓜湖始丽鱼(Archocentrus centrarchus)对应的氨基酸序列同源性最高、为96.70%。亲鱼进入抚育阶段后,dfprl在性腺和皮肤中的表达水平逐渐上升,抚育结束时表达水平下降。原位杂交结果显示,dfprl在皮肤粘液细胞中表达,且与七彩神仙鱼催乳素受体(dfprlr)表达位点重叠,在抚育早期阶段高表达。【结论】七彩神仙鱼dfprl基因高度保守,存在稳定的Hormone_1结构域。七彩神仙鱼dfprl基因在亲代抚育阶段的皮肤中高表达,且在亲代抚育阶段的皮肤粘液细胞中与dfprlr表达位点重合,表明dfprl可能通过作用于dfprlr进而促进七彩神仙鱼独特抚育行为的发生。 展开更多
关键词 七彩神仙鱼 催乳素 亲代抚育 皮肤粘液细胞 基因克隆
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体细胞克隆技术扩繁超细型二狼山绒山羊的应用研究
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作者 卿玉波 程雯 +9 位作者 角德灵 刘城 郝志强 郭建雄 李钰莹 张筱琳 李臻 李效宇 王文 魏红江 《中国畜牧兽医》 CAS CSCD 北大核心 2024年第4期1420-1427,共8页
【目的】通过体细胞克隆技术生产超细型二狼山绒山羊克隆个体,为该技术在地方优质羊品种资源扩繁及新品种培育中的应用提供理论依据。【方法】选择1只4岁龄羊绒细度为13.89μm的二狼山绒山羊(♂),采集耳组织并分离培养成纤维细胞,以其... 【目的】通过体细胞克隆技术生产超细型二狼山绒山羊克隆个体,为该技术在地方优质羊品种资源扩繁及新品种培育中的应用提供理论依据。【方法】选择1只4岁龄羊绒细度为13.89μm的二狼山绒山羊(♂),采集耳组织并分离培养成纤维细胞,以其为供体细胞,利用体细胞克隆技术开展超细型二狼山绒山羊的扩繁研究,通过克隆羊体重、羊绒细度、繁殖性能等指标评估该技术的可行性。【结果】试验成功建立了稳定的山羊卵母细胞体外成熟培养及体细胞克隆技术体系,卵母细胞成熟率为44.8%,孤雌激活卵裂率、囊胚率分别为83.0%、22.4%;二狼山绒山羊克隆胚的卵裂率、囊胚率分别为77.8%、13.7%。获得5只克隆羊个体,其生长发育正常,二狼山绒山羊母羊自然交配共获得16只健康的F1代个体,具有正常繁殖能力。克隆个体羊绒细度极显著低于同龄对照个体(P<0.01)。【结论】本研究通过体细胞克隆技术成功克隆了超细型二狼山绒山羊个体,其具备正常生产性能和繁殖性能,并保留了超细羊绒的特性,为地方优良羊种质资源扩繁和新品种培育应用研究奠定了基础。 展开更多
关键词 二狼山绒山羊 体细胞克隆 羊绒细度 繁殖性能
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戊地昔布抑制clone 26肿瘤细胞增生及机制 被引量:1
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作者 李军霞 师晨霞 +2 位作者 苏素文 张永健 王永利 《中国药学杂志》 CAS CSCD 北大核心 2007年第6期441-444,共4页
目的探讨戊地昔布对小鼠肠癌clone26细胞的生长抑制作用及机制。方法用噻唑蓝(MTT)法检测戊地昔布对clone26细胞生长的作用。用流氏细胞仪检测clone26细胞凋亡率和细胞周期分布。用Western blot检测clone26细胞caspase-3,PCNA和COX-... 目的探讨戊地昔布对小鼠肠癌clone26细胞的生长抑制作用及机制。方法用噻唑蓝(MTT)法检测戊地昔布对clone26细胞生长的作用。用流氏细胞仪检测clone26细胞凋亡率和细胞周期分布。用Western blot检测clone26细胞caspase-3,PCNA和COX-2的表达.结果①戊地昔布可抑制clone26细胞生长并呈时间和浓度依赖性。②50-400μmol·L^-1戊地昔布可明显提高clone26细胞的凋亡率,从对照的3.1%提高到4.4%~11.0%。给予戊地昔布后,细胞的增殖指数,S期的细胞比例和G2/M期细胞有下降趋势,但只有在400μmol·L^-1戊地昔布组时才有统计学意义。③给予戊地昔布后,细胞PCNA的表达降低,caspase-3的表达升高,COX-2的表达没有变化。结论戊地昔布通过诱导凋亡和细胞周期停滞而抑制clone26细胞生长,小剂量时主要与凋亡有关,大剂量时与诱导凋亡和细胞周期停滞有关、Caspase-3参与戊地昔布诱导的凋亡。 展开更多
关键词 戊地昔布 环氧化酶-2 小鼠肠癌clone26细胞 凋亡
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抗人Ⅰ型干扰素受体亚基1人源化单克隆抗体的制备和鉴定
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作者 裘霁宛 孔永 +4 位作者 陈卫 徐蕾 曹纯洁 陈涛 吴亦亮 《中国药科大学学报》 CAS CSCD 北大核心 2024年第3期404-411,共8页
Ⅰ型干扰素在系统性红斑狼疮(SLE)等自身免疫性疾病的发病机制中发挥重要作用,采用抗体阻断其信号转导通路具有潜在的治疗作用。本研究以人Ⅰ型干扰素受体亚基1(IFNAR1)重组蛋白为抗原免疫新西兰白兔,采用B细胞克隆技术筛选兔抗人IFNAR... Ⅰ型干扰素在系统性红斑狼疮(SLE)等自身免疫性疾病的发病机制中发挥重要作用,采用抗体阻断其信号转导通路具有潜在的治疗作用。本研究以人Ⅰ型干扰素受体亚基1(IFNAR1)重组蛋白为抗原免疫新西兰白兔,采用B细胞克隆技术筛选兔抗人IFNAR1单克隆抗体,经过人源化改造获得QX006N。体外研究结果显示,QX006N能特异性地结合人IFNAR1,亲和力约108 pmol/L,可阻断Ⅰ型干扰素信号通路及其介导的生物学效应。本研究为开发靶向干预Ⅰ型干扰素信号途径用于治疗SLE的抗体药物提供了坚实的基础。 展开更多
关键词 B细胞克隆 人源化 兔单克隆抗体 IFNAR1 Ⅰ型干扰素 系统性红斑狼疮
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雄激素受体调控的lncRNA ARLNC1对前列腺癌细胞增殖、克隆、迁移和细胞周期的影响
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作者 郑江婷 尹冶 +1 位作者 寸淑娥 王玉明 《现代肿瘤医学》 CAS 2024年第2期220-226,共7页
目的:探讨雄激素受体(androgen receptor,AR)调控的长链非编码RNA(long non-coding RNA,lncRNA)ARLNC1对前列腺癌细胞增殖、克隆、迁移和细胞周期的影响。方法:选取正常前列腺上皮细胞株WPMY-1和前列腺癌细胞株PC3、VCaP、22RV1、DU145... 目的:探讨雄激素受体(androgen receptor,AR)调控的长链非编码RNA(long non-coding RNA,lncRNA)ARLNC1对前列腺癌细胞增殖、克隆、迁移和细胞周期的影响。方法:选取正常前列腺上皮细胞株WPMY-1和前列腺癌细胞株PC3、VCaP、22RV1、DU145和LNCaP为研究对象,qRT-PCR分析lncRNA ARLNC1的表达水平;双氢睾酮(DHT)以时间和浓度依赖的方式刺激LNCaP细胞,分析lncRNA ARLNC1的转录水平;采用数据库预测和双荧光素酶报告基因系统分析雄激素受体与lncRNA ARLNC1的关系;将LNCaP细胞分为sh-NC和sh-lncRNA ARLNC1组,采用CCK-8分析细胞增殖,检测细胞克隆和迁移能力;流式细胞仪分析细胞周期变化,Western blot分析细胞周期蛋白CyclinD1、CDK6、p27的表达水平。结果:与正常前列腺上皮细胞相比,lncRNA ARLNC1在雄激素依赖性前列腺癌细胞株(LNCaP、VCaP、22RV1)中的表达水平明显增加,而在雄激素非依赖性前列腺癌细胞株(PC3、DU145)中几乎不表达(P<0.05),其中LNCaP细胞变化最显著,所以下续选用LNCaP细胞作为实验株。100 nmol/L DHT刺激LNCaP细胞0、6、12、18、24 h后,lncRNA ARLNC1的转录水平相对于对照组以时间依赖的方式逐渐升高(P<0.05);不同浓度(0、0.1、1、10、100 nmol/L)DHT刺激LNCaP细胞24 h后,lncRNA ARLNC1转录水平随浓度不断增加(P<0.05);JASPAR数据库预测到lncRNA ARLNC1启动子区域有AR结合的位点,双荧光素酶报告基因系统表明AR能结合在lncRNA ARLNC1启动子区域,并激活其转录(P<0.05);与sh-NC组相比,sh-lncRNA ARLNC1组细胞增殖、迁移和克隆能力减弱,细胞周期阻滞在S、G 2期(P<0.05);sh-NC组相对于sh-lncRNA ARLNC1组,CyclinD1、CDK6蛋白水平增加,而p27蛋白水平降低(P<0.05)。结论:AR调控的lncRNA ARLNC1作为癌基因在雄激素受体阳性的前列腺癌细胞系中高表达,能促进前列腺癌细胞的增殖、克隆、迁移和细胞周期进展。 展开更多
关键词 前列腺癌 长链非编码RNA 雄激素受体 增殖 克隆 迁移 细胞周期
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新西兰白兔BMP 15基因的真核表达载体构建、表达模式及其在卵巢组织的表达
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作者 陈孟娟 刘雨晴 +4 位作者 王智通 温佳乐 许会芬 于光晴 李明 《畜牧兽医学报》 CAS CSCD 北大核心 2024年第2期562-575,共14页
旨在获得新西兰白兔BMP 15基因序列及其组织表达规律,构建其真核表达载体,预测BMP15的生物学功能。本研究选择180日龄性成熟的健康新西兰白兔作为研究对象,采用RT-PCR技术扩增BMP 15基因CDS区序列,利用T4连接方法将目的片段连接至线性化... 旨在获得新西兰白兔BMP 15基因序列及其组织表达规律,构建其真核表达载体,预测BMP15的生物学功能。本研究选择180日龄性成熟的健康新西兰白兔作为研究对象,采用RT-PCR技术扩增BMP 15基因CDS区序列,利用T4连接方法将目的片段连接至线性化的Pmcherry-N1和pCMV-Myc空载体,构建真核表达载体,利用生物信息学分析其编码蛋白的性质及结构。采用实时荧光定量PCR(qRT-PCR)技术与Western blotting技术检测pCMV-Myc-BMP 15过表达情况。接下来,使用qRT-PCR检测BMP 15基因在不同组织中的表达水平。激光共聚焦方法检测BMP 15基因的亚细胞定位。利用免疫荧光技术检测内源BMP15在新西兰白兔卵巢组织定位情况。结果表明:克隆得到新西兰白兔BMP 15基因CDS区序列长1182 bp,PCR及测序结果表明pCMV-Myc-BMP 15和Pmcherry-N1-BMP 15真核表达载体构建成功。生物信息学分析显示,BMP 15基因编码393个氨基酸;BMP15蛋白不稳定系数为55.32,等电点大小为9.69,是一种稳定的碱性蛋白质。BMP15蛋白共有26个磷酸化位点,15个糖基化位点,存在信号肽,不存在跨膜结构域。系统进化树表明,新西兰白兔与猪亲缘关系最近,与鸡的亲缘关系最远。二级结构和三级结构分析结果表明,BMP15蛋白主要由α-螺旋(38.68%)、无规则卷曲(37.4%)、延伸链(15.52%)、β-转角(8.40%)组成,为混合型蛋白。蛋白互作关系预测发现,BMP15蛋白与FSHR、FIGLA、BMPR1B、AMHR2、NOBOX等卵巢生长发育的相关蛋白之间存在相互作用。不同组织表达分析显示,BMP 15基因在卵巢组织中特异性表达;激光共聚焦结果显示,BMP15主要存在于在细胞质中。将pCMV-Myc-BMP 15转染至HEK293T细胞内,BMP15的mRNA水平和蛋白水平均显著上调。卵巢组织免疫荧光检测结果显示,BMP15主要定位在卵巢颗粒细胞的细胞质中。本研究成功构建了BMP15的真核表达载体,对BMP 15基因及其编码的蛋白的理化性质和生物学特性进行了预测分析,在HEK293T细胞内成功过表达,并得到了该基因的亚细胞定位情况、组织表达情况及在卵巢组织的分布情况。为后续开展BMP 15基因在卵巢生长发育中的功能及机制研究提供了理论依据。 展开更多
关键词 新西兰白兔 BMP 15基因 克隆 组织表达 细胞定位
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简州大耳羊CKMT2基因克隆、生物信息学分析及时空表达研究
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作者 孙诗雨 李金岚 +4 位作者 邢佳妮 董耀徽 李艳艳 王友利 林亚秋 《中国畜牧兽医》 CAS CSCD 北大核心 2024年第6期2342-2353,共12页
【目的】克隆简州大耳羊肌节线粒体肌酸激酶2(creatine kinase mitochondrial 2,CKMT2)基因并探究其生物学特征,明确其在简州大耳羊不同组织以及不同分化阶段成肌细胞中的表达特性。【方法】采集简州大耳羊心脏、肝脏、脾脏、肾脏、背... 【目的】克隆简州大耳羊肌节线粒体肌酸激酶2(creatine kinase mitochondrial 2,CKMT2)基因并探究其生物学特征,明确其在简州大耳羊不同组织以及不同分化阶段成肌细胞中的表达特性。【方法】采集简州大耳羊心脏、肝脏、脾脏、肾脏、背最长肌、臂三头肌组织样品,利用PCR方法扩增简州大耳羊CKMT2基因并克隆测序,通过在线软件对其进行生物信息学分析。利用实时荧光定量PCR技术检测CKMT2基因在简州大耳羊不同组织及不同分化阶段成肌细胞中的表达水平。【结果】简州大耳羊CKMT2基因全长1 314 bp,其中包括CDS区1 259 bp,编码419个氨基酸,与绵羊和羚羊的亲缘关系最近。CKMT2蛋白是一种无信号肽及跨膜结构域的碱性亲水稳定蛋白,主要定位于线粒体、过氧化物类酶体及细胞质,共包含32个磷酸化位点、1个N-糖基化位点以及4个O-糖基化位点。蛋白二级结构主要包括α-螺旋(39.14%)、无规则卷曲(36.28%)、延伸链(16.23%)和β-转角(8.35%),三级结构与二级结构预测结果基本一致。蛋白互作分析结果显示,CKMT2蛋白与半胱氨酸和甘氨酸富含蛋白3(cysteine and glycine-rich protein 3,CSRP3)、M型肌酸激酶(creatine kinase M-type, CKM)、磷酸甘油酸变位酶2(phosphoglycerate mutase 2,PGAM2)等蛋白存在相互作用。组织表达谱结果显示,CKMT2基因在简州大耳羊心脏中表达量最高,显著高于其他组织(P<0.05);在背最长肌中表达量较高,显著高于脾脏和臂三头肌(P<0.05)。时序表达谱结果显示,简州大耳羊CKMT2基因在成肌细胞诱导分化4 d时表达水平达到峰值,显著高于0、6 d(P<0.05)。【结论】本研究成功克隆简州大耳羊CKMT2基因序列并明确了其分子特征,其在心脏、背最长肌以及成肌分化4 d时表达量较高。研究结果为进一步揭示CKMT2基因调控简州大耳羊成肌细胞增殖分化的分子机制奠定基础。 展开更多
关键词 简州大耳羊 CKMT2基因 克隆 组织表达 细胞分化
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家蚕glial cell missing(BmGcm)基因鉴定、表达、亚细胞定位和功能
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作者 张奎 潘光照 +4 位作者 苏晶晶 谈娟 徐曼 李钰添 崔红娟 《中国农业科学》 CAS CSCD 北大核心 2018年第7期1401-1411,共11页
【目的】鉴定、克隆家蚕(Bombyx mori)glial cell missing(Bm Gcm)基因,分析其m RNA表达及亚细胞定位特征。制备多克隆抗体,同时在细胞水平进行过表达,检测Gcm对细胞增殖和周期的影响,为探究Bm Gcm功能打下基础。【方法】利用RACE方法... 【目的】鉴定、克隆家蚕(Bombyx mori)glial cell missing(Bm Gcm)基因,分析其m RNA表达及亚细胞定位特征。制备多克隆抗体,同时在细胞水平进行过表达,检测Gcm对细胞增殖和周期的影响,为探究Bm Gcm功能打下基础。【方法】利用RACE方法克隆获得Bm Gcm全长c DNA序列,利用ORF Finder和SMART等在线工具对Bm Gcm基本序列特征和结构信息进行分析,运用Clustalx和MEGA 6.0等软件对多物种Gcm蛋白进行同源序列比对和进化分析。采用RT-PCR和q RT-PCR方法检测Bm Gcm的表达情况。利用原核表达系统获得重组蛋白,通过蛋白纯化和免疫小鼠制备多克隆抗体,运用Western blot对抗体进行检测。构建Bm Gcm表达载体,转染家蚕胚胎细胞系,分析其亚细胞定位情况,同时利用EDU细胞增殖标记和流式细胞仪对其功能进行探索。【结果】Bm Gcm(BGIBMGA006182)定位于4号染色体的nscaf2847上,其基因全长4 046 bp,包含4个外显子和3个内含子。其c DNA全长1 734 bp,包含166 bp的5′UTR、227 bp的3′UTR和1 341 bp的完整开放阅读框(ORF)。该基因编码446个氨基酸残基,预测蛋白分子量为50.61 k D,等电点5.557,含有典型的GCM结构域。多重比对结果显示GCM结构域在不同物种间具有高度的保守性,进化分析显示昆虫Gcm蛋白单独聚为一支,其中Bm Gcm蛋白与帝王蝶同源蛋白亲缘关系最为接近。表达分析结果显示Bm Gcm在胚胎发育第4天表达达到峰值,随后表达水平逐渐下调,而在幼虫阶段,Bm Gcm主要表达于中肠、精巢和卵巢。将Bm Gcm完整的开放阅读框序列构建至原核表达系统,经IPTG诱导和亲和层析纯化获得高纯度重组蛋白,通过免疫小鼠获得了多克隆抗体,Western blot检测该抗体可以特异性识别重组蛋白。在家蚕细胞系中过表达Bm Gcm蛋白,结果显示其定位于细胞核。在细胞水平,过表达Bm Gcm会明显抑制细胞增殖,将细胞周期阻滞于G1/S期。【结论】克隆鉴定得到Bmgcm全长序列,获得其表达和亚细胞定位信息。通过原核表达、蛋白纯化和免疫小鼠制备了可用的多克隆抗体。细胞实验发现Bm Gcm可以显著抑制增殖和影响正常的细胞周期进程。 展开更多
关键词 家蚕glial cell missing基因(BmGcm) 克隆 表达分析 抗体制备 过表达
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Lnc RNA SNHG14通过调节miR-206表达来促进卵巢癌的发展
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作者 崔静 贾志燕 +1 位作者 王立君 郭姝媛 《中国现代药物应用》 2024年第13期170-174,共5页
目的探讨长链非编码RNA(Lnc RNA)小核仁RNA宿主基因14(SNHG14)在卵巢癌组织中的表达,以及Lnc RNA SNHG14通过微小RNA(miR)-206影响卵巢癌的增殖、迁移和侵袭能力。方法收集58例行卵巢癌根治手术患者的癌组织及其邻近的癌旁组织(距离癌组... 目的探讨长链非编码RNA(Lnc RNA)小核仁RNA宿主基因14(SNHG14)在卵巢癌组织中的表达,以及Lnc RNA SNHG14通过微小RNA(miR)-206影响卵巢癌的增殖、迁移和侵袭能力。方法收集58例行卵巢癌根治手术患者的癌组织及其邻近的癌旁组织(距离癌组织>3 cm),将其进行细胞培养,待细胞融合度达到70%左右时进行细胞转染,分别转染阴性对照质粒(si-NC组)、SNHG14高表达质粒(si-SNHG14组);采用实时荧光定量聚合酶链式反应(qRT-PCR)检测卵巢癌组织与癌旁组织中SNHG14、miR-206表达量,采用细胞增殖活性检测试剂盒(CCK-8)实验、平板克隆形成实验、Matrigel侵袭实验分别检测SNHG14对卵巢癌细胞的增殖能力、克隆形成数、迁移及侵袭;双荧光素酶报告实验检测Lnc RNA SNHG14与miR-206的靶向关系。结果与癌旁组织的(1.01±0.08)、(1.00±0.07)比较,卵巢癌组织中SNHG14表达量(2.56±0.57)升高、而miR-206表达量(0.36±0.11)降低(P<0.05);通过Pearson法检测卵巢癌组织中SNHG14与miR-206的相关性显示,SNHG14与miR-206呈负相关(r=-0.803,P<0.01)。与si-NC组的(1.01±0.06)、(1.00±0.08)比较,si-SNHG14组的SNHG14表达量(0.27±0.07)降低、miR-206表达量(2.96±0.45)升高(P<0.05);si-SNHG14组的吸光度(OD值)(0.29±0.05)较si-NC组的(0.79±0.06)降低,克隆形成数(52.35±7.37)较si-NC组的(120.51±19.34)减少(P<0.05)。si-SNHG14组的迁移细胞数(43.11±5.87)、侵袭细胞数(49.66±11.01)均较si-NC组的(117.11±10.48)、(122.30±12.97)明显减少(P<0.05)。采用starbase预测与SNHG14可互补结合的miR-206。转染miR-206 mimics可降低含有野生型载体的卵巢癌细胞的荧光素酶活性(P<0.05),而未能抑制含有突变型载体的卵巢癌细胞的荧光素酶活性(P>0.05)。结论卵巢癌组织和卵巢癌细胞中Lnc RNA SNHG14的表达会升高,Lnc RNA SNHG14可靶向miR-206,从而调节卵巢癌细胞的增殖、克隆形成、迁移和侵袭。 展开更多
关键词 长链非编码RNA 小核仁RNA宿主基因14 微小RNA-206 卵巢癌细胞 增殖 克隆形成 迁移 侵袭
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Stem cell pluripotency and transcription factor Oct4 被引量:38
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作者 GUANGJINPAN ZENGYICHANG +1 位作者 HANSR.SCHOELER DUANQINGPEI 《Cell Research》 SCIE CAS CSCD 2002年第5期321-330,共10页
Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of ... Mammalian cell totipotency is a subject that has fascinated scientists for generations. A long lasting question whether some of the somatic cells retains totipotency was answered by the cloning of Dolly at the end of the 20th century. The dawn of the 218t has brought forward great expectations in harnessing the power of totipotentcy in medicine. Through stem cell biology, it is possible to generate any parts of the human body by stem cell engineering. Considerable resources will be devoted to harness the untapped potentials of stem cells in the foreseeable future which may transform medicine as we know today. At the molecular level, totipotency has been linked to a singular transcription factor and its expression appears to define whether a cell should be totipotent. Named Oct4, it can activate or repress the expression of various genes. Curiously, very little is known about Oct4 beyond its ability to regulate gene expression. The mechanism by which Oct4 specifies totipotency remains entirely unresolved. In this review, we summarizere the structure and function of Oct4 and address to Oct4 function in maintaining totipotency or pluripotency of embryonic stem cels. 展开更多
关键词 干细胞 全能性 复制 翻译 蛋白因子
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Bcl-1 Rearrangement and Cyclin D1 Protein Expression in Multiple Myeloma Precursor Cells 被引量:3
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作者 刘新月 唐泽海 邹萍 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2000年第2期128-131,136,共5页
The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL... The rearrangement of Bcl-1 gene (Bcl-1/IgH rearrangement) and expression of cyclin D1 in multiple myeloma (MM) precursor cells were studied and the role of cyclin D1 in the pathogenesis of MM was investigated. The BCL-1 rearrangement and cyclin D1 protein expression in 15 cases of MM were detected. By using hemi-nested polymerase chain reaction (PCR) the genomic DNA from fresh peripheral blood and bone marrow was amplified and the expression of cyclin D1 in the smears was detected by using immunohistochemical method. Ten volunteer with normal bone marrow served as control group. The results showed Bcl-1 rearrangement was detectable in 3/15 (20 %) MM patients and cyclin D, expression in 4/15 (27 % ) MM patients. BeLl-1 rearrangement and cyclin D1 protein expression were also detected in MM precursor cells. No overexpression of cyclin D1 or the rearrangement of the BeL-1 gene was found in the 10 volunteers. It was concluded that Bel-1 rearrangement and cyclin D1 protein overexpression were detected in MM precursor cells, speculating that overexpression of cyclin D1 protein may play an initial (critical) role in the pathogenesis of MM. 展开更多
关键词 multiple myeloma BCL-1/IgH rearrangement clone cell cyclin D1 protein
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Embryonic stem cells generated by nuclear transfer of human somatic nuclei into rabbit oocytes 被引量:57
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作者 YINGCHEN ZHIXuHE +19 位作者 AILIANLIU KAIWANG WENWEIMAO JIANKINCHU YONGLU ZHENGFUFANG YINGTANGSHI QINGZHANGYANG DAYUANCHEN MINKANGWANG JINSONGLI SHAOLIANGHUANG XIANGYINKONG YAOZHOUSHI ZHIQIANGWANG JIAHuIXIA ZHIGAOLONG ZHIGANGXUE WENXIANGDING HUIZHENSHENG 《Cell Research》 SCIE CAS CSCD 2003年第4期251-263,共13页
To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the ... To solve the problem of immune incompatibility, nuclear transplantation has been envisaged as a means to produce cells or tissues for human autologous transplantation. Here we have derived embryonic stem cells by the transfer of human somatic nuclei into rabbit oocytes. The number of blastocysts that developed from the fused nuclear transfer was comparable among nuclear donors at ages of 5, 42, 52 and 60 years, and nuclear transfer (NT) embryonic stem cells (ntES cells) were subsequently derived from each of the four age groups. These results suggest that human somatic nuclei can form ntES cells independent of the age of the donor. The derived ntES cells are human based on karyotype, isogenicity, in situ hybridization, PGR and immunocytochemistry with probes that distinguish between the various species. The ntES cells maintain the capability of sustained growth in an undifferentiated state, and form embryoid bodies, which, on further induction, give rise to cell types such as neuron and muscle, as well as mixed cell populations that express markers representative of all three germ layers. Thus, ntES cells derived from human somatic cells by NT to rabbit eggs retain phenotypes similar to those of conventional human ES cells, including the ability to undergo multilineage cellular differentiation. 展开更多
关键词 胚胎干细胞 细胞核移植 卵母细胞 免疫排斥
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Activating interactions of sulfanilamides with T cell receptors 被引量:1
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作者 Stephan Watkins Werner J. Pichler 《Open Journal of Immunology》 2013年第3期139-157,共19页
Activation and expansion of drug reactive T cells are key features in drug hypersensitivity reactions. Drugs may interact directly with immune receptors such as the human leukocyte antigens (HLA) or the T-cell recepto... Activation and expansion of drug reactive T cells are key features in drug hypersensitivity reactions. Drugs may interact directly with immune receptors such as the human leukocyte antigens (HLA) or the T-cell receptors (TCR) itself, the pharmacological interaction [p-i] concept. To analyze whether the drug sulfamethoxazole (SMX) interacts directly with the TCR and thereby contributing to signaling and T cell activation, we analyze two SMX specific T cell clones (TCC “1.3”and “H13”). Proliferation to SMX and 11 related sulfanilamides, Ca++ influx in drug stimulated T-cells and the inhibitory effect of non-reactive sulfanilamides on SMX stimulation were analyzed. In silico docking of SMX and related sulfanilamide to the TCR were used to identify possible drug binding sites, and correlated to in vitro data to find the correct docking. In Ca++ influx assays, reactions occurred as early as 14 sec after adding SMX to TCC and APC. The broadly reactive clone (“H13”) was stimulated by 5 additional sulfanilamide, while one TCC (“1.3”) was reactive exclusively with SMX but not other sulfanilamides. Competition experiments with sulfanilamide inhibited SMX induced Ca++ influx and proliferation of the TCC1.3 ina dose dependent way. Docking experiments with SMX and related sulfanilamides confirmed and explained the in vitro data as docking localized binding sites for SMX and the 5 stimulating sulfanilamides on the CDR2β domain of the clone H13, while the 6 non-stimulatory SA failed to bind. In TCC 1.3, SMX could be docked on the CDR3α of the TCR. The other, non-stimulatory but inhibitory SA could also be docked to the same site. The combined analysis of in vitro and in silico data show that sulfanilamide can bind directly to TCRs. It shows that TCR, like other receptors, appear to be reamenable to manipulations by small molecules. 展开更多
关键词 p-i Concept Drug HYPERSENSITIVITY SULFAMETHOXAZOLE TCR Signaling TCR SPECIFICITY TCR-Modelling T cell cloneS
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