三氧化二砷对小鼠肝脏祖细胞生物学特性的影响  

Effect of arsenic trioxide on biological behaviors of hepatic progenitor cells in mice

作　　者：张钰[1] 王洪山[1] 锁涛[1] 付亮[1] 鲁超[1] 宋陆军[1] 

机构地区：[1]复旦大学附属中山医院普外科,上海200032

出　　处：《中华实验外科杂志》2013年第10期2079-2081,共3页Chinese Journal of Experimental Surgery

摘　　要：目的 观察三氧化二砷（As2O3）对小鼠肝脏祖细胞（AHPCs）增殖及代谢的影响.方法 应用改良Seglen二步法原位灌注结合机械离心获取AHPCs,体外培养并持续观察超过40 d.应用免疫荧光技术对AHPC及其形成的克隆进行白蛋白、甲胎蛋白（AFP）和细胞角蛋白19 （CK19）染色.取原代培养12d的细胞随机分为3组：对照组（不含As2O3）、低浓度组（含2μmol/L As2O3）和高浓度组（含4 μmol/L As2 O3）.干预48 h后,采用免疫荧光法观察细胞核增殖抗原（Ki-67）阳性细胞所占的比例,逆转录-聚合酶链反应（RT-PCR）技术检测Ki-67基因的表达,Western blot技术检测增殖细胞核抗原（PCNA）蛋白的表达.酶联免疫吸附试验（ELISA）检测细胞上清液中尿素浓度的变化.结果 体外培养的AHPC可持续扩增超过40 d,培养第1天强阳性表达白蛋白,培养第5天细胞克隆开始表达AFP,第35天表达CK19.阴性对照组所含Ki-67阳性细胞的比例较高（25.1±2.8）％,而As2O3（2μmol/L）组[（18.8±1.0）％]和As2O3（4μmol/L）组[（11.8±2.0）％]的Ki-67阳性细胞比例逐渐降低.Ki-67 mRNA的表达量在As2 O3（2μmol/L）组（0.823 ±0.012）明显下降,并以As2O3（4μmol/L）组（0.309 ±0.002）降低最为明显.随着As2O3浓度的增加,PCNA表达逐渐下降,并呈剂量-效应关系.阴性对照组细胞代谢产物尿素浓度较高[（0.586 ±0.056） mmol/L]随着As2O3浓度增加As2O3（2μmol/L）组[（0.506±0.058） mmol/L]与As2O3 （4 μmol/L）组[（0.410±0.045） mmol/L]尿素浓度逐渐下降.结论 成功建立AHPC体外培养模型.As2O3能抑制原代AHPC的增殖,并降低其代谢能力.Objective To examine the inhibitory effect of arsenic trioxide on hepatic progenitor cells （HPCs） cultured in vitro.Methods HPCs were isolated by two-step collagenase perfusion and mechanical centrifugation,and observed under the phase contrast microscopy for over 40 days.The albumin,alpha fetal protein （AFP） and cytokeratin 19 （CK19） staining was performed on adult HPCs and their colonies by immunonuorescence.At 12th day,primary cultured adult HPCs were randomly divided into three groups：control group （no treatment）,As2O3 （2 μmol/L） group and As2O3 （4 μmol/L） group.After treatment for 48 h,immunofluorescence and reverse transcriptase-polymerase chain reaction （RT-PCR）were performed to analyze the protein and mRNA expression of proliferation cell nuclear antigen （Ki-67）.The expression of proliferating cell nuclear antigen （PCNA） was detected by using Western blotting.Urea released in the supernatant was determined by using enzyme linked immunosorbent assay （ELISA）.Results Adult HPCs could expand continually more than 40 days.Albumin was expressed in adult HPCs at 24 h after attachment.Within adult HPC colonies,AFP was expressed at day 5,and CK19 was positive at day 35.The rate of Ki-67 postive cells in control group [（25.1 ±2.8）％] was higher than that in As2O3 （2 μmol/L）group [（18.8 ± 1.0）％] and As2O3 （4 μmol/L） group [（11.8 ± 2.0）％].The mRNA expressed in control group was higer than that in As2O3 （2 μ mol/L） group [（0.823 ±0.012）] and As2O3 （4 μmol/L） group [（0.309 ± 0.002）].The concentration-dependent decrease of PCNA protein was observed by Western blotting.In addition,the level of urea concentration in control group [（0.586 ± 0.056） mmol/L] was higher than that in As2O3（2 μmol/L） group [（0.506±0.058） mmol/L] and As2O3（4 μmol/L） group [（0.410 ± 0.045） mmol/L].Conclusion Mouse adult HPCs cultured in vitro have been successfully established.Arsenic trioxide can inhibit the proliferation and met

关 键 词：三氧化二砷 肝脏祖细胞 增殖 细胞代谢 

分 类 号：R965[医药卫生—药理学]