摘要
目的观察染矽尘大鼠肺泡上皮细胞(AEC)能否诱导骨髓间充质干细胞(BMMSCs)分化为AEC。方法随机取6只无特定病原体级健康雄性SD大鼠,以全骨髓贴壁法培养BMMSCs。随机取同批大鼠6只,其中3只以1.0 m L质量浓度为40 g/L的矽尘混悬液一次性气管内注入制作染矽尘模型,另3只注入0.9%氯化钠溶液1.0 m L,上述2种大鼠均采用免疫粘附纯化法培养AEC。设置实验组(BMMSCs与染矽尘大鼠AEC共培养)、对照A组(BMMSCs和正常大鼠AEC共培养)和对照B组(BMMSCs单独培养),于共培养第4和8天,以倒置显微镜观察各组BMMSCs形态变化,对BMMSCs进行水通道蛋白5(AQP5)和表面活性蛋白C(SP-C)免疫荧光双重染色,分别采用倒置荧光显微镜(IFM)和激光扫描共聚焦显微镜(LSCM)观察其荧光表达,采用Image-pro plus 6.0图像分析软件分析2种蛋白的荧光积分光密度(IOD)。结果细胞共培养后,实验组和对照A组的BMMSCs逐渐从长梭形向立方形和多边形转化,第8天时变化较第4天更明显,对照A组BMMSCs形态变化无实验组明显,对照B组BMMSCs的形态无明显变化。IFM和LSCM下,在共培养第4和8天,实验组、对照A组BMMSCs均可见呈绿色荧光的AQP5和红色荧光的SP-C表达,对照B组BMMSCs均可见少量AQP5绿色荧光表达,均无SP-C红色荧光表达。2种显微镜下均观察到,于共培养第4和8天,实验组BMMSCs的2种蛋白荧光IOD值均较同时间点对照A组和对照B组增强(P<0.01),对照A组BMMSCs的2种蛋白荧光IOD值均较同时间点对照B组增强(P<0.01);实验组和对照A组BMMSCs在共培养第8天时间点2种蛋白的荧光IOD值均较同组第4天时间点增强(P<0.01)。结论染矽尘大鼠AEC能有效诱导BMMSCs分化成AEC。
Objective To observe whether bone marrow mesenchymal stem cell（ BMMSC） could be induced by alveolar epithelial cell（ AEC） of rats exposed to silica dust or not. Methods BMMSCs were isolated and cultivated from 6specific pathogen free healthy male SD rats through bone marrow adherent method. The AECs from other 6 rats randomly selected from the same batch were cultivated by immune adherent purification method. Three rats were treated with 1. 0 m L（ 40 g/L mass concentration） of silicosis dust suspension by one time intratracheal injection as silicosis dust exposure model,and the other 3 rats were given 0. 9% sodium chloride solution as normal. Experimental group was the co-culture of BMMSCs and AECs from silicosis dust exposure rats. Control group A was the co-culture of BMMSCs and AECs from normal rats. Control group B was the culture of BMMSCs alone. The morphology changes were observed by the inverted phase contrast microscope at the time points of the 4th and the 8th day. Double immunofluorescence staining using aquaporin 5（ AQP5） and surfactant protein C（ SP-C） was performed on the treated BMMSCs. The fluorescence staining was observed using the inverted fluorescence microscope（ IFM） and laser scanning confocal microscope（ LSCM）. Integral optical density（ IOD） analysis was conducted on fluorescence of 2 kinds of proteins by Image-pro plus 6. 0 graphic analysis software. Results After the co-culture,the BMMSCs in experimental group and control group A changed from long spindle shape to cubic and polygonal shape,the variation of morphology on day 8 was more obvious than that on day 4,and the change in control group A was less obvious than that of experimental group. There was no obvious morphology change in BMMSCs of control group B. By IFM and LSCM,on day 4 and day 8,the expression of green fluorescence AQP5 and red fluorescence SP-C were all observed in BMMSCs of experimental group and control group A. The BMMSCs of control group B only showed a little green fluorescence expres
出处
《中国职业医学》
CAS
北大核心
2016年第1期1-7,共7页
China Occupational Medicine
基金
国家科技支撑计划项目(2014BAI12B01)
国家自然科学基金(81302396)
国家临床重点专科建设项目(2011-09)
广东省职业病防治重点实验室(2012A061400007)
