【摘要】 目的了解骨髓间充质干细胞(BMMSC)在染矽尘大鼠体内的分布情况。方法取7只无特定病原体级健康雄性SD大鼠采用全骨髓贴壁法分离培养BMMSC,经慢病毒载体介导的增强型绿色荧光蛋白(LV-e GFP)转染后,采用台盼蓝染色和CCK-8法检测转染前后细胞的活力及增殖能力。取同批大鼠随机分为对照组和染尘组,每组4只。染尘组以1.0mL质量浓度为40 g/L的矽尘混悬液气管内注入染尘,对照组气管内注入1.0mL生理氯化钠溶液。以转染后的BMMSC经尾静脉注入2组大鼠体内,分别于移植后第1、2、3和4周取肺、肝、脾、心、肾脏和脑组织冰冻切片,荧光显微镜下观察转染LV-e GFP的BMMSC植入后在上述脏器组织的荧光分布,采用Image-pro plus 6.0图像分析软件计算组织荧光强度。结果感染复数为50时,BMMSC转染率达80.00%,绿色荧光表达强烈、持续,转染后BMMSC形态无改变;转染与未转染LV-e GFP的BMMSC的细胞存活率差异无统计学意义[(97.67±0.60)%vs(98.03±0.56)%,P>0.05]。BMMSC增殖能力在LV-e GFP转染处理的主效应上无统计学意义(P>0.05)。植入转染LV-e GFP的BMMSC后第1周,2组大鼠各脏器组织切片镜下均可见荧光分布,其中染尘组大鼠肺脏组织切片各视野均可见较强荧光,持续至植入后第4周,以气管、血管及其周围组织荧光明显,肺泡及肺间质荧光也分布较多。植入转染LV-e GFP的BMMSC后,染尘组大鼠肺脏组织4个观察时间点荧光强度均高于同时间点对照组(P<0.01),且随观察时间的延长而减弱(P<0.01);染尘组大鼠肝脏和心脏组织4个观察时间点以及肾脏组织第2、3和4周的荧光强度均低于同时间点对照组(P<0.01);染尘组大鼠脾脏组织第1和2周的荧光强度均高于同时间点对照组(P<0.01),第3和4周的荧光强度均低于同时间点对照组(P<0.01);染尘组大鼠脑组织第1周的荧光强度高于同时间点对照组(P<0.01)。染尘组大鼠肺脏组织第1和2周荧光强度均高于同组同时间点肝脏、肾脏和脑组织(P<0.01),低于脾脏组织(P<0.01);但第3和4周荧光强度均高于同组同时间点其余5种器官组织(P<0.01)。结论 BMMSC进入染矽尘大鼠体内后可归巢并定植到受损肺部。更多还原

【Abstract】 Objective To investigate the distribution of bone marrow mesenchymal stem cell(BMMSC) in rats exposed to silica dust.Methods BMMSCs were isolated from 7 specific pathogen free healthy male SD rats through bone marrow adherent method and transfected using lentiviral vector-enhanced green fluorescence protein(LV-e GFP).Then Trypan blue staining and CCK-8 were applied to assay the cellular survival rate and cell proliferation activity before and after transfection,while 8 rats of the same kind were randomly divided into control group and silicotic group,with 4 rats in each group.Silicotic group was injected intratracheally with 1.0mL silica suspension(mass concentration was 40 g/L) while control group was given 1.0mL physiological sodium chloride solution.The transfected BMMSCs were intravenously injected into the caudal veins of rats of the two groups.The lung,liver,spleen,heart,kidney and brain were taken and observed under the fluorescence microscopy by frozen section 1,2,3 and 4 weeks respectively after transplantation.The fluorescence distribution and of BMMSCs transplanted with LV-e GFP were detected,the fluorescence intensity of every organ was calculated by Image-pro plus 6.0.Results When the multiplicity of infection was 50,the transfection efficiency of BMMSCs was 80.00%,the green fluorescence expression was powerful and sustained,and there was not any change found in morphological characteristics of BMMSC after transfection.The respective cellular survival rates had no statistical significant difference between transfected and untransfected BMMSCs with LV-e GFP [(97.67 ± 0.60) % vs(98.03 ± 0.56) %,P > 0.05].Cell proliferation activity had no statistical significant difference in main effects of transfection treatment(P > 0.05).In the first week after transplantation of BMMSCs transfected with LV-e GFP,the fluorescence expression was observed in tissue sections of each organ in rats of two groups,the stronger fluorescence was observed in all field visions in the lung tissue sections in silicotic group until 4 weeks after transplantation,especially the bronchus and blood vessels and their surrounding tissues were obvious,as well as the fluorescence distribution of alveoli and pulmonary mesenchyme.After transplantation of BMMSCs transfected with LV-e GFP,the fluorescence intensities at 4observation time points in lung tissues of silicotic group were significantly higher than those at the same time points in the control group(P < 0.01),and became weakened with time extension(P < 0.01).Fluorescence intensities of 4observation time points in liver and heart tissues,and the fluorescence intensities of kidney tissue in the 2nd,the 3rd and the 4th week in silicotic group were significantly lower than those of the control group at the same time points(P < 0.01).The fluorescence intensities of spleen tissue in the 1st and the 2nd week in silicotic group were significantly higher than those of the control group at the same time points(P < 0.01),while the fluorescence intensities in the 3rd and the 4th week were significantly lower than those of the control group at the same time points(P < 0.01).In the 1st week of silicotic group,the fluorescence intensity of brain tissue was significantly higher than that of the control group at the same time points(P < 0.01).The fluorescence intensities in lung tissue of silicotic group in the 1st and the 2nd week were significantly higher than those in liver,kindney and brain tissues(P < 0.01),but lower than that of spleen tissue(P <0.01); the fluorescence intensities in the 3rd and the 4th week were all higher than those of other 5 kinds of organ tissues in the same group at the same points(P < 0.01).Conclusion After BMMSCs enter into the bodies of rats exposed to silica dust,they may perform homing and planting to the impaired lung,LV-e GFP is a stable and efficient tracer agent,which could be used in bone marrow MSC for in vivo tracing study in the silicosis animals.更多还原