摘要
目的研究聚乙烯亚胺(polyethylenimine,PEI)包被的超顺磁性氧化铁粒子(superparamagnetic iron oxid,SPIO)对透明软骨细胞增殖和分泌Ⅱ型胶原蛋白功能的影响,探讨其标记软骨细胞的适宜标记浓度和适宜孵育时间。方法取1～3月龄巴马小香猪后膝关节透明软骨体外培养的第2代细胞,以2、4、6、8、10、12、14μg/mL浓度的PEI-SPIO标记液标记的软骨细胞为实验组,没有PEI-SPIO标记的软骨细胞作为对照组,分别在37℃培养箱中培育6、12、18、24、30 h,通过普鲁士蓝染色、透射电镜、细胞内铁含量、WST-1和半定量RT-PCR法检测,筛选出PEI-SPIO的最佳标记浓度和最佳孵育时间。结果 6、8、10、12、14μg/mL组标记后的普鲁士蓝染色显示90%以上的细胞被铁粒子标记;细胞内铁含量的测定结果显示标记时间超过24 h,各组细胞内铁含量基本不再增加;WST-1检测磁标记的细胞毒性结果显示,对照组与实验组无统计学差异(P>0.05)。8μg/mL标记的软骨细胞,其Ⅱ型胶原蛋白RT-PCR条带的灰度值比值与未标记软骨细胞比较有明显降低[(13.04±0.60)%vs(18.89±1.26)%,P<0.05];6μg/mL标记的软骨细胞,其条带的灰度值比值与未标记软骨细胞比较无明显差异[(27.32±1.02)%vs(28.56±1.15)%,P>0.05]。结论 PEI-SPIO可达到安全有效标记软骨细胞的要求,PEI-SPIO标记液为6μg/mL,标记24 h是PEI-SPIO标记软骨细胞的适宜标记浓度和标记时间,既可达到标记软骨细胞的目的,又不对软骨细胞的活性、增殖和Ⅱ型胶原蛋白基因的表达造成影响。
Objective To investigate the effect of superparamagnetic iron oxid （SPIO） coated with amphiphilic polyethylenimine （PEI） on the proliferation and type Ⅱ collagen secretion of labeled articular chondrocytes, and to determine the optimal labeling concentration and incubation time of PEI-SPIO. Methods Chondrocytes from knee joints of Bama mini-pigs （ 1 - 3 months old） were harvested and cultured. Ceils in passage two that were labeled with PEI-SPIO at the concentrations of 2, 4, 6, 8, 10, 12 and 14 μg,/mL were enrolled as experiment groups, and the unlabeled cells served as control. All the cells were incubated in 37 % for 6, 12, 18, 24 and 30 h, respectively. Prussian blue staining, transmission electron microscopy, intracellular iron content test, WST-1 test and semi-quantitative RT-PCR were applied to evaluate the effect of PEI-SPIO on articular chondrocytes for screening the optimal labeling concentration and incubation time. Results Prussian blue staining showed that more than 90% of chondrocytes were labeled in 6, 8, 10, 12 and 14 μg/mL of PEI-SPIO. Intracellular iron content increased slowly and became steady after 24 h. There was no statistically significant difference in cell viability and proliferation between the labeled and unlabeled ceils （P 〉 0.05 ）. Type Ⅱcollagen expression in the 8 μg/mL group was significantly lower than that in the control group [ （ 13.04 ± 0.60） % vs （ 18.89± 1,26 ） %, P 〈 0.05 ], while the difference of type Ⅱ collagen expression was not significant between the 6 μg/mL group and the group [ （27.32 ± 1.02 ）% vs （28.56 ± 1. 15 ）%.P 〉 O. 05 ]. Conclusion PEI-SPIO can effectively and safely label articular chondrocytes. In this study, the optimal labeling concentration and incubation time of PEI-SPIO are determined as 6 Ixg/mL and 24 h, which do not affect the viability, proliferation and type Ⅱ collagen secretion of articular chondrocytes.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2013年第4期284-287,共4页
Journal of Third Military Medical University
基金
国家高技术研究发展计划(863计划
2012AA020504)
国家自然科学基金重点项目(31130021)
国家自然科学基金面上项目(30870639)~~
关键词
SPIO
透明软骨细胞
磁标记
superparamagnetic iron oxid
hyaline cartilage cells
magnetic marker
