CAJ | 학술논문

目的：利用磁共振成像技术动态监测超顺磁性氧化铁（superparamagnetic iron oxide ，SPIO ）标记的兔脂肪干细胞（adipose‐derived stemcells ，ADSCs）在兔体内的变化及其对兔膝关节软骨缺损修复的进程。方法：用不同浓度（0、12．5、25、50、100μg／mL）的超顺SPIO标记ADSCs。采用普鲁士蓝染色法测定细胞的标记情况，并计算细胞标记率。用细胞计数试剂盒（CCK‐8）测定不同浓度的SPIO对细胞活性的影响。将不同浓度SPIO标记的不同数量的 ADSCs快速均匀地重悬于盛有1％琼脂糖的冷冻管中进行体外M RI成像，测量T2*图像信号强度。将50μg／mL的SPIO标记的ADSCs接种至聚乳酸‐羟基乙酸共聚酶[Poly （lactic‐co‐glycolic acid），PLGA]支架材料后，回植入兔（n＝5）膝关节软骨直径为3 mm的软骨缺损处，分别于术后1、4、8和12周进行M RI T 2*序列动态扫描，监测干细胞在体内的变化及关节软骨的修复情况。结果：原代培养的ADSCs呈成纤维细胞样外观，培养7 d后细胞呈聚集样生长。普鲁士蓝染色见细胞质内有明显的蓝色致密颗粒，随着SPIO浓度的升高，蓝色致密颗粒增多；当SPIO的浓度≥50μg／mL时，细胞的标记率可达100％。CCK‐8检测显示，SPIO的浓度越高，细胞的活性越低，当SPIO的浓度大于50μg／mL时，细胞的活性明显降低（P＜0．05）。体内MRI显示，术后1周和4周可见颗粒状低信号区域，信号强度明显低于对照组。术后8周，颗粒状低信号范围逐渐缩小，信号强度接近周围组织，说明S PIO标记的ADSCs在动物体内存活良好，并正常分裂增殖，形成正常关节软骨结构。结论：M RI联合SPIO可以动态监测细胞在体内的分布、生长和转归，有望作为一种动态监测组织工程修复关节软骨缺损效果的方法。
목적：이용자공진성상기술동태감측초순자성양화철（superparamagnetic iron oxide ，SPIO ）표기적토지방간세포（adipose‐derived stemcells ，ADSCs）재토체내적변화급기대토슬관절연골결손수복적진정。방법：용불동농도（0、12．5、25、50、100μg／mL）적초순SPIO표기ADSCs。채용보로사람염색법측정세포적표기정황，병계산세포표기솔。용세포계수시제합（CCK‐8）측정불동농도적SPIO대세포활성적영향。장불동농도SPIO표기적불동수량적 ADSCs쾌속균균지중현우성유1％경지당적냉동관중진행체외M RI성상，측량T2*도상신호강도。장50μg／mL적SPIO표기적ADSCs접충지취유산‐간기을산공취매[Poly （lactic‐co‐glycolic acid），PLGA]지가재료후，회식입토（n＝5）슬관절연골직경위3 mm적연골결손처，분별우술후1、4、8화12주진행M RI T 2*서렬동태소묘，감측간세포재체내적변화급관절연골적수복정황。결과：원대배양적ADSCs정성섬유세포양외관，배양7 d후세포정취집양생장。보로사람염색견세포질내유명현적람색치밀과립，수착SPIO농도적승고，람색치밀과립증다；당SPIO적농도≥50μg／mL시，세포적표기솔가체100％。CCK‐8검측현시，SPIO적농도월고，세포적활성월저，당SPIO적농도대우50μg／mL시，세포적활성명현강저（P＜0．05）。체내MRI현시，술후1주화4주가견과립상저신호구역，신호강도명현저우대조조。술후8주，과립상저신호범위축점축소，신호강도접근주위조직，설명S PIO표기적ADSCs재동물체내존활량호，병정상분렬증식，형성정상관절연골결구。결론：M RI연합SPIO가이동태감측세포재체내적분포、생장화전귀，유망작위일충동태감측조직공정수복관절연골결손효과적방법。
Objective:To detect dynamically in vivo superparamagnetic iron oxide‐labeled rabbit adipose‐derived stem cells (ADSCs) for treatment of carticular cartilage defect with MRI tracking technique .Methods:Rabbit ADSCs were firstly labeled with different concentrations of SPIO (0 μg/mL ,12 .5 μg/mL ,25 μg/mL ,50 μg/mL ,and 100 μg/mL) .The labeled ADSCs were stained with Prussian blue and the labeling rates were calculated .The activities of different concentration SPIO‐labeled ADSCs were determined by CCK‐8 . Different amounts of different concentration SPIO‐labeled ADSCs were dissolved homogeneously in cryovial with 1% agarose and T2*‐weighted MRI was used to evaluate their signal intensity in vitro .Then 50 μg/mL SPIO‐labeled ADSCs and unlabeled ADSCs were seeded in PLGA [Poly (lactic‐co‐glycolic acid )] scaffold , respectively .Following that ,the cells‐PLGA scaffold compounds were implanted in the rabbit (n=5) articular cartilage defect with average diameter 3 mm .At 1 ,4 ,8 ,and 12 weeks post‐operatively ,the change of implanted‐ADSCs and the repain of articular cartilage defect were dynamically detected by T2*‐weighted MRI .Results:Primarily cultured rabbit ADSCs presented fibroblast‐like appearance ,and ADSCs proliferated with aggregations after 7 days .Prussian blue staining showed amount of blue dense granules in cytoplasm .With the increasing of SPIO concentration , the count and region of blue dense granules increased .When SPIO concentration reached 50 μg/mL ,the labeling rate of ADSCs reached 100% .The measure of CCK‐8 showed that the activities of ADSCs decreased with the increasing of SPIO concentration .When the SPIO concentration was greater than 50 μg/mL ,the activities of ADSCs were obvious low (P<0 .05) .In vivo ,MRI showed postoperative 1 week and 4 weeks visible granules with low signal area ,and the signal strength was significantly lower than the control group .Until 8 weeks post‐surgery ,granules with low signal area gradually narrowed and signal strength was similar to the control group . SPIO‐labeled ADSCs lived with a normal division growth and performed normal cartilage articular .Conclusions:MRI combined SPIO is a potential method to dynamically detect the distribution ,growth and prognosis of cells in the body ,and can also be used to determine the respiration of articular cartilage defect .