CAJ | 학술논문

目的探讨精氨酸-甘氨酸-天冬氨酸(RGD)修饰的聚己内酯-左旋聚乳酸共聚物(PCL-b-PLLA)与犬骨髓基质干细胞(BMSCs)的生物相容性.方法应用密度梯度离心法及贴壁筛选法分离培养犬BMSCs,观察细胞形态及超微结构,流式细胞仪鉴定其表面标记物.将第3代犬BMSCs接种于RGD纳米胶束修饰的PCL-b-PLLA膜L培养,作为实验组,对照组为BMSCs与未修饰的PCL-b-PLLA膜复合培养,3 d后Hoechst 33342荧光染色、倒置显微镜及扣描电镜观察细胞形态,MTT法检测细胞毒性.分别于2、4、6 h细胞计数仪计数检测黏附的细胞数,计算黏附率.结果原代及传代培养的BMSCs呈梭形外观,具有较强的增殖能力.BMSCs超微结构显示其胞体较小,细胞核/浆比例大,胞浆少,染色质较疏松.细胞表而扰原CD29、CD44表达阳性,CD34表达阴性.Hoechst 33342荧光染色示实验组细胞核形态正常,核质均染,细胞数较对照组明显增多,未见明显凋亡及坏死细胞.扫描电镜示实验组较对照组细胞数量多,延展好,伪足相互接触紧密.两组材料对细胞均无毒性作用.随时间延长两组材料上细胞黏附率逐渐增高,各时间段实验组细胞黏附率显著高于对照组(P＜0.01).结论 RGD修饰的PCL-b-PLLA不仅与犬BMSCs具有良好的生物相容性,而且可显著促进BMSCs黏附、生长,是一种较为理想的组织工程支架材料.
목적 탐토정안산-감안산-천동안산(RGD)수식적취기내지-좌선취유산공취물(PCL-b-PLLA)여견골수기질간세포(BMSCs)적생물상용성.방법 응용밀도제도리심법급첩벽사선법분리배양견BMSCs,관찰세포형태급초미결구,류식세포의감정기표면표기물.장제3대견BMSCs접충우RGD납미효속수식적PCL-b-PLLA막L배양,작위실험조,대조조위BMSCs여미수식적PCL-b-PLLA막복합배양,3 d후Hoechst 33342형광염색、도치현미경급구묘전경관찰세포형태,MTT법검측세포독성.분별우2、4、6 h세포계수의계수검측점부적세포수,계산점부솔.결과 원대급전대배양적BMSCs정사형외관,구유교강적증식능력.BMSCs초미결구현시기포체교소,세포핵/장비례대,포장소,염색질교소송.세포표이우원CD29、CD44표체양성,CD34표체음성.Hoechst 33342형광염색시실험조세포핵형태정상,핵질균염,세포수교대조조명현증다,미견명현조망급배사세포.소묘전경시실험조교대조조세포수량다,연전호,위족상호접촉긴밀.량조재료대세포균무독성작용.수시간연장량조재료상세포점부솔축점증고,각시간단실험조세포점부솔현저고우대조조(P＜0.01).결론 RGD수식적PCL-b-PLLA불부여견BMSCs구유량호적생물상용성,이차가현저촉진BMSCs점부、생장,시일충교위이상적조직공정지가재료.
Objective To investigate the biocompatibility between Arg-Gly-Asp (RGD) peptide modified poly (ε-caprolactone)-block-poly (L-lactic acid) (PCL-b-PLLA) film and canine bone marrow stroreal cells (BMSCs) in vitro.Methods The canine BMSCs were isolated and euhured in vitro by density gradient centrifagation and adherence screening methods.The morphology of BMSCs was observed by inverted phase contrast microscopy and Giemsa stain.The uhrastructure of BMSCs was observed by transmission electron microscopy (TEM) and the flow cytometry was used to detect surface antigens of BMSCs.The passage 3 BMSCs were seeded onto the PCL-b-PLLA films (control group) and RGD modified PCL-b-PLLA films (experiment group) and cultured in DMEM.Cellular morphological changes were observed by Hoechst 33342 fluorometry,inverted phase contrast microscopy and scanning electron microscopy at day 3.The cytotoxicity was measured by MTT assay.The ratio of cell adhesion was detected by cell counting method at 2 h,4 h and 6 h,respectively.Results The BMSCs appeared to be spindle-shaped in morphology and showed active proliferative capacity in primary and passage cultures.The cell size was small and the ratio of cellular nucleus versus cytoplasm was high.TEM also showed cytoplasm was scanty and chromatin was loose.Flow cytometry analysis indicated that BMSCs were universally positive to CD29 and CD44,but negative to CD34.Hoechst 33342 fluorometry showed the phenotype of nucleus was normal.No apeptosis or necrotic cells were observed in the 2 groups.SEM showed that cells extended well and connected tightly with each other through pseudopod in the films.There were more cells on the RGD modified films.MTT assay showed no films in the 2 groups were cytotoxinic.The cell adhesion ratio between the experiment and control groups increased with time.There were significant differences between the 2 groups at each time point (P＜0.01,for all).Conclusion Since the RGD modified PCL-b-PLLA film has a satisfactory biocompatibility with canine BMSCs and can remarkably promote adhesion and growth of BMSCs,it can he used as a fine scaffold in tissue engineering.