CAJ | 학술논문

背景:生长分化因子5是软骨与骨组织形成、发育的重要调解因子,在诱导软骨形成,促进骨、软骨、肌健韧带损伤修复方面发挥重要作用.目的:小鼠骨髓基质干细胞体外转染真核表达质粒pcDNA 3.1(+),生长分化因子5,检测与软骨形成分化相关的细胞外基质及蛋白多糖的表达.设计、时间及地点:细胞学体外观察,于2008-03/12在武汉协和医院中心实验室完成.材料:雄性昆明种小鼠20只,由华中科技大学同济医学院实验动物中心提供.生长分化因子5真核表达质粒pCDNA 3.1(+)/生长分化因子5为自备.方法:全骨髓贴壁法体外分离培养小鼠骨髓基质干细胞,取传至第3代细胞接种到6孔板,在细胞生长到90%融合时开始转染.设立3组:转染组采用Lipofectamine<'TM>2000进行脂质体介导pcDNA3.1(+)/生长分化因子5重组质粒瞬时转染;空质粒组转染窄质粒pcDNA 3.1(+);空白对照组只加入等量脂质体,其余步骤相同.主要观察指标:转染后72 h通过RT-PCR及免疫细胞化学检测生长分化因子5基因与蛋白的表达鉴定转染是否成功,同法检测软骨基质Ⅱ型胶原的表达,转染后14 d阿尔辛蓝染色检测蛋白聚糖的表达.结果:转染组有一大小为219 bp的特异性扩增条带,骨髓基质干细胞胞浆内呈棕色阳性染色;而空质粒组、空白对照组均未发生生长分化因子5基因转录,无特异性扩增条带,且细胞胞浆未见明显染色.转染组町检测到Ⅱ型胶原基因的表达,基因大小225 bp,Ⅱ型胶原细胞胞浆中可见棕黄色染色;空质粒组、空白对照组均未检测到Ⅱ型胶原基因的表达,SP染色均无明显染色.阿尔辛蓝染色后转染组细胞呈蓝染,空质粒组、空白对照组均未见明显异染性着色.结论:pcDNA3.1(+)/生长分化因子5转染骨髓基质干细胞能显著增加Ⅱ型胶原及蛋白聚糖的表达,促进骨髓基质干细胞向软骨细胞方向分化.
배경:생장분화인자5시연골여골조직형성、발육적중요조해인자,재유도연골형성,촉진골、연골、기건인대손상수복방면발휘중요작용.목적:소서골수기질간세포체외전염진핵표체질립pcDNA 3.1(+),생장분화인자5,검측여연골형성분화상관적세포외기질급단백다당적표체.설계、시간급지점:세포학체외관찰,우2008-03/12재무한협화의원중심실험실완성.재료:웅성곤명충소서20지,유화중과기대학동제의학원실험동물중심제공.생장분화인자5진핵표체질립pCDNA 3.1(+)/생장분화인자5위자비.방법:전골수첩벽법체외분리배양소서골수기질간세포,취전지제3대세포접충도6공판,재세포생장도90%융합시개시전염.설립3조:전염조채용Lipofectamine<'TM>2000진행지질체개도pcDNA3.1(+)/생장분화인자5중조질립순시전염;공질립조전염착질립pcDNA 3.1(+);공백대조조지가입등량지질체,기여보취상동.주요관찰지표:전염후72 h통과RT-PCR급면역세포화학검측생장분화인자5기인여단백적표체감정전염시부성공,동법검측연골기질Ⅱ형효원적표체,전염후14 d아이신람염색검측단백취당적표체.결과:전염조유일대소위219 bp적특이성확증조대,골수기질간세포포장내정종색양성염색;이공질립조、공백대조조균미발생생장분화인자5기인전록,무특이성확증조대,차세포포장미견명현염색.전염조정검측도Ⅱ형효원기인적표체,기인대소225 bp,Ⅱ형효원세포포장중가견종황색염색;공질립조、공백대조조균미검측도Ⅱ형효원기인적표체,SP염색균무명현염색.아이신람염색후전염조세포정람염,공질립조、공백대조조균미견명현이염성착색.결론:pcDNA3.1(+)/생장분화인자5전염골수기질간세포능현저증가Ⅱ형효원급단백취당적표체,촉진골수기질간세포향연골세포방향분화.
BACKGROUND: Growth differentiation factor 5 (GDF-5) is an important factor to regulate the formation and development of the cartilage and bone, it plays a crucial role on the promotion of repairing bone, cartilage and tendon ligament injury. OBJECTIVE: To transfect eukaryotic expression plasmid pcDNA 3.1(+)/GDF-5 to bone marrow stroma stem cells of mouse and to check the expression of extracellular matrix and proteoglycan which relates with the cartilage formation and differentiation. DESIGN, TIME AND SETTING: An in vitro observation regarding cells was performed in the central laboratory of Wuhan Union Hospital between March and December in 2008.MATERIALS: Twenty Kunming specimen male mice were offered by Experimental Animal Center of Tongji Medical College of Huazhong University of Science and Technology. The eukaryotic expression plasmid pcDNA 3.1(+)/GDF-5 was preserved at the laboratory.METHODS: The man-ow stroma stem cells were isolated from mouse bone marrow and cultured in vitro with whole bone marrow adherence method. Passage 3 cells were incubated on 6-well plate and began to transfect when they were 90% confluent. Experiment was assigned into three groups: trensfection group underwent transient transfection of liposome-mediated pcDNA 3.1(+)/GDF-5 using LipofectamineTM2000; blank plasmid group was transfected with blank plasmid pcDNA 3.1(+); control group was added with equal volume of liposome and other protocols were the same as above. MAIN OUTCOME MEASURES: The transfection efficacy was identified success by the expression of GDF-5 gene and protein using RT-PCR and immunocytochemistry at 72 heurs following transfection, cartilage matrix Ⅱ collagen expression was determined as above methods. Then marrow stroma stem cells were cultured for additional two weeks to check expression of proteoglycan with alcian blue staining.RESULTS: In the transfection group, a 219-bp specific amplification band was visible, there were brown positive stain in the cytoplasm of marrow stroma stem calls; In blank plasmid group and control group, no GDF-5 trensfection, specific amplification band or obvious stain of cytoplasm was observed. In the transfection group, the collagen Ⅱ gene was detected to express at 225 bp, with brown yellow stain in cytoplasm; in the blank plasmid group and control group, no collagen Ⅱ gene expression or SP ～ stain was observed. Alcian blue staining results showed the transfected cells were stained blue while those in the blank plasmid group and control group were not metachromasia stained.CONCLUSION: Gene trensfection of pcDNA 3.1 (+)/GDF-5 to marrow stroma stem cells can significantly raise expression of collagen II and proteoglycan, and promote the chondrogenic differentiation of marrow stroma stem cells.