摘要
目的研究BMP-14与软骨细胞共同诱导脂肪来源干细胞(adipose-derived stem cells,ADSCs)向软骨细胞分化是否具有协同作用,优化软骨组织工程种子细胞来源。方法取2只雄性新西兰大白兔(体重1.5、2.0 kg)关节软骨和皮下脂肪组织,分离、培养软骨细胞和ADSCs,取第3代细胞进行实验。对ADSCs行成骨(茜素红染色)、成软骨(阿利新蓝染色)和成脂(油红O染色)诱导鉴定。测定Ad-巨细胞病毒-BMP-14-内部核糖体进入位点-人源化海肾绿色荧光蛋白1转染的最佳感染复数(multiplicity of infection,MOI)并以其转染ADSCs。实验分为5组:A组,采用Transwell小室将BMP-14转染的ADSCs与软骨细胞共培养(1∶1);B组,采用Transwell小室将未转染的ADSCs与软骨细胞共培养(1∶1);C组,单纯BMP-14转染的ADSCs培养;D组,单纯软骨细胞培养;E组,单纯ADSCs培养。培养3周后,取细胞采用阿利新蓝法检测糖胺聚糖(glycosaminoglycan,GAG)含量,Western blot检测BMP-14和Ⅱ型胶原蛋白表达,RT-PCR检测Sox-9基因表达。结果培养细胞经鉴定提示为ADSCs。倒置荧光显微镜观察示,MOI值为150时转染效果最好。A、C组GAG含量、BMP-14和Ⅱ型胶原蛋白表达以及Sox-9基因表达均明显高于其余3组,A组高于C组,差异均有统计学意义(P<0.05);B、D组均明显高于E组(P<0.05),B、D组间差异均无统计学意义(P>0.05)。结论 BMP-14与软骨细胞能共同诱导并促进兔ADSCs向软骨细胞分化,两者之间具有协同作用。
Objective To evaluate the synergistic effect of bone morphogenetic protein 14 （BMP-14） and chondrocytes co-culture on chondrogenesis of adipose-derived stem cells （ADSCs） so as to optimize the source of seed cells for cartilage tissue engineering. Methods ADSCs and chondrocytes were isolated and cultured respectively from articular cartilage and subcutaneous fat of 2 male New Zealand white rabbits （weighing, 1.5 kg and 2.0 kg）. The cells at passage 3 were harvested for experiment. ADSCs were identified by osteogenic induction （alizarin red staining）, chondrogenic induction （alcian blue staining）, and adipogenic induction （oil red O staining）. The optimum multiplicity of infection （MOI） of transfection of adenovirus-cytomegalovirus （CMV）-BMP-14-internat ribosome entry site （IRES）-human renilla reniformis green fluorescent protein 1 （hrGFP-1） was determined and then ADSCs were transfected by the optimum MOI. The experiment was divided into 5 groups： group A, co-culture ofADSCs transfected by BMP-14 and chondrocytes （1：1 in Transwell chambers）; group B, co- culture of ADSCs and chondrocytes （1 ： 1 in Transwell chambers）; group C, culture ofADSCs transfected by BMP-14; group D, simple chondrocytes culture; and group E, simple ADSCs culture. After 3 weeks, the glycosaminoglycan （GAG） content was detected by alcian blue staining; the expressions of collagen type II and BMP-14 protein were detected by Western blot; expression of Sox-9 gene was detected by RT-PCR. Results The cultured cells were proved to be ADSCs by identification. Inverted fluorescence microscope showed optimum transfection effect when MOI was 150. GAG content, expressions of collagen type II and BMP-14 protein, expression of Sox-9 gene were significantly higher in groups A and C than in the other 3 groups, in group A than in group C （P 〈 0.05）, and groups B and D were significantly higher than group E （P 〈 0.05）, but no significant difference was found between groups B and D （P 〉 0.05）. Conclusion It can promote differentiation of ADSCs into chondrocvtes bv BMP-14 co-culture with chondrocvtes, and thev have a synergistic effect.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2013年第3期353-357,共5页
Chinese Journal of Reparative and Reconstructive Surgery
关键词
软骨组织工程
脂肪来源干细胞
BMP-14
软骨细胞
共培养
兔
Cartilage tissue engineering Adipose-derived stem cells Bone morphogenetic protein 14Chondrocytes Co-culture Rabbit
