兔肌源性干细胞的分离及诱导分化为成骨细胞的观察

Isolation and induced differentiation of muscle derived stem cells in rabbits

目的:探讨兔肌源性干细胞的分离、鉴定和诱导分化的方法。方法:取2月龄幼兔臀肌1g,采用胶原酶,dispase,胰蛋白酶分步消化,针头过滤,差速贴壁法分离肌源性细胞。用锥虫蓝染色细胞计数描记生长曲线,MTT法间接反映细胞增殖活性。作Bcl-2,Desmin免疫细胞化学染色鉴定肌源性细胞。用分化培养基(DMEM+5mL/LFBS+50mL/LHS+10mg/LBMP2+5g/L维生素C+10mmol/Lβ-甘油磷酸钠+10mg/L地塞米松+10g/L青霉素/链霉素)诱导细胞分化,用Osteocalcin,碱性磷酸酶,Ⅰ型胶原免疫细胞化学染色鉴定成骨前体细胞。结果:用上述方法分离的肌源性细胞呈短梭形或多角形,体积明显小于肌细胞,24h贴壁,接种后36h开始增殖,生长有明显的方向性,原代肌源性干细胞对数生长期为7～9d,16d汇合。第3代肌源性干细胞接种后第3～5天为对数生长期,约8d汇合。细胞汇合后少部分细胞出现双核或三核,甚至出现核链,显示肌细胞分化倾向。约60%～70%的原代肌源性干细胞Bcl-2,Desmin免疫细胞化学染色呈阳性。用成骨细胞分化培养基中培养2周后,大部分细胞Osteocalcin,碱性磷酸酶,Ⅰ型胶原免疫细胞化学染色呈阳性,显示其成骨前体细胞分化。结论:用上述方法分离的肌源性细胞具有干细胞特征,并能在体外分化为成骨前体细胞和肌起源细胞,是一种组织特异性干细胞。

AIM:To explore the method of isolation, identification and induced differentiation of muscle derived stem cells (MDSC) in rabbits. METHODS:Gluteus of a two month old rabbit(1 g) was obtained,digested with collagenase,dispase and trypsin.Residual tissue blocks were filtered with a stainless sieve.The cells were further isolated with preplating technique.Viable cells were counted by trypan blue staining and the growth curve was plotted.The isolated cells were identified by immunocytochemical staining for Desmin and Bcl 2.The viability of MDSCs were indirectly assessed by MTT method.The cells differentiation were induced with differentiation medium(DMEM+5 mL/L FBS+50 mL/L HS+10 mg/L BMP2+5 g/L VitC+10 mmol/L β Sodium Glycerophosphate +10 mg/L DXM +10 g/L Benzylpenicillin/PST).The precursor were identified by Osteocalcin, alkaline phosphatase, type Ⅰcollagen immunocytochemical staining. RESULTS:The muscle derived cells isolated by the above methods were short fusiform shaped or multi angular shaped,which were significantly smaller than skeletal muscle cells. The cells adhered to the flasks within 24 hours, and began to grow 36 hours after plating. The cells grew in a certain oriented manner. The logarithmic growth phase of primary MDSCs was within 7-9 days after plating, and they grew to completely confluence by 16 days. The MDSCs of the third generation grew in logarithmic manner within 3-5 days after plating, and they obtained confluence by 8 days. Two or three nuclei, and even nucleus chain were observed in some of the cells after confluence, showing a trend of muscle cell differentiation. Positive staining for Desmin and Bcl 2 was observed in 60%-70%of primary muse derived cells.After growing in osteogenic differentiation medium for 2 weeks, most of the cells showed positive staining for Ostepcalcin,alkaline phosphatase and type I collagen showing osteogenic differentiation. CONCLUSION:The isolated muscle derived cells possess the characteristics of stem cells, and they can differentiate into myogenic and osteogenic cells in vitro.Therefore, these cells were tissue specific stem cells, which were called MDSCs.