猪骨髓间充质干细胞分离培养和诱导分化脂肪细胞

Isolation,culturation and differentiation of porcince bone marrow mesenchymal stem cells into adipocytes

采用全骨髓培养法分离猪骨髓间充质干细胞(Mesenchymal stem cells,MSCs)并传代培养;取第4代纯化的MSCs在成脂诱导培养基中诱导分化;分化的成脂细胞用形态学和油红O染色法进行鉴定;用实时荧光定量PCR(Real-time PCR)检测成脂分化标志基因PPARγ2和LPL mRNA的表达情况。结果显示,分离培养的猪MSCs细胞经连续传代形态上无明显改变;MSCs在成脂分化培养液中诱导分化2d开始有少量脂滴出现,油红O染色成阳性,诱导18d成脂转化率可达59.8%;在诱导分化第5、10、15天时,PPARγ2mRNA相对表达量分别是(5.065±0.159)、(6.268±0.340)、(9.277±0.261),LPL mRNA的相对表达量分别是(10.995±1.473)、(13.130±0.712)、(15.762±0.934)。结果表明,用本诱导条件诱导猪MSCs向脂肪细胞分化,经形态学和油红O染色鉴定,成脂细胞分化率可达60%,且随分化时间的延长,脂肪细胞标志基因表达增加。

To investigate the identification method of adipogenic differentiation and differentiated degree of pig bone marrow mesenchymal stem cells(MSCs),the pig MSCs were isolated by whole bone marrow culture method and subcultured.The fourth generation of purified MSCs were induced to differentiate in adipogenic differentiation medium.The differentiated adipocyte were identified by morphology and oil red O staining.Expressions of adipogenic marker genes PPARγ2and LPL mRNA were measured by quantitative real-time PCR.The isolation and cultivation of pig MSCs have no obvious change in morphology after continuous passaging.Some lipid appeared at 2d after the MSCs induced and differentiated,with for oil red O staining positive.The adipogenic conversion rate could reach 59.8%in the induction of 18 days.At 5,10 and 15days,the expressions of PPARγ2mRNA were(5.065±0.159),(6.268±0.340)and(9.277±0.261)and the expressions of LPL mRNA were(10.995±1.473),(13.130±0.712)and(15.762±0.934),respectively.The rate pig MSCs differentiation into adipocyte reached nearly 60% by identification using morphology and oil red O staining methods in this induction conditions.And the expressions of adipogenic differentiation marker genes were increased when extended the induced differentiation time.