Objective To screen out the stable plan with the highest ratio of directed differentiation of murine embryonic stem cells (MSCs) into osteoblasts in vitro using BMP - Smad signaling pathway. Methods Murine MSCs were obtained via suspension culture form embryoid bodies (EBs). On the 14th to 21st day, the cells were assigned into 4 groups, Group A (culture supplemented with 50 μg/mL ascorbic acid + 50mmol/L β - glycerophosphate + 10-8 mol/ L dexamethasone), Group B (50 μg/mL ascormc acid + 10 mmol/L β - glycerophosphate + 1 μmol/L dexamethasone), Group C (50 μg/mL ascorbic acid + 50 mmol/L β - glycerophosphate + 1 μmol/L dexamethasone), and Group D (no inducer, as control group). The alkaline phosphonate (ALP) acivity was assessed on Day 7, 14, 21 and 28, On Day 22, the osteoblasts were determined by the 1 ％ Alizarin red staining. The expressions of BMP2, BMPR2, Smad1 and Smad5 were determined by real time - PCR and western blot. Results Compared with the control group, the activities of ALP in the other 3 groups were significantly increased from Day 7 after adherence, and reached the peak on Day 21. After Alizarin red staining, orange mineralized nodules of varying size was observed in osteoblasts. The osteoblast ratio in Group A, B, C and D were 15.34％, 23.71％, 29.12％ and 0.66％, respectively. Although the expression of BMP2, BM-PR2, Smad1 and Smad5 were increased or reduced in Group A and B, when compared with Group D, they were all significantly increased in Group C (P＜0.05). Conclusion The osteoblast ratio in Group C is the highest, suggesting the optimal osteoblast induction.%目的 利用BMP-Smad信号通路在成骨分化中的作用,筛选出较稳定且成骨率较高的小鼠胚胎干细胞体外定向诱导分化成骨细胞的方案.方法 小鼠胚胎干细胞经悬浮培养形成拟胚体(embryoidbodies, EBs),用胰酶消化成单个细胞接种至6孔板中,在EBs单细胞贴壁生长的第14～21天,A组(方案1)添加50 μg/mL维生素C +50 mmol/L β - 磷酸甘油 + 10-8mol/L地塞米松;B组(方案2)添加50 μg/mL维生素C + 10 mmol/L β - 磷酸甘油+ 1 μmol/L地塞米松;C组(方案3)添加50 μg/mL维生素C + 50 mmol/L β - 磷酸甘油+1 μmol/L地塞米松;D组为正常对照组,不添加诱导剂.各组均在在贴壁第7、14、21、28天检测碱性磷酸酶(ALP)活性,第22天用1％茜素红染色鉴定成骨细胞.采用荧光定量PCR(q-PCR)和Western blot检测BMP-Smad信号通路中的BMP2、BMPR2、Smadl和Smad5等关键mRNA和蛋白的表达组平.结果 与正常对照组相比,各组均在EBs贴壁第7天后ALP活力逐渐增强,且均在第21天增至最强.茜素红染色结果 显示,阳性细胞被染成橘红色、呈大小不等的结节状.计算成骨细胞诱导形成率,A组、B组、C组和D组成骨细胞形成率分别为15.34％、23.71％、29.12％和0.66％.Q - PCR和Western blot检测结果 显示,与正常对照组相比,各组BMP2、BMPR2、Smad1和Smad5的mRNA和蛋白表达水平有不同程度的升高和降低,其中C组(方案3)的BMP2、BMPR2、Smad1和Smad5的相对表达量均升高,且差异有统计学意义(P＜0.05).结论 添加50 |xg/mL维生素C +50 mmol/Lβ-磷酸甘油+ 1 μmol/L地塞米松的方案成骨细胞诱导形成率最多,可最大量诱导骨结节的形成.
展开▼
