脂肪来源干细胞向成骨分化及褪黑素干预细胞生物学活性的变化

Osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells

背景:研究表明褪黑素能促进脂肪来源干细胞向神经元转化,但对脂肪来源干细胞成骨分化后细胞的作用报道较少。目的:观察脂肪来源干细胞向成骨分化及褪黑素对成骨分化后细胞生物学活性的影响。方法:1取昆明种小鼠附睾处脂肪利用Ⅰ型胶原酶消化法和差速贴壁法获取、纯化脂肪来源干细胞,应用CD44免疫组化染色对细胞进行鉴定。2取第2代脂肪来源干细胞,加入成骨诱导培养基培养,行碱性磷酸酶染色和von Kossa法检测细胞分化情况。3取成骨诱导后的细胞,加入不同浓度的褪黑素,利用MTT法检测24,48 h时细胞增殖情况。4取成骨诱导后的细胞,加入最佳浓度的褪黑素,分别检测加入药物3,6 d时的碱性磷酸酶活性。结果与结论:1接种48 h后大多数细胞贴壁,接种后4 d细胞呈现多种细胞形态,并形成大小不一的细胞集落,传代后的细胞形态趋于稳定,均称长梭型,培养细胞CD44呈阳性表达,阳性颗粒位于胞膜,呈棕黄色。2成骨诱导后的细胞应用von Kossa法染色呈阳性,黑色沉积物在细胞外的基质中呈网格状分布;碱性磷酸酶染色呈阳性,细胞内可见棕黑色颗粒。3在24 h和48 h时间点,1,10,100μmol/L组的吸光度值明显大于空白组(P<0.05),选择这3个浓度作为最佳的褪黑素浓度。4各组成骨诱导后细胞内碱性磷酸酶活性随时间延长明显增加,差异有显著性意义(P<0.05)。褪黑素组(1,10,100μmol/L)在3 d时的碱性磷酸酶活性与空白组相比无明显差异(P>0.05),在6 d时较空白组明显增加(P<0.05)。提示褪黑素可以增强脂肪来源干细胞成骨分化后细胞增殖,并提高细胞内碱性磷酸酶活性。

BACKGROUND： Studies have indicated that melatonin can promote the differentiation of adipose-derived stem cells into neurons, and the effect of melatonin on the osteoblasts form adipose-derived stem cells is rarely reported. OBJECTIVE： To observe the osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells. METHODS：（1） Adipose-derived stem cells were isolated and purified from the inguinal fat of Kunming mice by type I collagenase digestion and differential adhesion method, respectively. Immunohistochemical staining of CD44 was used as a quality control.（2） Osteogenic induction medium was added to induce osteogenic differentiation of passage 2 adipose-derived stem cells. Alkaline phosphatase staining and von Kossa method were combined to evaluate differentiation condition.（3） Melatonin at variable concentrations was added to treat mature osteocytes originated from adipose-derived stem cells and MTT was applied to determine their viability at 24 and 48 hours after culture respectively to find out optimal condition of melatonin treatment.（4） Melatonin at the optimal concentration was used to treat differentiated cells and detect alkaline phosphatase activity after 3 days and 6 days respectively. RESULTS AND CONCLUSION：（1） After seeding for 48 hours, most cells were adherent, and after 4 days, the cells displayed multiple shapes and colonies of different sizes formed. After subculture, cell morphology homogenized as spindle shape. Cells positive for CD44 were brownish yellow, and localized mainly on the cell membrane.（2） Differentiated cells were positive for von Kossa staining and black sediments scattered in the extracellular matrix. Alkaline phosphatase expressed positively, and brown-black particles, appeared within cells.（3） Melatonin supplement improved the viability of differentiated cells; and 1, 10 and 100 μmol/L was observed as the optimal concentrations both at 24 and 48 hours.（4） The intracellular alkaline phosphatatse activity was increased with time in all the groups（P〈0.05）. Compared with the blank group, the intracellular alkaline phosphatase activity in Melatonin groups（1, 10 and 100 μmol/L） had no changes at 3 days, but significantly increased at 6 days（P〈0.05）. These findings indicate that melatonin can enhance the proliferation of osteocytes differentiated from adipose-derived stem cells, and improve the activity of intracellular alkaline phosphatase.