背景：降钙素基因相关肽已被证实具有诱导成骨细胞分化作用，但其是否可使三维培养下的脂肪干细胞向成骨细胞分化构建组织工程骨的相关报道少见。 目的：探讨外源性降钙素基因相关肽诱导兔脂肪干细胞复合藻酸钙凝胶三维培养成骨分化的可行性。 方法：取新西兰兔双侧腹股沟区皮下脂肪垫，Ⅰ型胶原酶消化离心贴壁法分离培养脂肪干细胞，取第3代与海藻酸钠混合制备凝胶，于24孔板分组培养：对照组加入含10-2 mol/Lβ-甘油磷酸钠、10-7 mol/L地塞米松、50 mg/L抗坏血酸、体积分数10%胎牛血清的DMEM/F-12骨诱导培养基，实验组在此基础上再加入1.5µg/L降钙素基因相关肽进行诱导培养。于诱导不同时间点MTT法检测细胞增殖，RT-PCR法检测诱导细胞Ⅰ型胶原和骨钙素mRNA的表达，并检测碱性磷酸酶及钙离子浓度。 结果与结论：兔脂肪干细胞的增殖曲线呈“S”型，实验组诱导1，3，5，7，14，21 d 的 A 值高于对照组(P<0.05)；诱导2周后两组细胞碱性磷酸酶、茜素红染色均阳性，但实验组钙结节较对照组明显增多。实验组诱导7，14 d的Ⅰ型胶原和骨钙素mRNA表达均强于对照组。实验组诱导1，2，3，4周的碱性磷酸酶活性及钙离子浓度均高于对照组(P<0.05)。结果表明降钙素基因相关肽能诱导复合藻酸钙凝胶的脂肪干细胞向成骨细胞分化。%BACKGROUND:Calcitonin gene-related peptide has been confirmed to induce osteogenic differentiation, but whether it can induce the osteogenic differentiation of adipose-derived stem cells under three-dimensional culture to construct tissue-engineered bone is rarely reported. OBJECTIVE:To investigate the feasibility of exogenous calcitonin gene-related peptide to induce osteogenic differentiation of adipose-derived stem cells combined with calcium alginate gel in three-dimensional condition. METHODS:Adipose-derived stem cells were gained by col agenase I digestion of the subcutaneous adipose tissue of both inguinal regions of New Zealand rabbits. Passage 3 cells were mixed with sodium alginate to prepare calcium alginate gel, and then the cells were assigned into two groups and cultured in 24-wel plates. Adipose-derived stem cells in the control group were cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 supplemented with 10-2 mol/Lβ-glycerophosphate sodium, 10-7 mol/L dexamethasone, 50 mg/L ascorbic acid, 10%fetal bovine serum. While, adipose-derived stem cells in the experimental group were incubated with the same medium as above, but 1.5 µg/L calcitonin gene-related peptide was added. The cells proliferation and the mRNA expressions of col agen I and osteocalcin were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and reverse transcription-PCR respectively, and alkaline phosphatase and calcium concentration were detected at different induction time. RESULTS AND CONCLUSION:The cellproliferation curves were S shaped. The absorbance values of the experimental group were higher than those of the control group at1, 3, 5, 7, 14, 21 days after osteogenic induction (P<0.05). Alkaline phosphatase and alizarin red staining of adipose-derived stem cells was al positive, but golden round nodes became more and bigger in the experimental group compared with the control group after 2 weeks. At 7 and 14 days, col agen I and osteocalcin mRNA expressions were higher in the experimental group than in the control group. Alkaline phosphatase activity and calcium concentration of the experimental group were higher than those of the control group at 1, 2, 3, 4 weeks after osteogenic induction (P<0.05). Results showed that the calcitonin gene-related peptide can induce the osteogenic differentiation of adipose-derived stem cells combined with calcium alginate gel.
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