1,25(OH)2D3对脂肪干细胞分泌Ⅰ型胶原的影响及作用机制

摘要

BACKGROUND: Adipose-derived mesenchymal stem cells (ADMSCs) have been reported to improve wound healing. However, type I collagen secreted by ADMSCs will contribute to scar formation. Therefore, inhibiting type I collagen secretion from ADMSCs will strengthen its clinical application. OBJECTIVE: To investigate the effect of 1,25(OH)2D3on secretion of type I collagen by ADMSCs and its mechanism. METHODS: Human ADMSCs were isolated by collagenase digestion, and identified by flow cytometry. ADMSCs at passage 4 were cultured in DMEM/F12 medium containing different concentrations of 1,25(OH)2D3(10-7, 10-8, 10-9, 10-10and 0 mol/L) respectively for 4 days. Then, the concentration of type I collagen in cell supernatant was measured by ELISA. Real-time PCR and western blot were used to detect the expression of Smad3 at mRNA and protein levels and phosphorylated protein Smad3 level in ADMSCs cultured with and without 1,25(OH)2D3. To analyze the contribution of Smad3 to the effect of 1,25(OH)2D3, Smad3 inhibitor was added to culture medium 30 minutes before adding 1,25(OH)2D3, and type I collagen in cell supernatant was detected by ELISA at 4 days after addition of SMAD3 inhibitor. RESULTS AND CONCLUSION: 1,25(OH)2D3inhibited the secretion of type I collagen by ADMSCs in a dose-dependent manner. The results of real-time PCR and western blot showed that the expression of Smad3 was upregulated by 1,25(OH)2D3, and the results of western blot showed that the phosphorylated Smad3 protein level in ADMSCs was significantly increased by 1,25(OH)2D3. Moreover, the inhibition of type I collagen secretion by 1,25(OH)2D3could be blocked by Smad3 inhibitor. These results indicate that 1,25(OH)2D3can inhibit the secretion of type I collagen from ADMSCs by up-regulating the expression of Smad3.%背景:脂肪间充质干细胞具有促进创伤修复的作用,但其分泌的Ⅰ型胶原会促进瘢痕形成,因此,寻找抑制脂肪间充质干细胞分泌胶原的方法将进一步增强其应用价值.目的:观察1,25(OH)2D3对脂肪间充质干细胞分泌Ⅰ型胶原的影响及其作用机制.方法:①利用胶原酶消化法分离培养人脂肪间充质干细胞,将第4代脂肪间充质干细胞分别培养在含不同浓度(10-7,10-8,10-9,10-10,0 mol/L)1,25(OH)2D3的DMEM/F12培养基中干预4 d,收集细胞培养上清,采用ELISA方法检测Ⅰ型胶原水平;②选取1,25(OH)2D3抑制Ⅰ型胶原分泌作用最明显的浓度对脂肪间充质干细胞进行干预,通过Real-time PCR检测1,25 (OH)2D3干预前后细胞内Smad3的mRNA表达,Western blot检测1,25(OH)2D3干预前后细胞内Smad3总蛋白和磷酸化蛋白表达;③在1,25(OH)2D3干预基础上加入SMAD3抑制剂SIS3对脂肪间充质干细胞进行干预,4 d后收集细胞培养上清,采用ELISA方法检测Ⅰ型胶原水平,以分析Smad3信号通路在其中的作用.结果与结论:①1,25(OH)2D3对人脂肪间充质干细胞分泌Ⅰ型胶原的影响表现为剂量依赖性抑制作用;②Real-time PCR和Western blot结果显示1,25(OH)2D3干预后Smad3表达水平有一定升高;Western blot结果显示磷酸化Smad3蛋白含量显著升高;③Smad3抑制剂可阻断1,25(OH)2D3对Ⅰ型胶原分泌的抑制作用;④这些结果表明,1,25(OH)2D3可能通过上调Smad3的表达及活性来抑制人脂肪间充质干细胞分泌Ⅰ型胶原.