直接重编程人体脂肪间质血管细胞到棕色脂肪细胞并研究其特性

Brown fat can increase energy expenditure and protect against obesity through uncoupled respiration.To promote energy expenditure through adipocyte browning is a new development in anti-obesity research and development. We have hypothesized that human adipose stromal vascular cells are amenable to be reprogrammed into brown adipocytes and have been able to convert human adSVCs into BAs through overexpression of browning nuclear factors PPARγ,C/EBPβ,PGC1α and PRDM16.And qPCR results showed that UCP1 gene is induced about 320-fold by all four factors compared to GFP control in our previous study. We have constructed a luciferase reporter for human UCP1 promoter activity. The reporter and brown adipocyte reprogramming rivuses will be used for infecting human adSVCs and the resulting cells will be used for quantitive and qualitative analysis of the browning activity.We further aim to directly reprogram human adipose stromal vascular cells into brown adipocytes,characterize brown adipocytes in vitro and in vivo. We will characterize the reprogrammed cells in respect of glucose update, fatty acid metabolism and gene expression. In addition, we will characterize reprogrammed cells in vivo by transplanting the cells into immunocompromised NSG mice. A successful execution of the project will lead to the development of methodology to promote browning of human adipose tissue by reprogramming and/or small molecules in near future.

棕色脂肪以非耦联呼吸形式增加能量消耗，抵抗肥胖。通过脂肪细胞棕色化来促进燃脂，成为肥胖研究领域的新进展。我们前期实验通过短暂过表达转录因子PPARγ、C/EBPβ、PGC1α和PRDM16，使人体脂肪间质血管细胞（ASVCs）中的棕色脂肪细胞特异性标记分子UCP1表达明显增高，即首次将ASVCs通过重编程转化为棕色脂肪细胞；并通过连接有荧光素酶报告基因和绿荧光蛋白的UCP1启动子，检测了棕色脂肪细胞的分化效率。进一步研究将通过葡萄糖转运分析、脂解和脂肪酸氧化分析等证明所得细胞具有与棕色脂肪细胞相似的特性，并将在免疫缺陷小鼠体内研究其在调节代谢方面的功能。该项目的成功实施将对肥胖及其相关疾病治疗的靶点及药物筛选奠定重要基础。

棕色脂肪组织是肥胖研究领域的热点，其特征性生物学标记分子为解偶联蛋白1（UCP1），可以通过非耦联呼吸形式产热，从而增加代谢活性，促进机体代谢。近年来有文献报道，尽管棕色脂肪细胞和白色脂肪细胞的生理作用截然不同，但它们的分化通路有类似之处，例如都需要转录因子过氧化物酶增殖物激活型受体γ（PPARγ），但目前白色脂肪棕色化的具体调控机制尚不明确。我们在前期研究基础上，构建MyoD-PPARγ（M3-PPARγ）真核表达载体并转染人脂肪血管间质细胞（ASVCs）, 通过荧光素酶报告基因，qPCR和免疫组化等方法，发现重编程的ASVCs诱导分化后细胞中UCP1表达显著增加，细胞产热和代谢增加，提示M3-PPARγ是重编程ASVCs到棕色脂肪样细胞的强效的转录因子。此外，我们利用pull down技术筛选出20种与M3-PPARγ结合的共激活因子，为下一步进行其功能研究创造了条件。同时我们也探索了可能影响脂肪组织棕色化的药物及具体分子机制，在细胞水平我们发现唑来膦酸可以显著增加脂肪细胞内UCP1表达，促进棕色化。进一步借助ITRAQ、PCR和WB等技术，我们发现这一效应可能可以通过活化β肾上腺素受体，激活PRDM16-PGC1α通路的激活发挥。该研究为肥胖的临床治疗提供了新的理论技术手段和药物干预靶点。