造血微环境结构和功能异常导致造血抑制是骨髓衰竭性疾病的主要病理机制，BM-MSCs成骨/成脂定向分化失衡是其造血支持功能缺失的关键环节。本室前期研究显示，成脂诱导后，部分BM-MSCs并不触发成脂而表现出成脂抵抗。细胞亚克隆分析显示该群细胞上触发早期成脂分化的C/EBPβ基因表达显著降低，提示其在BM-MSCs上的异质性表达导致成脂抵抗，但对其进行调控的上游信号通路不明。传统方法只能检测细胞群体的基因表达平均水平，无法精准确定对C/EBPβ进行调控的上游调节蛋白及二者的确切关系。本项目拟应用一种新的微流体单细胞基因检测技术，筛选差异性表达C/EBPβ的BM-MSCs，行单细胞基因测序结合细胞生物信息学分析，确定成脂抵抗BM-MSCs上调控C/EBPβ的关键蛋白及信号通道，阐明BM-MSCs差异性表达C/EBPβ影响其成脂分化的分子机制，为成脂分化异常所致骨髓衰竭性疾病的临床救治提供新思路。

Hematopoietic suppression caused by structural changes and dysfunction of hematopoietic micro-environment is the main pathological mechanisms of bone marrow failure disorders in patients, such as acute hematopoietic radiation syndrome. Bone marrow mesenchymal stem cells (BM-MSCs) play a crucial supporting role in hematopoiesis by maintaining the balance between adipogenic and osteogenic differentiation. Our previous studies suggested that after adipogenic induction, part of a cell does not trigger the adipogenic differentiation, in which showed adipogenic resistance. Cell sub-clone analysis showed that the expression of the C/EBPβ gene decreased significantly in those cells, which suggesting that C/EBPβ might be a key molecule involved in adipogenic resistance. However, the molecular mechanism of this pathway remains elusive. Traditional methods could only detect average gene expression level of cell population, but could not accurately determine the exact relationship between the upstream regulator and C/EBPβ.This study aims to screen the single BM-MSCs which could heterogeneous expressed C/EBPβ, analyse and screen the key signal channel regulator of C/EBPβ which related to the adipogenic resistant BM-MSCs through the nano micro fluid single cell platform technique combined with analysis of cell biological information, clarify the molecular mechanism of heterogeneous expression of C/EBPβ in BM-MSCs would influence its adipogenic differentiation. We would provide a new idea for the clinical treatment of bone marrow failure diseases induced by abnormal adipogenic differentiation.

造血微环境结构和功能异常导致造血抑制是骨髓衰竭性疾病的主要病理机制，BM-MSCs成骨/成脂定向分化失衡是其造血支持功能缺失的关键环节。本室前期研究显示，成脂诱导后，部分BM-MSCs触发早期成脂分化的C/EBPβ基因表达显著降低，在BM-MSCs上的异质性表达导致成脂抵抗，但对其进行调控的上游信号通路不明。多发性骨髓瘤（multiple myeloma，MM）是一种以骨髓内多发性恶性浆细胞沉积为特征的疾病，MM中最显著的症状是由于破骨细胞的骨重吸收增加而引起的骨疾病，这使骨基质变弱并使骨高度易于骨折。最近， BM-MSCs诱导分化为成骨细胞以恢复骨密度并抵消骨疾病的有害后果，显示出治疗MM的希望。我们通过构建体外培养诱导BM-MSCs成脂成骨模型，采用改造优化后的微流体单细胞基因检测技术，对目标细胞进行单细胞分选及RNA测序，对目标细胞在成脂成骨不同阶段基因表达进行精确分析，发现成骨分化诱导7天和18天基因表达与对照组有明显差异特别是成骨关键基因Runx2，但第7天和第18天诱导分化细胞之间差异不大。Ingenuity通路分析（IPA®）结果显示：M-MSC分化为成熟成骨细胞的过程中，PI3K信号通路、PKA信号通路、整合蛋白途径、雌激素和ERK / MAPK通路相关的基因都是涉及BM-MSC向成骨细胞的分化重要的信号。骨矿化所需要的Wnt抑制剂DKK1和βcatenin1有明显差异但DKK2并没有明显差异。另外我们还发现：整合素调节Graf1，Fak和Pyk2，其活性可分别直接调节MSC粘着和成骨分化。这些数据表明，细微的基因表达水平的差异可以对MSC迁移和成骨能力有显著作用；倾斜骨髓微环境中的平衡，有利于BM-MSC向成骨细胞的分化，可能潜在地改善MM的临床结果。迄今为止，本课题共发表论著2篇，其中SCI收录1篇(IF=24.008)；获重庆市科技进步一等奖1项；培养博士研究生2名，硕士研究生4名。