干细胞是一类具有自我更新、高度增殖和多向分化潜能的细胞群体，是研究细胞生物学基础科学问题的理想细胞模型。早期胚胎来源的全能干细胞具有无限扩增和分化为多种类细胞的能力。成体组织来源的多能干细胞也具有远比人们开始预料的大的多的分化潜能。本实验室从成人和胎儿的多种组织中均成功分离获得具有三胚层分化潜能的Flk-1⁺CD31⁻CD34⁻的间充质干细胞，其在体外具有很强的克隆形成和扩增能力，因此很可能成为组织工程中理想的种子细胞，用于治疗组织损伤和一些遗传及退行性疾病。 以microRNA为代表的非编码小RNA领域是目前的国际研究热点，越来越多的研究结果表明，小RNA广泛参与生物体各种生物学过程，包括细胞的增殖、分化、凋亡、肿瘤形成和生物体发育等。 本研究的第一部分旨在研究小RNA在间充质干细胞向造血细胞分化过程中所扮演的角色，寻找Flk-1⁺CD31⁻CD34⁻骨髓间充质干细胞(bMSCs)向造血分化过程中具有决定意义的小RNA，并研究其作用机制。首先根据人基因组序列，应用计算机软件预测筛选了500条待验证的小RNA，定制小RNA芯片，检测并获得了人Flk-1⁺CD31⁻CD34⁻ bMSCs和人CD133⁺造血干细胞(HSCs)的小RNA差异表达谱； 3种小RNA在bMSCs中的表达显著低于HSCs中的表达；定量RT-PCR实验证实了芯片结果；小RNA克隆测序证实小RNA ZK-249为真实存在于人细胞中的内源性小RNA;利用逆转录病毒载体，上调ZK-249在bMSCs中的表达；免疫组织化学实验表明高表达ZK-249的bMSCs群体中，有少量CD45⁺造血细胞的存在；将细胞种植到CFU MIX中，表明这些细胞具有造血克隆形成能力；将shR-337高表达的人bMSCs从尾静脉注射到亚致死量照射的NOD SCID鼠体内，检测人源细胞在小鼠骨髓中向造血细胞分化能力，组织化学和流式细胞术检测人源细胞表面CD13、CD3、CD45、 CD34、CD71和CD19等造血标志抗原的表达情况，结果显示，shR337能够促进bMSCs向淋巴细胞和粒细胞的分化；定量PCR检测结果表明ZK-249高表达的bMSCs中，造血相关转录因子RUNX1的表达显著增加；计算机软件预测表明EID1可能是shR-337的靶基因，一系列验证实验，包括应用RNA干扰技术进行的功能验证实验，都证实了预测的结果。综上，小RNA ZK-249通过调节靶基因EID1的表达，促进了Flk-1⁺CD31⁻CD34⁻ bMSCs向造血细胞的分化。 本研究的第二部分研究成人脂肪来源的Flk-1⁺CD31⁻CD34⁻间充质干细胞(AMSC)向上皮、内皮和神经细胞体外定向诱导分化后，mRNA和小RNA表达谱的变化，从而从小RNA水平及基因水平揭示AMSC自我更新及分化机制。研究结果提示，在基因水平上，TGF β和Wnt信号通路参与了AMSC “干性”的维持，而Notch和BMP4信号的激活则会促进AMSC的分化。在小RNA水平上，尽管目前对于小RNA的功能的了解还非常有限，还是根据芯片的检测结果，归纳总结了可能和AMSC “干性”相关以及与AMSC向上皮、神经和内皮细胞定向分化相关的小RNA，并对小RNA的调控机制作了初步推测。另外对AMSC细胞中小RNA的克隆测序从另外一个角度获得了 AMSC的小RNA表达谱，对测序获得的小RNA与已鉴定的microRNA数据库及我们的预测小RNA数据库进行比对，结果表明，对于已知的microRNA而言，克隆测序结果中所显示的高拷贝往往对应着芯片检测结果中的高信号值，说明这两种检测方法获得的结果是平行可信的；另外还获得了21种目前没有报道的小RNA,其中一些与之前的预测结果结果相符，芯片结果显示的这些新的小RNA在AMSC分化前后的变化，为小RNA的功能研究作了初步提示。 关键词 Flk-1⁺CD31⁻CD34⁻间充质干细胞 小RNA 诱导细胞分化 造血 芯片检测小RNA克隆测序

 Stem cell research is one of the most fascinating areas of biology today. In our lab, Flk-1⁺CD31⁻CD34⁻MSCs could be isolated and purified from various fetal or adult tissues, which have been proven with extensive clone-forming and expansion capacity and the potential to differentiate into cells of all three layers, including hematopoietic differentiation, although at a very low frequency. Small non-coding RNAs (sRNA) study is also one of the most fascinating areas recently. MicroRNAs, as the most famous sRNAs, have been demonstrated to have important foles in development, cell proliferation, differentiation, apoptosis, cell cycle progression, tumorigenesis and many other physiological or pathological processes. In the first part of this study, we aims to find the highly functional sRNAs during the differentiation of bone marrow derived Flk-1⁺CD31⁻CD34⁻ MSCs (BMSCs) to hematopoietic cells. The results indicted that sRNA ZK-249, a newly identified short hairpin RNA (shRNA) could enhance the hematopoietic differentiation of BMSCs. BMSCs transduced with ZK-249 can differentiate into hematopoietic stem cells (HSCs) more efficiently and their descendant multipotent progenitor cells that have the capacity of further differentiating into blood cells in vitro. On transplantation into sublethally irradiated NOD/SCID mice, transduced human BMSCs engrafted and differentiated into all hematopoietic lineages including lymphocytes and myelocytes. Furthermore we find that this new shRNA alters BMSC fate by repressing the translation of EID1. Thus our findings establish a model that the endogenous shRNA can confer definitive tropism to BMSCs and unveil an intrinsic roadmap for programming hematopoiesis-related genes in HSCs. In the second part, adipose tissue-derived MSCs (ADAS) were induced to differentiate into endothelial cells, neural cells and epithelial cells respectively. And then mRNA microArray and sRNA microArray were used to detect the differential expression profiles of mRNA and sRNA. The results showed that the major signaling pathways involved in the "stemness" and differentiation of ADAS to three layers are TGFβ, Wnt, BMP4 and Notch pathways. The role of TGFβ and Wnt pathways is to maintain the stemness of ADAS, and the other pathways mainly to enhance the differentiation. Comparing to the mRNA level, sRNA alterations during the differentiation of ADAS are more the phenomena than elucidation due to the limit understanding of sRNA function.The sRNAs altering during the differentiation are classified into several categories, for example, the down-regulation sRNAs in all the three layers differentiation are listed together, which are proposed to be associated with the "stemness" of ADAS. In addition, all the sRNAs ＜30nt were cloned and sequenced, which provides the sRNA expression patterns of ADAS. And by alignment with the microRNA database, the microRNA expression profiles in ADAS were acquired as well as several novel sRNA requiring the further identification. Key words: Flk-1⁺CD31⁻CD34⁻ MSCs, differentiation, hematopoiesis, shRNA, gene chip, sRNA microArray, gene expression profile, microRNA expression profile, sRNA cloning and sequencing