研究背景：  

可变剪接作为提高基因转录组复杂性和蛋白质多样性的关键因素，对于调控个体生长发育至关重要。目前越来越多的研究表明，参与可变剪接的基因在造血调控方面发挥重要作用，可变剪接失调和剪接因子突变可引起造血系统紊乱最终导致血液系统疾病。然而，可变剪接对于早期造血发育是否有调控功能仍不清楚。课题前期研究发现在单层造血分化过程中存在广泛动态的可变剪接事件并且从APLNR⁺中胚层细胞分化成内皮祖细胞（endothelial progenitor cell，EPC）的过程存在剪接因子转换；抑制剪接发生过程可以改变剪接因子的表达模式，并且阶段特异性地影响内皮祖细胞的产生。在剪接因子发生转换的过程中，发现SR蛋白家族成员的剪接因子SRSF2表达变化明显。SRSF2对于调控血液系统疾病已有多种报道，但是在早期造血发育过程中的功能尚不清楚。SRSF1也属于SR蛋白家族经典成员，在造血分化过程中与SRSF2表达模式类似，其对于调控造血分化的功能也需要进一步研究。

研究目的：  

探索剪接因子SRSF2和SRSF1在人胚胎干细胞(human embryonic stem cell，hESC)早期造血发育过程中的功能和调控机理。

研究方法：  

为了探索剪接因子SRSF2和SRSF1在人胚胎干细胞早期造血发育过程中的功能，我们首先通过建立诱导性过表达人胚胎干细胞株，在APLNR⁺中胚层细胞分化形成内皮祖细胞的阶段特异性高表达SRSF2和SRSF1；通过流式检测CD31⁺CD34⁺内皮祖细胞的比例，来探究过表达SRSF2和SRSF1对造血分化的影响。之后通过建立诱导性敲低SRSF2的稳定细胞株，在造血分化过程中阶段性添加DOX诱导SRSF2敲低，day 5流式检测CD31⁺CD34⁺内皮祖细胞以探究阶段性敲低SRSF2对EPC的影响。此外，通过RNA干扰技术在EPC生成阶段添加siRNA敲低SRSF1，流式检测EPC的生成以分析阶段性敲低SRSF1对EPC的作用。最后通过可变剪接报告系统验证剪接因子SRSF2和SRSF1调控EPC生成的分子机制。

研究结果：

成功构建诱导性过表达SRSF2和SRSF1的人胚胎干细胞株，APLNR⁺中胚层细胞分化为EPC阶段过表达SRSF2抑制EPC的生成；而APLNR⁺中胚层细胞分化为EPC阶段过表达SRSF1对EPC的产生起促进作用。

成功构建诱导性敲低SRSF2的稳定细胞株，APLNR⁺中胚层细胞分化为EPC阶段敲低SRSF2促进EPC的生成。

通过RNA干扰技术在APLNR⁺中胚层细胞分化为EPC阶段敲低SRSF1抑制EPC的生成。

在分子机制上，SRSF2通过促进NUMB长的剪接异构体的产生调控EPC；而SRSF1通过促进NUMB短的剪接异构体的产生调控EPC。

研究结论：

剪接因子SRSF2和SRSF1通过调控NUMB转录本的可变剪接来调控人胚胎干细胞早期造血发育过程中EPC的生成。

Research background:

Alternative splicing, as a key factor to increase the complexity of the gene transcriptome and protein diversity, is essential for regulating individual growth and development. More and more studies have shown that the components involved in alternative splicing play an important role in regulating hematopoiesis. Alternative splicing disorders and splicing factor mutations can cause hematopoietic system disorders and eventually hematologic diseases. However, the regulatory effect of alternative splicing on early hematopoietic development remains unclear. The preliminary research of the subject found that there are extensive and dynamic alternative splicing events in the process of monolayer hematopoietic differentiation, and the splicing factor switch exists in the process of differentiation from APLNR⁺ mesoderm cells into endothelial progenitor cells. Inhibiting alternative splicing changes the expression pattern of splicing factors. And it specifically affects the generation of EPC (endothelial progenitor cell). During the switch process of the splicing factor, it was found that the expression of the splicing factor SRSF2, which is a member of the SR protein family, changed significantly. There have been many reports of SRSF2 for regulating blood system diseases, but its function in early hematopoietic development is still unclear. SRSF1 is also a classic member of the SR protein family, and its expression pattern is similar to that of SRSF2 in the process of hematopoietic differentiation. The function of regulating hematopoietic differentiation also needs further research.

Research objective:

Explore the function and regulation mechanism of splicing factors SRSF2 and SRSF1 in the early hematopoietic development of hESC ( human embryonic stem cell).

Research methods:

In order to explore the functions of splicing factors SRSF2 and SRSF1 in the early hematopoietic development of human embryonic stem cells, we first established inducible overexpression stable cell lines, and specifically overexpressed SRSF2 and SRSF1 at the stage of APLNR⁺ mesoderm cell differentiation to EPC. Flow cytometry was used to detect CD31⁺CD34⁺ endothelial progenitor cells to explore the effect of overexpression of SRSF2 and SRSF1 on hematopoietic differentiation. Then, by establishing stable cell lines with inducible knockdown of SRSF2, DOX was added periodically to induce knockdown during hematopoietic differentiation, and CD31⁺CD34⁺ endothelial progenitor cells were detected by flow cytometry on day 5 to explore the effect of staged knockdown of SRSF2 on EPC. In addition, RNA interference technology was used to add siRNA to knockdown SRSF1 in the EPC generation stage, and flow cytometry to detect the generation of EPC. Finally, the alternative splicing reporter system was used to verify the molecular mechanism of splicing factors SRSF2 and SRSF1 regulating EPC production.

Research results:

Successfully constructed stable cell lines that induced overexpression of SRSF2 and SRSF1. The overexpression of SRSF2 in APLNR⁺ mesoderm cells inhibits the generation of EPC, while the overexpression of SRSF1 promotes the production of EPC.

Successfully constructed stable cell lines with inducible knockdown of SRSF2. The knockdown of SRSF2 in APLNR⁺ mesoderm cells promotes the generation of EPC.

Knock down SRSF1 to inhibit the production of EPC at the stage of differentiation of APLNR⁺ mesoderm cells into EPC by RNA interference technology.

In terms of molecular mechanism, SRSF2 regulates EPC by promoting the production of the long isoform of NUMB; and SRSF1 regulates EPC by promoting the production of the short isoform of NUMB.

Research conclusion:

Splicing factors SRSF2 and SRSF1 regulate the production of EPC during the early hematopoietic development of human embryonic stem cells by regulating the alternative splicing of NUMB.