背景：造血系统是辐射敏感靶器官，受照后可能导致造血干细胞发生长期损伤、衰老和髓系分化偏移，临床常被忽略。此外髓系分化偏移在老年人以及骨髓恶性肿瘤患者中同样存在，但其作用机制仍有待阐明。MS4A3是有四个跨膜区域的蛋白亚型AMS4A家族的一个成员，密切参与细胞周期调节和分化，近年来被认为是造血早期髓系分化的重要标志物，但其在造血干细胞衰老中的作用及调节机制有待阐明。

方法：本研究首先通过流式检测K562细胞中MS4A3阳性表达细胞的分化水平，明确MS4A3对细胞分化的影响；随后构建MS4A3不同表达水平的细胞及MS4A3基因敲除小鼠，通过进行细胞活力检测、ROS水平检测、细胞周期、细胞凋亡、流式检测和PCR、Western技术观察MS4A3表达变化对细胞周期，分化的影响及调节机制；小鼠体内脏器指数、外周血细胞分型，骨髓细胞分型检测，造血干细胞移植等方法，观察MS4A3表达敲除对小鼠造血干细胞功能及分化的影响；利用照射及ROS、TGFβ与p38抑制剂，探讨MS4A3对辐射诱导造血干细胞髓系分化的作用机制。

结果：在第一部分MS4A3对细胞分化影响及机制研究中，我们发现MS4A3阳性的K562细胞髓系分化增加，进一步通过构建MS4A3不同表达水平的细胞确认MS4A3高表达有助于髓系分化偏移，结合ROS、TGFβ与p38抑制剂研究发现K562细胞发生髓系分化偏移与ROS的激活，p38蛋白与TGFβ蛋白的表达相关。在第二部分MS4A3经由TGFβ-P38MAPK通路在辐射诱导细胞髓系分化偏移中的作用及其机制研究中，在体外细胞实验中发现，辐射会诱导K562细胞中MS4A3表达水平升高，而MS4A3高表达的K562细胞具有较强的辐射敏感性，同时高表达MS4A3的K562细胞中TGFβ与p38蛋白也有较高的表达水平。利用构建的不同MS4A3表达的K562细胞株，进一步明确照射会引起K562细胞发生髓系分化偏移。结合ROS、TGFβ与p38抑制剂，我们发现，抑制 ROS、TGFβ与p38的表达可有效逆转辐射导致的K562的髓系分化偏移；而照射未引起MS4A3过表达的K562细胞髓系分化偏移，这可能与过表达MS4A3的K562细胞中TGFβ与p38蛋白有较高的表达水平有关。在体内实验中发现，MS4A3基因敲除的小鼠心脏、肝脏、肺、肾脏和胸腺的脏器指数与对照组小鼠没有明显的下降趋势，造血祖细胞、造血干细胞、红系祖细胞以及单核-巨噬系祖细胞比例有轻微变化，但均不显著。相比于照射组小鼠，受照MS4A3基因敲除小鼠中外周血中的B220在照射后发生明显下降，小鼠骨髓细胞分型检测实验中也呈现相同的结果。进一步检测小鼠骨髓造血细胞分化发现，MS4A3基因敲除小鼠的造血祖细胞、造血干细胞、红系祖细胞以及单核-巨噬系祖细胞对辐射的敏感度要略低于对照小鼠，但移植实验结果显示MS4A3基因敲除的小鼠HSC再生能力未发现显著改变，提示其功能可能被其它通路代偿。

结论： MS4A3表达升高会导致细胞发生髓系分化偏移；辐射会引起MS4A3表达升高；MS4A3通过激活ROS，进而激活TGFβ-p38MAPK信号通路在辐射诱导造血干细胞髓系分化偏移中发挥作用。

Background: The hematopoietic system is a radiation sensitive target organ. Radiation exposure to the hematopoietic system may lead to long-term injury of hematopoietic stem cells, senescence, and myeloid differentiation skewing, which is often ignored in clinic. In addition, myeloid differentiation skewing also exists in the elderly and patients with bone marrow malignancies, but its mechanism remains to be elucidated. MS4A3 is a member of the AMS4A protein subtype family with four transmembrane regions, which is closely involved in cell cycle regulation and differentiation. In recent years, MS4A3 has been considered as an important marker of early hematopoietic myeloid differentiation, but its role and regulatory mechanism in hematopoietic stem cell aging remain to be clarified.

Methods: In this study, the differentiation level of MS4A3 positive expression cells in K562 cells was detected by flow cytometry to determine the effect of MS4A3 on cell differentiation. Subsequently, cells with different expression levels of MS4A3 and mice with MS4A3 gene knockout were constructed, and the influence of the change of MS4A3 expression on cell cycle, cell differentiation and its regulatory mechanism on cells were observed by cell viability testing, ROS level testing, cell cycle and cell apoptosis, flow cytometry and PCR, Western techniques. The effect of MS4A3 expression knockout on the function and differentiation of mouse hematopoietic stem cells was observed by means of organ index, peripheral blood cell typing, bone marrow cell typing testing and hematopoietic stem cell transplantation. To investigate the mechanism of MS4A3 on radiation induced myeloid differentiation of hematopoietic stem cells by using ROS, TGFβ and p38 inhibitors.

Result: In the first part of the study (study on the effect and mechanism of MS4A3 on cell differentiation), we found that MS4A3-positive K562 cells had increased myeloid differentiation, and further confirmed that high expression of MS4A3 contributed to the migration of myeloid differentiation by constructing cells with different expression levels of MS4A3. Combining ROS, TGFβ and p38 inhibitors, it was found that the process of myeloid differentiation and migration in K562 cells was related to the activation of ROS, and the expression of p38 and TGFβ was related. In the second part of the study (the role of MS4A3 in radiation-induced myeloid differentiation and migration via TGFβ-P38MAPK pathway and its mechanism), it was found in vitro cell experiments that radiation induced increased expression level of MS4A3 in K562 cells, and K562 cells with high expression of MS4A3 had strong radiation sensitivity. Meanwhile, TGFβ and p38 proteins in K562 cells with high expression of MS4A3 also had higher expression levels. Using constructed K562 cell lines with different MS4A3 expression, it was further determined that irradiation could induce myeloid differentiation skewing in K562 cells. Combining ROS, TGFβ and p38 inhibitors, we found that inhibiting the expression of ROS, TGFβ and p38 could effectively reverse the radiation induced myeloid differentiation skewing of K562. However, irradiation did not induce myeloid differentiation skewing in MS4A3 overexpressed K562 cells, which may be related to the higher expression levels of TGFβ and p38 proteins in MS4A3 overexpressed K562 cells. In vivo test, it was found that the organ indexes of the heart, liver, lung, kidney, and thymus of MS4A3 knockout mice showed no obvious downward trend compared with the control mice, and the proportions of hematopoietic progenitor cells, hematopoietic stem cells, erythroid progenitor cells and mononuclear macrophage progenitor cells had slight changes, but no significant changes. Compared with mice in the irradiation group, B220 in peripheral blood of mice with MS4A3 gene knockout was significantly decreased after irradiation, and the same result was also shown in the mouse bone marrow cell typing testing. By further testing the differentiation of mouse bone marrow hematopoietic cells, it was found that hematopoietic progenitors, hematopoietic stem cells, erythroid progenitors, and mononuclear macrophage progenitors of MS4A3 gene knockout mice were less sensitive to radiation than control mice. However, transplantation experiments showed that the regeneration ability of HSC in MS4A3 knockout mice was not significantly changed, suggesting that its function may be compensated by other pathways.

Conclusion: The increased expression of MS4A3 can cause myeloid differentiation skewing, and the increased expression of MS4A3 can be induced by radiation. MS4A3 plays a role in radiation induced myeloid differentiation skewing of hematopoietic stem cells by activating ROS and thereby activating TGFβ-p38MAPK signaling pathway.