研究背景： 造血干细胞(Hematopoietic stem cells, HSCs)是一种成体干细胞，具有自我更新 和多向分化潜能，也是机体整个生命周期中各种终末分化的血液细胞的来源。通常， 在生理状态下，骨髓中大多数的 HSCs 处于静息期，少数 HSCs 通过增殖和分化生 成各种幼稚或者成熟的血细胞，并组成了机体中分层（等级不同）和分类（功能不 同）明确的不同血细胞群，且各群细胞在血液系统中所占比例较为恒定，维持着一 种体内造血稳态。在急性感染和炎症等病理性应激状态下以及机体衰老过程中，造 血微环境会发生改变，会促使更多的造血干/祖细胞进入增殖和分化阶段（包括一些 HSCs 从静息期进入分裂期），增强和维持骨髓中急性造血的发生，导致上述体内造 血稳态发生改变。目前，有关机体中的 HSCs 的命运抉择（增殖和分化或衰老）仍 然是造血调控研究的热点研究之一，还需要更加全面和深入的研究。 蛋白酶 3（Proteinase 3, PRTN3）是存在于中性粒细胞中的一种丝氨酸蛋白酶， 它在中性粒细胞中的杀菌、细胞因子的翻译后修饰和调控中性粒细胞的自发死亡等 方面已被广泛研究。近期，我们的单细胞转录组测序结果显示 Prtn3 基因在年轻小 鼠造血干/祖细胞中高表达，该结果与公共数据库 Chambers 和 Hyatt 报道的数据基 本一致，此外，上述数据库还揭示了 Prtn3 基因在人的 HSC 和急慢性髓系白血病患 者的白血病细胞内也存在高水平转录表达，但该基因在上述造血干/祖细胞和髓系白 血病细胞中具体起着怎样的作用还不十分清楚。基于以上的研究背景，我们开展了 Prtn3 基因在造血干/祖细胞中的作用及机制研究，以期为今后“HSCs 的命运抉择” 相关研究提供新的调控靶点。 研究目的：明确 Prtn3 基因在造血干/祖细胞中的表达和分布，探究 Prtn3 基因在造 血干/祖细胞中的作用及其作用机制。 研究方法：采用单细胞转录组测序方法分析 Prtn3 基因在野生型小鼠（WT）血细胞 中的表达；采用实时定量 PCR 方法、Western Blot、免疫荧光等方法检测造血细胞 中 PRTN3 蛋白的表达；构建 Prtn3 基因敲除小鼠(Prtn3-/- )，并采用流式细胞术和血 常规分别分析 4 周、8 周、24 周、48 周及 72 周 Prtn3-/-小鼠骨髓中的分化成熟细胞 及 LT-HSC、ST-HSC、MPP、CMP、GMP、MEP 的数目和比例；体外干/祖细胞单 细胞和集落培养法分析 LSK 细胞的增殖和髓系分化潜能。竞争移植实验分析 8 周、 24 周及 48 周小鼠 Prtn3-/-小鼠 LSK 细胞的竞争能力以及外周血、脾脏和骨髓中各种 血细胞的数目和比例，免疫荧光法分析 8 周、24 周及 48 周小鼠 Prtn3-/-小鼠脾脏和 骨髓中的供体血细胞分布；体外观察 G-CSF 因子刺激下 Prtn3-/-小鼠 LSK 细胞向髓 中国医学科学院 北京协和医学院 硕士研究生学位论文 2 系的分化情况及转录组测序分析基因表达和潜在参与的分子机制并验证。 研究结果： （1）单细胞转录组测序结果显示 Prtn3 基因在 LSK、GMP、CMP 细胞中高表 达，实时定量 PCR 结果显示 LSK 细胞中 Prtn3 的 mRNA 含量是中性粒细胞的 5 倍 左右，同时 ELISA、Western Blot 实验结果表明 LSK 细胞 PRTN3 蛋白有表达，免 疫荧光结果表明 PRTN3 在 LSK 细胞中存在且散在分布。以上结果表明 Prtn3 基因 在小鼠造血干/祖细胞中持续高表达，且散在分布在胞浆里。 （2）Prtn3 基因缺失促进造血干/祖细胞向髓系分化且导致 LSK 和 LK 细胞比 例显著增加， LT-HSC、ST-HSC、MPP、CMP 及 GMP 在骨髓细胞中的比例均显著 增加（MEP 除外）（P<0.05）。这些结果提示我们生理条件下 Prtn3 基因缺失促进造 血干/祖细胞的增殖分化。 （3）条件敲除小鼠髓系细胞中 Prtn3 基因对造血干/祖细胞的增殖分化无显著 性影响，这表明上述 Prtn3 基因全敲鼠中造血干/祖细胞的增殖和分化能力显著增强 只与造血干/祖细胞中的 Prtn3 基因敲除具有相关性，与成熟髓系细胞中的 Prtn3 基 因敲除无相关性。 （4）竞争移植实验结果表明：与对照鼠相比，不同周龄 Prtn3-/-小鼠来源的 LSK 细胞的竞争移植能力显著降低，并表现出倾向髓系分化的特点（P<0.05）。 （5）单个 LSK 细胞克隆形成能力结果表明 Prtn3-/-小鼠的 LSK 细胞分裂产生 的细胞数目特别是小克隆形成的数量显著高于对照组（P<0.05）。体外多细胞集落 形成实验结果显示 Prtn3-/-小鼠 LSK 细胞的 G、M、GM 集落形成数量与对照组相比 显著增加（P<0.05）。 （6）体外 LSK 细胞髓系分化实验表明相比于 WT 小鼠的 LSK 细胞，在体外促 粒系分化因子 G-CSF 诱导下，Prtn3-/-小鼠的 LSK 细胞向髓系分化能力增强 （P<0.05）。RNA-seq 和 Western blot 结果表明，Prtn3 通过负调控 ERK1/2 的磷酸化 参与 MAPK 信号通路影响造血干/祖细胞的稳态维持。 研究结论：Prtn3 基因缺失促进造血干/祖细胞的增殖、分化。Prtn3 基因缺失导致造 血干/祖细胞稳态失衡并主要表现出髓系细胞分化增强，且在竞争性移植时出现竞争 能力下降的现象。初步的作用机制研究结果表明：Prtn3 基因可以通过调控 ERK1/2 的磷酸化参与 MAPK 信号通路，参与造血干/祖细胞的增殖和分化的调控。本研究 将为今后“HSCs 的命运抉择”相关研究提供了一个新的调控靶点。

Background: Hematopoietic stem cells (HSCs) are a kind of adult stem cells with the potential of self-renewal and multi-differentiation, which are also the source of various terminally differentiated blood cells in the whole life cycle of the organism. Generally, under physiological conditions, most of the HSCs in the bone marrow are in the resting period, while a few HSCs generate various naive or mature blood cells through proliferation and differentiation, and form different blood cell groups with clear stratification (different grades) and classification (different functions) in the body, and each group of cells accounts for a relatively constant proportion in the blood system. It maintains a homeostasis in the body. In pathologic stress states such as acute infection and inflammation and in the aging process of the body, the hematopoietic microenvironment will be changed, which will promote more hematopoietic stem/progenitor cells to enter the proliferation and differentiation stage (including some HSCs from the resting period to the division period), enhance and maintain the occurrence of acute hematopoietic in the bone marrow, and lead to the above changes in the in vivo hematopoietic homeostasis. At present, the fate of HSCs in the organism (proliferation, differentiation or senescence) is still one of the hot topics in the research of hematopoietic regulation, and more comprehensive and in-depth studies are needed. Proteinase 3 (PRTN3) is one of the main members of the serine protease family of neutrophils. It has been widely studied for its bactericidal role in neutrophils, posttranslational modification of cytokines, and regulation of spontaneous death of neutrophils. Recently, our single-cell transcriptome sequencing results showed that Prtn3 gene was highly expressed in young mouse hematopoietic stem/progenitor cells, which was consistent with the data reported by Chambers and Hyatt in the public databases. The above database also revealed that Prtn3 gene is also highly transcribed in human HSC and leukemia cells of patients with acute and chronic myeloid leukemia, but the specific role of this gene in the above hematopoietic stem/progenitor cells and myeloid leukemia cells is not very clear. Based on the above research background, we conducted a study on the role and mechanism of Prtn3 gene in hematopoietic stem/progenitor cells, in order to provide new regulatory targets for future studies on "fate choice of HSCs". Purpose: To clarify the expression and distribution of Prtn3 gene in hematopoietic stem/progenitor cells, and explore the role and mechanism of Prtn3 in hematopoietic stem/progenitor cells. Methods: The expression of Prtn3 gene in wild-type mouse (WT) blood cells was analyzed by single-cell transcriptome sequencing. Real-time quantitative PCR, Western Blot and immunofluorescence were used to detect the expression of PRTN3 protein in hematopoietic cells. Prtn3 knockout mice (Prtn3-/- ) were constructed, and the number and proportion of differentiated mature cells, LT-HSC, ST-HSC, MPP, CMP, GMP and MEP in bone marrow of Prtn3-/- mice at 4, 8, 24, 48 and 72 weeks were analyzed by flow cytometry and blood routine. The proliferation and myeloid differentiation potential of LSK cells were analyzed by in vitro dry/progenitor cell single cell and colony culture. The competitive transplantation experiment of Prtn3-/- mouse LSK cells at 8 weeks, 24 weeks and 48 weeks and the number and proportion of various blood cells in peripheral blood, spleen and bone marrow were analyzed. The distribution of donor blood cells in spleen and bone marrow of Prtn3-/- mice at 8 weeks, 24 weeks and 48 weeks were analyzed by immunofluorescence method. The effect of G-CSF on myeloid differentiation of Prtn3-/- mouse LSK cells was observed in vitro, and the gene expression and potential molecular mechanism involved were analyzed and verified by transcriptional sequencing. Results： (1) Single-cell transcriptome sequencing results showed that Prtn3 gene was highly expressed in LSK, GMP and CMP cells, and real-time quantitative PCR results showed that the mRNA content of Prtn3 in LSK cells was about 5 times that of neutrophils. Meanwhile, ELISA and Western Blot results showed that PRTN3 protein was expressed in LSK cells, and immunofluorescence results showed that PRTN3 existed and distributed in LSK cells. These results indicated that Prtn3 gene was consistently highly expressed in mouse hematopoietic stem/progenitor cells and scattered in the cytoplasm. (2) The deletion of Prtn3 promoted hematopoietic stem/progenitor cells to differentiate into the myeloid system and significantly increased the proportions of LSK and LK cells. The proportions of LT-HSC, ST-HSC, MPP, CMP and GMP in bone marrow cells were significantly increased (except MEP) (P<0.05). These results suggest that Prtn3 deletion promotes hematopoietic stem/progenitor cell proliferation and differentiation under physiological conditions. (3) Conditional knockout of Prtn3 gene in mouse myeloid cells had no significant effect on the proliferation and differentiation of hematopoietic stem/progenitor cells, suggesting that the proliferation and differentiation of hematopoietic stem/progenitor cells in Prtn3 gene knockout mice were significantly enhanced only with Prtn3 gene knockout in hematopoietic stem/progenitor cells. There was no correlation with Prtn3 gene knockout in mature myeloid cells. (4) The results of competitive transplantation experiment showed that, compared with control mice, the competitive transplantation ability of Prtn3-/- mouse derived LSK cells of different weeks of age was significantly decreased, and showed a tendency to myeloid differentiation (P<0.05). (5) The results of single LSK cell clonogenesis showed that the number of LSK cell division in Prtn3-/- mice, especially the number of small clones, was significantly higher than that in the control group (P<0.05). The results of in vitro multicellular colony formation experiment showed that the number of G, M and GM colony formation of Prtn3-/- mouse LSK cells was significantly increased compared with the control group (P<0.05). (6) In vitro experiments on myeloid differentiation of LSK cells showed that compared with LSK cells of WT mice, LSK cells of Prtn3-/- mice were more capable of myeloid differentiation under the induction of granulotropic differentiation factor G-CSF in vitro (P<0.05). The results of RNA-seq and Western blot showed that Prtn3 affected the homeostasis of hematopoietic stem/progenitor cells by negatively regulating the phosphorylation of ERK1/2 in MAPK signaling pathway. Conclusion: Deletion of Prtn3 gene promotes proliferation and differentiation of hematopoietic stem/progenitor cells. The deletion of Prtn3 gene resulted in the imbalance of hematopoietic stem/progenitor cell homeostasis and mainly showed enhanced differentiation of myeloid cells and decreased competitiveness during competitive  transplantation. The preliminary mechanism of action showed that Prtn3 could participate in the regulation of hematopoietic stem/progenitor cell proliferation and differentiation through the regulation of ERK1/2 phosphorylation and MAPK signaling pathway. This study will provide a new regulatory target for future research on "fate choice of HSCs".