Gadd45a基因对小鼠造血干细胞功能的影响

目的 Gadd45a基因对小鼠造血干细胞功能的影响。方法流式细胞仪分选小鼠骨髓造血干细胞、体外单克隆培养,竞争性骨髓移植,放射线照射观察生存曲线。结果 Gadd45a基因缺失的小鼠造血干细胞克隆形成能力增强,短期造血重建能力无差异,8.5Gy放射线照射后生存情况无差异。结论 Gadd45a基因对小鼠造血干细胞功能起重要作用。     

IL-33转基因小鼠骨髓造血干／祖细胞向髓系细胞分化的能力增强

目的：利用IL-33转基因小鼠研究IL-33对造血干/祖细胞的增殖和分化影响。方法利用流式细胞仪分析IL-33转基因小鼠及同窝野生对照小鼠的外周血、脾脏、骨髓细胞的免疫表型及造血干细胞分化不同阶段细胞的数量变化;利用体外成克隆实验和细胞周期分析研究IL-33对于造血干细胞增殖能力的影响。结果与野生型小鼠相比,IL-33转基因小鼠B细胞和T细胞在外周血中都明显降低,粒细胞在外周血和骨髓中都有明显增加;IL-33转基因小鼠的骨髓造血干细胞和多能祖细胞数量减少,共同淋系祖细胞数量减少,共同髓系祖细胞和粒单系祖细胞数量增加;IL-33转基因小鼠的造血干细胞处于S-G2-M的细胞增多;体外单克隆实验发现IL-33转基因小鼠造血干细胞形成的集落数增加。结论 IL-33转基因小鼠造血干细胞增殖能力增强,更易向髓系细胞分化。     

X线全身照射对2型糖尿病KKAy小鼠造血免疫系统功能的影响

目的观察X线全身照射对2型糖尿病模型KKAy小鼠的造血免疫系统功能的损伤作用,并与对照C57小鼠进行比较。方法KKAy小鼠,分为对照组和照射组,照射组小鼠经X线全身照射,剂量4Gy,C57小鼠作为对照。照射后15d检测小鼠的外周血常规,流式细胞术检测骨髓中造血祖细胞、造血干细胞和长期造血干细胞的比例,脾中B细胞和T细胞的比例,胸腺中CD4CD8双阳性T细胞、CD4单阳性T细胞和CD8单阳性T细胞的比例。通过粒细胞集落形成能力实验评价小鼠造血祖细胞的功能。结果照射前KKAy小鼠的HSC和LT-HSC的比例低于C57小鼠。4Gy全身照射后,KKAy小鼠的外周血WBC、RBC、PLT、HGB和LYM%分别下降了68.42%、12.17%、8.78%、30.12%、70.84%;骨髓中HPC、HSC和LT-HSC的比例分别下降了34.02%、29.49%、35.74%;脾B细胞和T细胞的比例分别下降了57.85%、58.81%;胸腺CD4CD8双阳性细胞的比例下降了51.70%。KKAy小鼠的骨髓HSC、LT-HSC、外周血RBC和HGB的降低幅度显著低于C57小鼠。结论4Gy全身照射损伤KKAy小鼠的造血免疫系统功能,KKAy小鼠可能比C57小鼠表现出对电离辐射较强的耐受性。     

Exo-1基因对端粒酶缺陷小鼠造血干细胞植入效率的影响

目的利用不同的造血干细胞移植方式,探讨Exo-1基因缺失对端粒酶基因敲除小鼠的造血干细胞植入效率的影响。方法以CD45.1小鼠的骨髓细胞或骨髓造血干细胞为供体,以端粒酶基因敲除小鼠或Exo-1基因和端粒酶基因双敲除小鼠为受体,在给予不同剂量X线照射或不照射的情况下,重复进行静脉注射全骨髓细胞或分选的骨髓造血干细胞(c-kit+、Sca-1+、lineage-,KSL),于移植后1个月取外周血,流式分析嵌合率。结果未经X线照射及1 Gy、2 Gy照射情况下,端粒酶基因缺陷受体小鼠的外周血中供体来源的细胞嵌合率较低;6 Gy照射后,供体来源的外周血细胞嵌合率仍低于50%,而且端粒酶基因缺陷受体小鼠在移植后1个月内死亡较多;3 Gy照射可形成较高嵌合率,Exo-1基因缺失对端粒酶缺陷小鼠的造血干细胞植入效率的影响不显著。结论以端粒酶缺陷小鼠作为衰老模型研究造血干细胞植入效率时,3 Gy X线照射能够有效地形成较高的外周血供体细胞的嵌合率,但是Exo-1基因没有进一步提高造血干细胞在端粒酶敲除小鼠的植入效率。     

p18INK4C基因缺失增强造血干细胞在亚致死剂量照射小鼠体内长期植入能力

观察p18INK4C(p18)基因缺失对造血干细胞(HSC)在亚致死剂量照射小鼠体内长期植入的影响. 供体为p18基因缺失型(p18(/()纯系C57BL/6小鼠(CD45.2表型), 竞争性细胞来源于C57BL/6-Ly5.1(CD45.1/2)双表型小鼠, 受体为野生型(p18+/+)C57BL/6-Ly5.1(CD45.1)小鼠. 竞争性骨髓移植(cBMT)实验根据受体小鼠照射剂量的不同分为3个剂量组(10 Gy, 5 Gy和1 Gy). 供体细胞和竞争性细胞1:1混合后移植, 移植后采集外周血和骨髓细胞用流式细胞仪检测各细胞比例. 造血恢复移植实验: 移植后检测外周血白细胞计数评价移植后造血恢复速度. 10和5 Gy照射剂量组, 供体细胞和竞争性细胞成功植入, 而1 Gy照射剂量组无供体细胞植入. 无论在10 Gy或是5 Gy照射剂量情况下, 供体细胞在受体内的比例均高于竞争性细胞. 移植后6周, 10和5 Gy照射剂量时外周血中供体细胞比例分别为竞争性细胞的1.46±0.21倍和1.64±0.43倍, 14周时分别为竞争性细胞的1.84±0.25倍和2.00±0.49倍, 26周时分别为竞争性细胞的3.13±0.79倍和3.24±1.33倍. 移植后6个月, 10 Gy照射剂量时骨髓细胞中供体细胞比例为竞争性细胞的7.68±4.42倍, 5 Gy照射剂量时为竞争性细胞的10.83±2.98倍. 移植后6个月, 在10和5 Gy照射剂量组之间骨髓中造血细胞植入率相当, 分别为(85.53±8.71)%和(80.87±2.87)% (P = 0.457). p18(/(细胞与p18+/+细胞相比, 移植后造血恢复的速度相当. p18基因缺失可以显著增强HSC在亚致死剂量照射小鼠体内的长期植入能力.     

RunX3对小鼠骨髓造血干细胞功能的影响

目的研究RunX3基因对造血干细胞自我更新和分化能力的影响。方法流式细胞术测定小鼠骨髓干细胞和外周血单个核细胞的比例;通过竞争性骨髓移植实验检测RunX3转基因小鼠骨髓干细胞的功能。结果移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血细胞占总外周血细胞的比例与野生对照鼠相比无明显差异,移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血中髓系细胞占总外周血髓系细胞的比例较野生型对照鼠高。结论RunX3基因缺失对骨髓造血干细胞的自我更新没有影响,但其可能参与了骨髓造血干细胞的分化过程。     

MKP1基因对小鼠造血干细胞功能的影响

目的通过构建MKP1转基因小鼠模型,研究MKP1基因对造血干细胞自我更新能力的影响。方法运用显微注射法建立MKP1转基因小鼠;PCR和RT-PCR检测MKP1基因在转基因小鼠的表达水平;流式细胞术测定小鼠骨髓干细胞和外周血单个核细胞的比例;通过竞争性骨髓移植实验检测MKP1转基因小鼠骨髓干细胞的功能。结果建立了MKP1转基因小鼠;MKP1转基因小鼠骨髓干细胞数量减少;竞争性骨髓移植实验显示MKP1转基因骨髓干细胞来源的外周血细胞总数、B细胞、粒细胞显著减少（P〈0.001）,提示MKP1转基因小鼠骨髓干细胞的功能下降。结论在MKP1转基因小鼠模型中,MKP1基因的过表达影响了小鼠的骨髓干细胞的功能。     

造血干细胞的浓集及其功能研究

应用细胞毒剂和非连续密度梯度离必法可以提高小鼠骨髓CFU-S的相对浓度。实验研究表明，骨髓CFU-S是维持正常机体恒定造血或对照射小鼠促进造血恢复的主要细胞成分。在对750拉德γ线照射小鼠注射相同细胞数量的基础上，治疗效果随CFU-S浓集程度的提高而增强。小鼠注射细胞毒剂有影响骨髓CFU-S生理状态的作用，促进处于砌期的CFU-S转入增殖活动。因此，对照射小鼠移植经细胞毒剂或合并非连纹密度梯度离心后的造血干细胞，它将在CFU-S浓集的基础上迸一步提高其促进照射动物造血功能的恢复。     

Cramp过表达对小鼠骨髓造血干细胞功能的影响

目的研究Cramp蛋白过表达对小鼠骨髓造血干细胞自我更新和分化能力的影响。方法应用流式细胞仪分析Cramp过表达转基因小鼠及同龄野生型小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例;分选骨髓造血干细胞,体外培养,观察其克隆形成能力。结果与野生型小鼠相比,Cramp过表达转基因小鼠的骨髓、脾脏、胸腺等组织器官中各种细胞的比例、骨髓造血干细胞的克隆形成能力等均无明显变化。结论本研究中,Cramp过表达转基因小鼠骨髓造血干细胞的分化能力、克隆形成能力无明显变化。     

Cramp基因敲除对小鼠骨髓造血干细胞的影响

目的研究Cramp基因敲除在衰老过程中对小鼠造血干细胞的作用。方法应用流式细胞仪分析3月龄及12月龄Cramp基因敲除小鼠及同窝野生型小鼠的骨髓造血干细胞的比例及不同发育阶段B淋巴细胞的比例。结果与野生型小鼠相比,12月龄Cramp基因敲除小鼠的骨髓长期造血干细胞增多,多潜能造血祖细胞减少;前体B淋巴细胞和未成熟B淋巴细胞减少,成熟B淋巴细胞增多。结论在衰老过程中,Cramp基因敲除对骨髓造血干细胞及B淋巴细胞发育有重要影响。     

The potential benefits of nicaraven to protect against radiation-induced injury in hematopoietic stem/progenitor cells with relative low dose exposures

Nicaraven, a hydroxyl radical-specific scavenger has been demonstrated to attenuate radiation injury in hematopoietic stem cells with 5 Gy γ-ray exposures. We explored the effect and related mechanisms of nicaraven for protecting radiation injury induced by sequential exposures to a relatively lower dose γ-ray. C57BL/6 mice were given nicaraven or placebo within 30 min before exposure to 50 mGy γ-ray daily for 30 days in sequences (cumulative dose of 1.5 Gy). Mice were victimized 24 h after the last radiation exposure, and the number, function and oxidative stress of hematopoietic stem cells were quantitatively estimated. We also compared the gene expression in these purified stem cells from mice received nicaraven and placebo treatment. Nicaraven increased the number of c-kit⁺ stem/progenitor cells in bone marrow and peripheral blood, with a recovery rate around 60–90% of age-matched non-irradiated healthy mice. The potency of colony forming from hematopoietic stem/progenitor cells as indicator of function was completely protected with nicaraven treatment. Furthermore, nicaraven treatment changed the expression of many genes associated to DNA repair, inflammatory response, and immunomodulation in c-kit⁺ stem/progenitor cells. Nicaraven effectively protected against damages of hematopoietic stem/progenitor cells induced by sequential exposures to a relatively low dose radiation, via complex mechanisms.     

Interferon regulatory factor-2 induces megakaryopoiesis in mouse bone marrow hematopoietic cells

Megakaryopoiesis is associated with inflammatory reactions. To investigate the role of interferon regulatory factors (IRFs) in inflammation-associated megakaryopoiesis, mouse bone marrow hematopoietic stem cells (HSCs) were analyzed. IFN-γ treatment induced IRF-2 expression as well as the expression of CD41 and IRF-1 in HSCs. An in vitro clonogenic assay showed that IRF-2- but not IRF-1-overexpressing cells increased the number of megakaryocytic colonies. IRF-2 transfection up-regulated CD41 promoter activity in hematopoietic cell lines. The number of CD41-positive bone marrow cells increased in mice injected with IRF-2-expressing bone marrow cells. These findings suggest that IRF-2 plays an important role in megakaryopoiesis in inflammatory states.     

GPX4 and vitamin E cooperatively protect hematopoietic stem and progenitor cells from lipid peroxidation and ferroptosis

Ferroptosis, a newly defined mode of regulated cell death caused by unbalanced lipid redox metabolism, is implicated in various tissue injuries and tumorigenesis. However, the role of ferroptosis in stem cells has not yet been investigated. Glutathione peroxidase 4 (GPX4) is a critical suppressor of lipid peroxidation and ferroptosis. Here, we study the function of GPX4 and ferroptosis in hematopoietic stem and progenitor cells (HSPCs) in mice with Gpx4 deficiency in the hematopoietic system. We find that Gpx4 deletion solely in the hematopoietic system has no significant effect on the number and function of HSPCs in mice. Notably, hematopoietic stem cells (HSCs) and hematopoietic progenitor cells lacking Gpx4 accumulated lipid peroxidation and underwent ferroptosis in vitro. α-Tocopherol, the main component of vitamin E, was shown to rescue the Gpx4-deficient HSPCs from ferroptosis in vitro. When Gpx4 knockout mice were fed a vitamin E-depleted diet, a reduced number of HSPCs and impaired function of HSCs were found. Furthermore, increased levels of lipid peroxidation and cell death indicated that HSPCs undergo ferroptosis. Collectively, we demonstrate that GPX4 and vitamin E cooperatively maintain lipid redox balance and prevent ferroptosis in HSPCs.Subject terms: Cell biology, Physiology, Stem-cell research     

A small molecule inhibitor of p53 stimulates amplification of hematopoietic stem cells but does not promote tumor development in mice

It has been shown that genetic inhibition of p53 leads to enhanced proliferation of hematopoietic stem cells (HSCs). This could, in theory, contribute to the increased frequency of tumor development observed in p53-deficient mice and humans. In our previous work, we identified chemical p53 inhibitors (PFTs) that suppress the transactivation function of p53 and protect cultured cells and mice from death induced by gamma irradiation (IR). Here we found that when applied to bone marrow cells in vitro or injected into mice, PFTb impeded IR-induced reduction of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) population sizes. In addition, we showed that PFTb stimulated HSC and HPC proliferation in the absence of IR in vitro and in vivo and mobilized HSCs to the peripheral blood. Importantly, however, PFTb treatment did not affect the timing or frequency of tumor development in irradiated p53 heterozygous mice used as a model for determination of carcinogenicity. Thus, although PFTb administration led to increased numbers of HSCs and HPCs, it was not carcinogenic in mice. These findings suggest that chemical p53 inhibitors may be clinically useful as safe and effective stimulators of hematopoiesis.     

Radioprotective effect of misoprostol on mouse spermatogonial stem cells

The radioprotective effects of misoprostol, a synthetic stable analogue of prostaglandin E1, on spermatogonial stem cells of C3H/HeH x 101/F1 hybrid mice (3H1) were analysed by establishing dose--response relationships for stem cell killing by X-rays in mice that were pretreated with misoprostol. Spermatogonial stem cell killing was studied through determination of the percentage of tubular cross-sections showing repopulation at 10 days after irradiation. In control mice, the D0 values ranged between 1.7 and 3.6 Gy, dependent on the stage of the cycle of the seminiferous epithelium the cells were in. As found previously, proliferating spermatogonial stem cells were much more radioresistant than quiescent stem cells. In the misoprostol-pretreated animals the spermatogonial stem cells were more radioresistant, the D0 values ranging from 3.6 to 5.0 Gy. Both proliferating and quiescent spermatogonial stem cells were protected by misoprostol. As the dose--response curves in control and misoprostol-pretreated mice showed about the same extrapolation number to the y-axis it was concluded that the misoprostol pretreatment did not alter the kinetics of the repopulation process.     

In Vivo Tracking and Comparison of the Therapeutic Effects of MSCs and HSCs for Liver Injury

Background

Mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) have been studied for damaged liver repair; however, the conclusions drawn regarding their homing capacity to the injured liver are conflicting. Besides, the relative utility and synergistic effects of these two cell types on the injured liver remain unclear.

Methodology/Principal Findings

MSCs, HSCs and the combination of both cells were obtained from the bone marrow of male mice expressing enhanced green fluorescent protein(EGFP)and injected into the female mice with or without liver fibrosis. The distribution of the stem cells, survival rates, liver function, hepatocyte regeneration, growth factors and cytokines of the recipient mice were analyzed. We found that the liver content of the EGFP-donor cells was significantly higher in the MSCs group than in the HSCs or MSCs+HSCs group. The survival rate for the MSCs group was significantly higher than that of the HSCs or MSCs+HSCs group; all surpassed the control group. After MSC-transplantation, the injured livers were maximally restored, with less collagen than the controls. The fibrotic areas had decreased to a lesser extent in the mice transplanted with HSCs or MSCs+HSCs. Compared with mice in the HSCs group, the mice that received MSCs had better improved liver function. MSCs exhibited more remarkable paracrine effects and immunomodulatory properties on hepatic stellate cells and native hepatocytes in the treatment of the liver pathology. Synergistic actions of MSCs and HSCs were most likely not observed because the stem cells in liver were detected mostly as single cells, and single MSCs are insufficient to provide a beneficial niche for HSCs.

Conclusions/Significance

MSCs exhibited a greater homing capability for the injured liver and modulated fibrosis and inflammation more effectively than did HSCs. Synergistic effects of MSCs and HSCs were not observed in liver injury.     

Mobilizing of haematopoietic stem cells to ischemic myocardium by plasmid mediated stromal-cell-derived factor-1alpha (SDF-1alpha) treatment

A concentration gradient of stromal-cell-derived factor-1alpha (SDF-1alpha) is the major mechanism for homing of haematopoietic stem cells (HSCs) in bone marrow. We tested the hypothesis that a gene therapy using SDF-1alpha can enhance HSCs recruiting to the heart upon myocardial infarction (MI). Adult mice with surgically induced myocardial ischemia were injected intramyocardially with either saline (n=12) or SDF-1alpha plasmid (n=12) in 50 microl volume in the ischemic border zone of the infarcted heart 2 weeks after myocardial infarction. Donor Lin-c-kit+ HSCs from isogenic BalB/c mice were harvested, sorted through magnetic cell sorting (MACS) and labeled with PKH26 Red. Three days after plasmid or saline injection, 1x10(5) labeled cells were injected intravenously (i.v.) into saline mice (n=4) and SDF-1alpha plasmid mice (n=4). The hearts and other tissue were removed for Western blot assay 2 weeks after plasmid or saline treatment. The labeled Lin-c-kit+ cells were identified with immunofluoresent staining and endogenous c-kit+ cells were identified by immunohistochemical staining. In mice killed at 1 month postinfarct, Western blot showed higher levels of SDF-1alpha expression in SDF-1alpha-treated mouse ischemic hearts compared to saline-treated hearts and other tissues. In the SDF-1alpha plasmid-treated hearts, SDF-1alpha is overexpressed in the periinfarct zone. The labeled stem cells engrafted to the SDF-1alpha positive site in the myocardium. There was also evidence for endogenous stem cell recruiting. The density of c-kit+ cells in border zone, an index of endogenous stem cell mobilization, was significantly higher in the SDF-1alpha-treated group than in the saline group (14.63+/-1.068 cells/hpf vs. 11.31+/-0.65 cells/hpf, P=0.013) at 2 weeks after SDF-1alpha or saline treatment. Following myocardial infarction, treatment with SDF-1alpha recruits stem cells to damaged heart where they may have a role in repairing and regeneration. The gene therapy with an SDF-1alpha vector offers a promising therapeutic strategy for mobilizing stem cells to the ischemic myocardium.     

Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition

Background

Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-differentiate into activated myofibroblasts, which produce extracellular matrix.Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study showed that succinate and its specific receptor, GPR91, are involved in the activation of HSCs and the overexpression of α-smooth muscle actin (α-SMA).Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model. However, the role of metformin in HSC activation and succinate-GPR91 signaling has not been clarified.

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1⁺Mac-1⁺ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca²⁺ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.