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31.
Mesenchymal stromal cells (MSCs) have proven useful for cell and immune therapy, but the molecular constituents responsible for their functionalities, in particular, those on the plasma membrane, remain largely unknown. Here we employed both gel and nongel based MS to analyze human MSCs' membrane proteome before and after adipogenesis. 2-DE of cells that were pretreated with membrane impermeable fluorescent dyes revealed that both the whole cell proteome and the cell surface subproteome were independent of donors. LC coupled with tandem MS analysis of the plasma membrane-containing fraction allowed us to identify 707 proteins, approximately half of which could be annotated as membrane-related proteins. Of particular interest was a subset of ectodomain-containing membrane-bound proteins that encompass most known surface markers for MSCs, but also contain a multitude of solute carriers and ATPases. Upon adipogenic differentiation, this proteomic profile was amended to include several proteins involved in lipid metabolism and trafficking, at the expense of, most noticeably, ectoenzymes. Our results here provide not only a basis for future studies of MSC-specific molecular mechanisms, but also a molecular inventory for the development of antibody-based cell isolation and identification procedures. 相似文献
32.
Jing Wu Fei Guan Wei Luo Zhi-Wei Yuan Rong-Qiong Chen Xin Gou Xin Shi Hai-Xiang Guo Ke-Wei Fang 《Journal of cellular physiology》2019,234(10):17800-17811
Urinary incontinence (UI) is known as a distressing condition particularly among older adults, and negatively associated with health-related quality of life in both males and females. Prelamin A accumulation has been found in all progeroid laminopathies and is obviously linked to cell and organism aging. Therefore, this study was expected to investigate the effect of prelamin A on detrusor on UI. Prelamin A expression in clinical and animal samples was detected. To investigate the degree of prelamin A accumulation and detrusor calcification/aging, the detrusor cells were subcultured separately into low and high passage. The low-passage subculture cells were treated with transfection of overexpressed prelamin A plasmid, and transfection of overexpressed prelamin A plasmid and application of farnesyl transferase inhibitor (FTIs) H-9279, respectively. Zmpste24, Icmt and lamin A/C expression were detected to explore how prelamin A affected detrusor calcification/aging. Prelamin A was overexpressed in aged detrusor cells, indicating prelamin A expression was positively related to the age of subjects. The degree of prelamin A accumulation and detrusor calcification/aging was higher in aged rats and high passage subculture cells. Zmpste24, Icmt and lamin A/C were poorly expressed in cells transfected with overexpressed prelamin A, as well as cell proliferation activity decreased and calcium deposition and apoptotic rate increased. Furthermore, we also found that the effect of overexpressed prelamin A was lost when cells were treated with H-9279. These findings provide evidence that prelamin A overexpression impairs degradation of its farnesylated form, thus causing prelamin A accumulation which induces detrusor calcification/aging in UI. 相似文献
33.
Gongmin Zhu Lijiao Pei Fan Lin Hubin Yin Xinyuan Li Weiyang He Nian Liu Xin Gou 《Journal of cellular physiology》2019,234(12):23736-23749
Renal ischemia/reperfusion (I/R) injury is the main reason for acute kidney injury (AKI) and is closely related to high morbidity and mortality. In this study, we found that exosomes from human-bone-marrow-derived mesenchymal stem cells (hBMSC-Exos) play a protective role in hypoxia/reoxygenation (H/R) injury. hBMSC-Exos were enriched in miR-199a-3p, and hBMSC-Exo treatment increased the expression level of miR-199a-3p in renal cells. We further explored the function of miR-199a-3p on H/R injury. miR-199a-3p was knocked down in hBMSCs with a miR-199a-3p inhibitor. HK-2 cells cocultured with miR-199a-3p-knockdown hBMSCs were more susceptible to H/R injury and showed more apoptosis than those cocultured with hBMSCs or miR-199a-3p-overexpressing hBMSCs. Meanwhile, we found that HK-2 cells exposed to H/R treatment incubated with hBMSC-Exos decreased semaphorin 3A (Sema3A) and activated the protein kinase B (AKT) and extracellular-signal-regulated kinase (ERK) pathways. However, HK-2 cells cocultured with miR-199a-3p-knockdown hBMSCs restored Sema3A expression and blocked the activation of the AKT and ERK pathways. Moreover, knocking down Sema3A could reactivate the AKT and ERK pathways suppressed by a miR-199a-3p inhibitor. In vivo, we injected hBMSC-Exos into mice suffering from I/R injury; this treatment induced functional recovery and histologic protection and reduced cleaved caspase-3 and Sema3A expression levels, as shown by immunohistochemistry. On the whole, this study demonstrated an antiapoptotic effect of hBMSC-Exos, which protected against I/R injury, via delivering miR-199a-3p to renal cells, downregulating Sema3A expression and thereby activating the AKT and ERK pathways. These findings reveal a novel mechanism of AKI treated with hBMSC-Exos and provide a therapeutic method for kidney diseases. 相似文献
34.
Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli 总被引:1,自引:0,他引:1
Kong X Liu Y Gou X Zhang H Wang X Zhang J 《Biochemical and biophysical research communications》2001,280(1):396-400
Trehalose is a nonspecific protective agent for biomacromolecules. Trehalose-6-phosphate synthase (OtsA)/phosphatase (OtsB), which is encoded by the gene operon otsBA located at -42 of the Escherichia coli genome, is the main enzyme system that catalyzes the synthesis of trehalose in E. coli. We cloned the operon and modified it by directed evolution. Unlike in the previously reported work, we modified the whole operon and screened the positive mutant simultaneously. Thus we believe that the gene complex solves the negative effects between two enzymes if one of them diversifies its structure or functions and finds the form most suitable for trehalose synthesis. It thus mimics the natural process, in which the functional improvement of organisms is related to alterations in coordinated enzymes. The evolution procedure was carried out in a sequence of error-prone PCR, shuffling PCR, and then strict screening of the mutants. After screening of a library of more than 4000 colonies, about 15 positive colonies were analyzed, resulting in a higher concentration of trehalose than control. One of them, E. coli TS7, shows 12.3-fold higher trehalose synthesis ability than E. coli DH5alpha. In contrast, we introduced the cDNA sequence of the tps1 gene from Saccharomyces cerevisiae, which has 54% identity with the gene otsA, as one of the templates in shuffling PCR. By hybrid evolution and screening, we obtained 10 positive colonies with higher concentrations of trehalose than control. E. coli TS22 appears to have 5.3-fold higher trehalose synthesis ability than E. coli DH5alpha and 1.6-fold more than E. coli DEF3(pOTS11). This result demonstrated that coevolution and hybrid evolution, as powerful protocols in protein engineering, are effective in modifying enzyme. It indicates that repeating the process of genomic evolution in nature is feasible. 相似文献
35.
36.
Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche 总被引:69,自引:0,他引:69
The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche. 相似文献
37.
Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock 总被引:3,自引:0,他引:3
Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP. 相似文献
38.
39.
Plasticity of rat bone marrow-derived 5-hydroxytryptamine-sensitive neurons: dedifferentiation and redifferentiation 总被引:7,自引:0,他引:7
Li TY Shu C Wong CH Lo PS Zhu H Lau MC Chan MY Tsang LL Gou YL Chung YW Chan HC 《Cell biology international》2004,28(11):801-807
Inducing cellular dedifferentiation has been proposed as a potential method for enhancing endogenous regeneration in mammals. Here we demonstrate that phenotypic and functional neurons derived from adult rat bone marrow stromal stem cells (MSCs) can be induced to undergo dedifferentiation, then proliferation and redifferentiation. In addition to morphological changes and expression of neuronal markers, neuron-specific enolase and neurofilament H, functional differentiation was monitored by intracellular Ca2+ mobilization in response to a ubiquitous neurotransmitter, 5-hydroxytryptamine (5-HT) at different stages. The neurons derived from rMSCs were found to have increased 5-HT response. This 5-HT sensitivity could be reversed to basal level similar to that found in rMSCs when neurons, up to 3 days after neuronal induction, were induced to undergo dedifferentiation. Increase in 5-HT-induced Ca2+ mobilization was again observed when rMSCs derived from dedifferentiated neurons were induced to redifferentiate into neurons again. Variation in 5-HT1A receptor immunoreactivity was observed in stem cells, differentiated neurons, dedifferentiated neurons and redifferentiation neurons, consistent with their respective 5-HT sensitivity. These results suggest that adult bone marrow-derived 5-HT sensitive neurons are capable of dedifferentiation, then proliferation and redifferentiation, indicating their plasticity and potential use in treatment of neural degenerative diseases. 相似文献
40.
人骨髓间充质干细胞在成年大鼠脑内的迁移及分化 总被引:29,自引:2,他引:27
骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞 相似文献