STAR Protocols
·
2021
·
IF 0
Zhen Xu,Yanxia Rao,Bo Peng
Summary Microglia are important immune cells in the central nervous system. Replacement of mutated microglia by wild-type cells through microglia replacement by bone marrow transplantation can correct gene deficiencies. However, the limited availability of bone marrow cells may restrict its potential of becoming a widely used clinical treatment. Here, we introduce a potentially clinical-feasible strategy achieving efficient microglia replacement by peripheral blood cells in mice, boosting the donor cell availability. We named it microglia replacement by peripheral blood (Mr PB). For complete details on the use and execution of this protocol, please refer to Xu et al. (2020) . The original abbreviation of this microglia replacement strategy is mrPB. We hereby change the name to Mr PB.
研究领域:
神经科学
·
免疫学
·
再生医学
✓ 已验证
DOI
PubMed
ANALYTICAL BIOCHEMISTRY
·
2021
·
IF 3.365
Yuanyuan Ding,Xiaoqian Li,Qingpeng Gao,Xinyan Dong,Liyun Kong,Shengli Han,Tao Zhang,Langchong He
Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH 2 groups for covalent immobilization of the capture antibody. Through amide condensation , a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody . We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.
研究领域:
医学
·
免疫学
·
生物化学
✓ 已验证
DOI
高影响因子
BIOMEDICAL MICRODEVICES
·
2017
·Vol. 19
·期 3
·1-8
·
IF 2.062
Li Yuan,Lu Yao,Chen Qiong,Kang Yuejun,Yu Ling
Retinopathy is a complication of diabetes that affects the eyes; it stems from damage to the microvasculature of the retina and eventually compromises vision. The diagnosis of retinopathy is difficult to make because there are no early symptoms or warning signs. Dysfunction of the retina’s microvascular networks is believed to be associated with inflammatory cytokines and tumor necrosis factor alpha (TNF-α). To investigate the effect of these cytokines, such as TNF-α, a polydimethylsiloxane (PDMS)/glass hydride microfluidic device reflecting the physiological structure of the retina’s microvasculature was developed. In this model, the bifurcations and tortuosity of branch vessels were based on photographs of the fundus and an endothelial cell layer (EA.hy926 cells) were reconstructed within the microfluidic network. The adhesion, spreading, and growth of cells was ensured by optimizing the conditions for cell seeding and perfusion. Fluorescent staining was used to visualize the cytoskeleton and measurement of the nitric oxide (NO) level proved that the endothelial EA.hy926 cells had spread in the direction of flow perfusion system, forming artificial vascular networks. The endothelial layer was further challenged by TNF-α perfusion. Cytokine treatment increased the anchoring of peripheral blood mononuclear cells (PBMCs) on the endothelial layer. The microfluidic device developed in this study provides a low-cost platform reflecting the physiological structures of the retina’s microvasculature. It is anticipated that this device will be useful in evaluating the diseased retina as well as in drug screening.
研究领域:
医学
·
生物工程
✓ 已验证
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2025
·
IF 8.2
Ying Yu,Yuan Cheng,Shiwei Xu,Wanyun Song,Mingming Wang,Liqiang Song,Kejia Zhu,Tao Zhang
Noninvasive prenatal diagnosis based on fetal cells (cell-based NIPD) offers a safer alternative to traditional invasive procedures but remains limited by the low abundance of fetal nucleated red blood cells (FNRBCs) in maternal circulation and the inefficiency of current isolation techniques. Here, we present a functionalized interventional system for in situ hematologic cell capture (FISHC) directly from peripheral blood, eliminating the need for blood withdrawal and ex vivo processing. FISHC features a carboxybetaine methacrylate (CBMA)-functionalized medical wire with immobilized FNRBC-specific antibodies, enabling high-specificity cell capture under physiological conditions. In vivo validation in a pregnant monkey model confirms its feasibility for efficient and minimally invasive fetal cell retrieval. FISHC represents a paradigm shift in cell-based NIPD and holds promise for broader biomedical applications, including circulating tumor cell detection and real-time liquid biopsy technologies.
研究领域:
医学工程
·
生殖健康
·
生物医学工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Journal of Extracellular Vesicles
·
2023
·Vol. 12
·期 9
·12364
·
IF 16
Jie He,Hengyu Li,John Mai,Yuqing Ke,Chunhui Zhai,Jiao Jiao Li,Lai Jiang,Guangxia Shen,Xianting Ding
Extracellular vesicle (EV) surface proteins, expressed by primary tumours, are important biomarkers for early cancer diagnosis. However, the detection of these EV proteins is complicated by their low abundance and interference from non-EV components in clinical samples. Herein, we present a MEmbrane-Specific Separation and two-step Cascade AmpLificatioN (MESS2CAN) strategy for direct detection of EV surface proteins within 4 h. MESS2CAN utilises novel lipid probes (long chains linked by PEG2K with biotin at one end, and DSPE at the other end) and streptavidin-coated magnetic beads, permitting a 49.6% EV recovery rate within 1 h. A dual amplification strategy with a primer exchange reaction (PER) cascaded by the Cas12a system then allows sensitive detection of the target protein at 10 EV particles per microliter. Using 4 cell lines and 90 clinical test samples, we demonstrate MESS2CAN for analysing HER2, EpCAM and EGFR expression on EVs derived from cells and patient plasma. MESS2CAN reports the desired specificity and sensitivity of EGFR (AUC = 0.98) and of HER2 (AUC = 1) for discriminating between HER2-positive breast cancer, triple-negative breast cancer and healthy donors. MESS2CAN is a pioneering method for highly sensitive in vitro EV diagnostics, applicable to clinical samples with trace amounts of EVs.
研究领域:
生物技术
·
医学诊断
·
癌症研究
·
分子生物学
✓ 已验证
DOI
PubMed
高影响因子
Cell Reports
·
2020
·
IF 8.109
Zhen Xu,Yanxia Rao,Yubin Huang,Tian Zhou,Rui Feng,Shanshan Xiong,Ti-Fei Yuan,Shan Qin,Yijie Lu,Xin Zhou,Xiaoyu Li,Bo Qin,Ying Mao,Bo Peng
Summary Microglia are important immune cells in the central nervous system (CNS). Dysfunctions of gene-deficient microglia contribute to the development and progression of multiple CNS diseases. Microglia replacement by nonself cells has been proposed to treat microglia-associated disorders. However, some attempts have failed due to low replacement efficiency, such as with the traditional bone marrow transplantation approach. In this study, we develop three efficient strategies for microglia replacement: microglia replacement by bone marrow transplantation (mrBMT), microglia replacement by peripheral blood (mrPB), and microglia replacement by microglia transplantation (mrMT). mrBMT and mrPB allow microglia-like cells to efficiently replace resident microglia in the whole CNS. On the other hand, mrMT achieves microglia replacement in brain regions of interest. In summary, the present study offers effective tactics for microglia replacement with diverse application scenarios, which potentially opens up a window on treating microglia-associated CNS disorders.
研究领域:
神经科学
·
免疫学
·
细胞治疗
✓ 已验证
DOI
PubMed
高影响因子
iScience
·
2023
·Vol. 26
·期 11
·108246-108246
·
IF 5.8
Peng Xie,Xiaoyue Yao,Zhenyu Chu,Yang Yang,Haifeng Li,Siyuan Tan,Haodong Tang,Jiahua Zhou,Wanqin Jin
The detection of circulating tumor cells (CTCs) in peripheral blood is a novel and accurate technique for the early diagnosis of cancers. However, this method is challenging because of the need for high collection efficiency due to the ultralow content and similar size of CTCs compared with other blood cells. To address the aforementioned issue, we proposed a homoporous polydimethylsiloxane (PDMS) membrane and its microfilter device to perform the ultrafast isolation and identification of CTCs directly from peripheral blood without any labeling treatment. The membrane pores can be homogenously controlled at a size of 6.3 μm through the cross-linking time of PDMS during a filtration-coating strategy. Within only 10 s, the designed device achieved a retention rate greater than 70% for pancreatic cancer cells, and it exhibited excellent cell compatibility to support cell proliferation. The isolated CTCs on this membrane can be easily observed and identified using a fluorescence microscope.
研究领域:
生物医学工程
·
癌症诊断
·
微流控技术
·
细胞分离
✓ 已验证
DOI
PubMed
高影响因子
Journal of Ocean University of China
·
2024
·Vol. 23
·期 6
·1615-1629
·
IF 1.4
Wang Wenwen,Wei Shu,Chen Yadong,Tan Suxu,Shi Kunpeng,Zang Shaoqing,Wang Minmin,Yang Teng,Yang Yingming,Cui Zhongkai,Sha Zhenxia
As an efficient immunostimulant, chitosan oligosaccharide (COS) can enhance the immunity of teleosts; however, the underlying molecular mechanisms still require elucidation. Competing endogenous RNAs (ceRNAs) are vital regulators in the immune response, but their roles in half-smooth tongue sole ( Cynoglossus semilaevis ) remain unclear. In this study, for the first time, we studied whole-transcriptome expression profiles and analyzed ceRNA networks in peripheral blood leukocytes of half-smooth tongue sole treated with COS. A total of 19 circRNAs (DE-circRNAs), 18 miRNAs (DE-miRNAs) and 50 previously identified lncRNAs (DE-lncRNAs) were differentially expressed after COS stimulation. The DE-lncRNAs and DE-miRNAs targeted numerous immunity-related genes, and were enriched in important pathways, including MAPK and Toll-like receptor signaling pathways, suggesting the immunoregulatory roles of COS. Furthermore, we constructed circRNA-miRNA-mRNA and lncRNA-miRNA-mRNA regulatory networks using DE-circRNAs, DE-miRNAs, DE-lncRNAs, and DE-mRNAs. Additionally, a ceRNA network with immunity-related DE-mRNAs was constructed, showing that 3 DE-circRNAs, 12 DE-lncRNAs, and 29 DEGs exhibited crosstalk through 9 DE-miRNAs. Intriguingly, a DE-miRNA in the ceRNA network, miR-144-3p, was targeted by DE-lncRNA tnrc6a , and negatively regulated the genes of inhibitor of nuclear factor kappa B kinases (IKKs) ( ikbkg , ikbkb , and ikbip ) and c3ar1 . Ikbkg , ikbkb , and c3ar1 were significantly up-regulated in macrophages stimulated by LPS. It could be inferred that ncRNAs participated in the immune and inflammatory response by acting as ceRNAs after COS stimulation in teleosts. These findings indicate that COS could enhance the immunity of teleosts by regulating ncRNAs, and lay the foundation for further practical application of COS in aquaculture.
研究领域:
免疫学
·
分子生物学
·
水产养殖
·
转录组学
✓ 已验证
DOI
高影响因子
STAR Protocols
·
2021
·
IF 0
Zhen Xu,Bo Peng,Yanxia Rao
Summary Mutations in microglia may cause brain disorders. Replacement of dysfunctional microglia by allogeneic wild-type microglia from bone marrow transplantation (Mr BMT) or peripheral blood can correct the gene deficiency at the brain-wide scale but cannot achieve precise replacement at specific brain regions. Here, we introduce a strategy with potential clinical relevance—microglia replacement by microglia transplantation (Mr MT), combining tamoxifen-induced ablation of Mr BMT cells and intracranial injection of microglia to mouse brain, to achieve region-sepcific microglia replacement. The original abbreviation of this microglia replacement strategy is mrMT. We hereby change the name to Mr MT. For complete details on the use and execution of this protocol, please refer to Xu et al. (2020) .
研究领域:
神经科学
·
细胞生物学
·
移植学
✓ 已验证
DOI
PubMed
JOURNAL OF INTERNATIONAL MEDICAL RESEARCH
·
2023
·
IF 1.6
Fang Wang,Xiu-Li Zhang,Jing-Ya Yang,Gang Zhou
ObjectiveOral lichen planus (OLP) is a T cell-mediated inflammatory condition in the oral cavity. Mucosal-associated invariant T (MAIT) cells are gaining more relevance in immune diseases because they can be activated by cytokines without T cell receptor stimulation. Herein, we tested the effect of interleukin-23 (IL-23) on the activation status of OLP MAIT cells.MethodsPeripheral blood mononuclear cells (PBMCs) isolated from OLP patients were stimulated by IL-23 in the absence or presence of phorbol myristate acetate (PMA) and ionomycin. The activation status of MAIT cells was analyzed by flow cytometry after staining for CD3, CD4, CD8, CD161, TCR Vα7.2, and CD69.ResultsThe fraction of MAIT cells in OLP peripheral blood was approximately 0.38% to 3.97%, and CD8+ subpopulations overwhelmed CD4+ cells. The mean percentages of OLP MAIT cells in PBMCs and CD8+MAIT cells in MAIT cells were approximately 40%. PMA and ionomycin significantly increased CD69 expression on OLP T cells, MAIT cells, and CD8+MAIT cells. Cells with enhanced activation had different responsiveness to exogenous IL-23, showing increased CD69 expression on OLP T cells, decreased CD69 on OLP CD8+MAIT cells, and no significant change on OLP MAIT cells.ConclusionsIL-23 showed different effects on the activation status of OLP MAIT cells and CD8+MAIT cells.
研究领域:
免疫学
·
皮肤科
·
口腔医学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MUSCLE & NERVE
·
2025
·Vol. 71
·期 3
·474-486
·
IF 3.1
Zhuajin Bi,Qing Zhang,Huajie Gao,Huizhen Ge,Jiayang Zhan,Mengge Yang,Bitao Bu
Introduction/Aims Tofacitinib, a first-generation Janus kinase (JAK) 1/3 inhibitor, is commonly used for treating ulcerative colitis and rheumatoid arthritis. However, its role in myasthenia gravis (MG) remains unclear. This study aimed to evaluate the immunomodulatory effects of tofacitinib on experimental autoimmune myasthenia gravis (EAMG) and peripheral blood mononuclear cells (PBMCs) from patients with MG. Methods Flow cytometry, enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blot were used to evaluate the effects of tofacitinib on T helper (Th) cell profiles, humoral immune responses, and the JAK-signal transducer and activator of transcription (STAT) pathway proteins. Results In vivo, tofacitinib significantly ameliorated EAMG severity in rats, reducing the proportions of Th1, Th17 and memory B cells, and anti-acetylcholine receptor (AChR) antibodies levels, while increasing the proportions of regulatory T (Treg) cells. In vitro, tofacitinib administration resulted in a significant decrease in the proportions of Th1 and IgG-secreting B cell, and a significant upregulation of Treg cells in mononuclear cells (MNCs) from EAMG rats, which was consistent with findings in PBMCs from MG patients. Further analysis revealed that tofacitinib inhibited CD4 + T cell differentiation into Th1 by decreasing phosphorylated STAT1 levels, while promoting Treg differentiation via increased phosphorylated STAT5 levels in MNCs from EAMG rats. Discussion Tofacitinib modulates Th cell profiles and humoral immune responses by targeting the JAK–STAT pathway, suggesting its potential as a therapeutic candidate for MG. Further clinical studies are warranted to evaluate the efficacy and safety of tofacitinib in MG patients.
研究领域:
免疫学
·
药理学
·
神经病学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
American Journal of Translational Research
·
2018
·
IF 3.061
Qin Zhu,Peifen Zheng,Xinyu Chen,Feng Zhou,Qiaona He,Yuefeng Yang
Ulcerative colitis (UC) is a chronic and nonspecific intestinal inflammatory disease, which may increase the risk of colon cancer. Andrographolide is a main active component of Andrographis paniculata . The anti-inflammatory ability of andrographolide suggested its potential therapeutic effect against UC. In the present study, elevated serum concentrations of proinflammatory factors, including (TNF)-α, interleukin (IL)-1β, IL-6 and IL-23, as well as increased percentages of Th17 cells (IL-17+CD4+ cells) in CD4+ cells were detected in UC patients compared to that in healthy donors. These data suggested that Th17 immune responses may involve in the pathogenesis of UC. Experimental colitis mouse model was then established. The results of hematoxylin and eosin staining demonstrated the therapeutic effect of andrographolide on colitis. Enzyme-linked immunosorbent assay (ELISA), flow cytometry and western blotting analyses showed that andrographolide could decreased the levels of proinflammatory factors TNF-α, IL-1β, IL-6 and IL-17A in the serum and in the colon tissues, reduced the percentages of Th17 cells in CD4+ cells, and suppressed the levels of IL-23, IL-17A, ROR-γt (key transcription factor of Th17 cells) and p-STAT3 in the colon tissues. Further, peripheral blood mononuclear cells (PBMCs) were isolated from UC patients and treated with various concentrations of andrographolide (0, 10, 20 and 30 μg/ml). Andrographolide also showed inhibitory effects on the levels of proinflammatory factors, the percentages of Th17 cells and the expression of relative proteins. Similar results were obtained in lipopolysaccharide-treated normal PBMCs. These data suggested that andrographolide may inhibit Th17 immune response via STAT3 signaling. In conclusion, we demonstrated that andrographolide inhibited the activity of IL-23/IL-17 axis and down-stream pro-inflammatory factors so as to suppress inflammation response, resulting in the relieving of UC.
研究领域:
免疫学
·
药理学
·
炎症性肠病
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）