30 November 2024, Volume 44 Issue 6

  

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  Salidroside improves keloid by targeting JAG1/Notch3 signaling pathway through miR-26a-5p

  CAI Xiang, QIU Bai-yi, DUAN Yao, WANG Wei, HE Ya-nan

  2024, 44(6): 465-473.

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  To investigate the mechanism by which salidroside improves keloid on regulating JAG1/Notch3 signaling pathway through miR-26a-5p, 10 keloid patients diagnosed by histopathology in Wuhan Hospital of Traditional Chinese Medicine were enrolled as the research objects from October 2019 to January 2021. The keloid tissues removed by surgery were used in the keloid group, and the adjacent healthy skin tissues were used in the normal group. Primary keloid fibroblasts were isolated and cultured, and the cells of generation 4~8 were divided into 8 groups: control group (normal culture), salidroside low-dose group (10 μmol/L salidroside), salidroside medium-dose group (20 μmol/L salidroside), salidroside high-dose group (40 μmol/L salidroside), miR-NC group (40 μmol/L salidroside+miR-NC), miR-26a-5p inhibitor group (40 μmol/L salidroside+miR-26a-5p inhibitor), sh-RNA group (40 μmol/L salidroside+miR-26a-5p inhibitor+sh-RNA) and sh-JAG1 group (40 μmol/L salidroside+miR-26a-5p inhibitor+sh-JAG1). Cells in each group were treated with salidroside for 24 h after transfection or directly treated with the corresponding dosage of salidroside for 24 h. The relative mRNA expressions of miR-26a-5p and JAG1  in tissues and cells were quantified using qRT-PCR. Cell proliferating viability was measured by the MTT assay and cell cloning capacity was detected using the plate cloning method. Cell apoptosis was detected by FACS and cell migration and invasion were assessed by Transwell method. Luciferase reporter system was utilized to examine the targeting interaction between miR-26a-5p and JAG1. Western blotting was used to detect the expressions of tissue hyperplasia-related proteins. The results showed that, compared to that of the normal group, the expression of miR-26a-5p in keloid group was significantly decreased (P＜0.05), while the JAG1  expression significantly increased (P＜0.05). Compared to the 0 μmol/L salidroside group, 10, 20, 40, 80, and 160 μmol/L salidroside treatment inhibited the survival rate of keloid fibroblasts in a concentration-dependent manner (all with P＜0.05). To ensure both the survival of keloid fibroblasts and the effect of salidroside, subsequent experiments were conducted with salidroside concentrations of 10, 20, and 40 μmol/L (It was proved that miR-26a-5p combines to JAG1). In the three salidroside groups, compared to those of the control group, the expression of miR-26a-5p and the apoptosis rate increased dependently, while the expression of JAG1, the numbers of cell clones, cell migrations, and invasions, and the expression of α-SMA, collagen Ⅰ, collagen Ⅲ, JAG1, and Notch3 proteins decreased significantly (all with P＜0.05). Compared to those of the salidroside high-dose group and the miR-NC group, the expression of miR-26a-5p and the cell apoptosis rate in the miR-26a-5p inhibitor group reduced significantly, while the expression of JAG1, the numbers of cell clones, migration and invasion, and the expression of α-SMA, collagen Ⅰ, collagen Ⅲ, JAG1 and Notch3 proteins increased significantly (all with P＜0.05). Inhibition of JAG1  expression suppressed the effect of miR-26a-5p inhibitor on promoting cell proliferation, migration and invasion, and on inhibiting cell apoptosis. In conclusion, the expression of miR-26a-5p is down-regulated and JAG1  is up-regulated in keloid tissue, salidroside can inhibit the JAG1/Notch3 signaling pathway and the proliferation, migration and invasion of keloid fibroblasts, while promoting cell apoptosis, which in turn improves keloid by up-regulating the expression of miR-26a-5p.
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  Study on the treatment of diabetic nephropathy mice with myeloid-derived suppressor cell-produced exosomes

  LIANG Li, ZHANG Yi-ke, SHAO Shuo-yun, XIE Jing-song, ZHU Dong-wei

  2024, 44(6): 474-481.

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  Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of bone marrow-derived cells that are abundantly accumulated in patients and mouse models of diabetic nephropathy (DN). Studies have shown that MDSC-derived exosomes (EXs) have obvious immuno-suppressive functions, which may inhibit the development of inflammation in DN. This study aims to investigate the immuno-suppressive capacity of EX derived from MDSC (MDSC-EX) and its effect on DN mice. DN mice were established through high-fat diet and intra-peritoneal injection of streptozotocin (STZ). Two subsets of MDSC, polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (MO-MDSC), were isolated from DN mice spleens by magnetic-activated cell-sorting (MACS). PMN-MDSC and MO-MDSC-derived EXs were prepared from the culture supernatants of PMN-MDSCs and MO-MDSCs and FACS was used to examine their inhibitory effect on CD3⁺T cell proliferation. PMN-MDSC-EX or MO-MDSC-EX were caudal vein injected into DN mice and the effects on fasting blood glucose (FBG）, body weight (BW), total protein in urine within 24 hours (TPU), urea nitrogen in serum (UNS), creatinine in serum (CS) and glomerular filtration rate (GFR) were observed. H-E staining of kidney was observed under microscope and the protein expressions of fibronectin, collagen type Ⅳ (collagen Ⅳ), and alpha smooth muscle actin (α-SMA) in kidney tissues were measured by western blotting. The results showed the successful establishment of  DN mouse model and the isolation of PMN-MDSC-EX and MO-MDSC-EX. Compared to MO-MDSC-EX, PMN-MDSC-EX had a greater capacity to inhibit the proliferation of CD3⁺T lymphocytes (P＜0.05). Compared to MO-MDSC-EX, PMN-MDSC-EX decreased the FBG, TPU, UNS, and CS in DN mice to a larger extent, and increased the BW and GFR significantly (all with P＜0.05). Intervention with both PMN-MDSC-EX and MO-MDSC-EX significantly alleviated kidney tissue damage in DN mice. Compared to MO-MDSC-EX, PMN-MDSC-EX presented a better inhibitory effect on glomerulosclerosis in model mice. In conclusion, PMN-MDSC-EX has a stronger protective effect on DN mice than MO-MDSC-EX, which highlights a novel path to DN treatment.
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  Effects of leonurine on renal protection, Th1/Th2 balance and immune function in mice with Henoch-Schonlein purpura nephritis

  YUAN Li, REN Xian-qing, ZHANG Wei-xing

  2024, 44(6): 482-487.

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  To investigate the effect of leonurine (LEO) on Henoch-Schonlein purpura nephritis (HSPN), mice were divided into the blank control group, the model group, the low (25 mg/[kg·d]), medium (50 mg/[kg·d]) and high (100 mg/[kg·d]) doses of LEO treatment groups. The 24-hour urinary albumin (Alb) content was measured by the Bradford method and urine red blood cells were counted. The serum creatinine (Cr) and blood urea nitrogen (BUN) were measured by the automatic biochemical analyzer. Immunofluorescence staining was used to detect IgA deposition in renal tissues. The histopathological changes in renal tissues were observed by H-E staining. ELISA was performed to detect serum IL-4,  IL-10,  IFN-γ，and TNF-α. Serum circulating immune complex levels were measured by polyethylene glycol precipitation turbidimetry, and the peripheral blood Th1/Th2 ratio was measured by flow cytometry. The results showed that compared to those of the blank control group, the 24-hour urinary Alb level, urine red blood cell count, and serum levels of Cr, BUN, IL-4, IL-10, and circulating immune complex of the model group were significantly higher, whereas the serum IFN-γ, TNF-α levels, and peripheral blood Th1/Th2 ratio were significantly lower (P<0.05). Compared to those of the model group, the 24-hour urinary Alb level, urine red blood cell count, serum Cr, BUN, IL-4, IL-10, and circulating immune complex levels were significantly reduced, while the serum IFN-γ, TNF-α levels, and peripheral blood Th1/Th2 ratio were significantly increased in all the LEO treatment mice (P<0.05). In addition, the effect of LEO was dose-dependent (P<0.05). Taken together, this study suggests that LEO treatment reduces renal tissue damage and improves renal function and renal protection in HSPN mice. The underlying mechanism may be related to the promotion of Th1/Th2 balance which helps to restore normal immune function.
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  Effects of Zhilong Tongluo capsule on renal fibrosis in rats with adriamycin-induced nephropathy through the TLR4/NF-κB/TGF-β1 signaling pathway

  2024, 44(6): 488-493.

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  Aiming to explore the underlying mechanism of Zhilong Tongluo capsule on protecting rats with adriamycin(ADR)-induced nephropathy from renal fibrosis, renal fibrosis rat model was established by right side nephrectomy and multiple caudal vein injections of 3 mg/kg ADR. After successful model establishment, rats were divided into model group, Zhilong Tongluo capsule group and Zhilong Tongluo capsule+activator group, and rats without treatment were used as control. Rats in Zhilong Tongluo capsule group were gavaged with Zhilong Tongluo capsule suspension, rats in Zhilong Tongluo capsule+activator group were additionally intraperitoneally injected with LPS (5 mg/kg), and rats in blank control group and model group were treated with same volume of saline. All treatments lasted for 8 weeks. The general conditions of rats and body weight (BW) changes were recorded. The 24 h urine protein (24 h UPO), creatinine (Cr), and blood urea nitrogen (BUN) were detected by urine protein quantification kit and ELISA kit. The rats were sacrificed and then renal tissues were collected for pathological study. The fibrosis of renal tissues was observed after Masson staining. The mRNA and protein levels of TLR4, NF-κB, and TGF-β1 were measured by qRT-PCR and immunohistochemistry. Compared to that of the blank control group, BW was decreased, while the collagen proportionate area (CPA), 24 h UPO, Cr, and BUN were increased in the model group. The mRNA and protein levels of TLR4, NF-κB, and TGF-β1  were also significantly increased (all with P＜0.05). Compared to those of the model group, the BW of rats was increased, and CPA, 24 h UPO, Cr, and BUN were decreased in the Zhilong Tongluo capsule group. The mRNA and protein levels of TLR4, NF-κB, and TGF-β1  were significantly decreased (all with P＜0.05). Compared to those of the Zhilong Tongluo capsule group, the BW was decreased, CPA, 24 h UPO, Cr, and BUN were increased in the Zhilong Tongluo capsule+activator group while the mRNA and protein levels of TLR4, NF-κB, and TGF-β1  were all significantly increased (all with P＜0.05). In conclusion, Zhilong Tongluo capsule may mitigate fibrosis of renal tissues and alleviate renal damage by inhibiting the expression of the TLR4/NF-κB/TGF-β1 signaling pathway.
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  D-limonene interferes with inflammatory and immune responses in COPD mice by inhibiting the NF-κB pathway

  ZHANG Jun, SHU Ting-ting, LI Han-yong, LUO Wei-dong, FAN Yan-bo, LIU Jing, WU Di, LI Xu-cheng, CUI Jin-tao

  2024, 44(6): 494-499.

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  To investigate the effects and mechanism of D-limonene, the active ingredient of Xiefeiqingganyin, on inflammatory and immune responses in mice with chronic obstructive pulmonary disease (COPD), 36 6-week-old male C57BL/6J mice were randomly divided into three groups: control group, model group, and D-limonene group, with 12 mice in each group. The COPD model were established by administration of LPS and smoking. Mice in D-limonene group were intraperitoneally injected with 100 mg/kg D-limonene daily from the 2nd day of modeling until the 28th day. The hair, movement, and mental state of mice in each group were observed, and the body mass was recorded. Pathological changes of mouse lung tissues were observed by H-E staining. Western blotting was used to detect the expression of NF-κB and p-NF-κB protein in lung tissues, and ELISA was used to detect the levels of IL-6, TNF-α, IL-1β, and IL-10 in serum and bronchoalveolar lavage fluid (BALF). FACS was used to detect the ratio of Th17/Treg in peripheral blood. The results showed that compared to control group, mice in model group showed more severe symptoms including weight loss, listless and decadent, dull coat and hair loss, reduced activity, abnormal alveolar structure, hyperplasia of bronchial epithelium, and increased infiltration of inflammatory cells around the lung tissues. In addition, the expression of p-NF-κB protein in lung tissue, the levels of IL-6, TNF-α, and IL-1β in serum and BALF, and the ratio of Th17/Treg in peripheral blood were all significantly increased (P<0.01), while IL-10 in serum and BALF was significantly decreased (P<0.01). These changes were significantly reversed after D-limonene administration (P<0.01). Altogether, this study suggests that D-limonene has a good therapeutic effect on COPD mice by restoring the Th17/Treg balance and reducing the expressions of inflammatory factors by inhibiting NF-κB pathway. 
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  Geniposide alleviates cognitive impairment induced by Japanese encephalitis virus and the exploration of the underlying mechanism

  JIN An-song, ZHONG Lian-mei, XU Chun-yan, MA Rui-fang, TANG Shu-guang, MA Zhi-ping

  2024, 44(6): 500-505.

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  The aim of this study is to investigate the potential protective effect and the underlying mechanism of geniposide (GE) on neurologic symptoms related to cognitive impairment induced by Japanese encephalitis virus (JEV), which will provide new ideas for the treatment of Japanese encephalitis (JE). Eighty male BALB/c mice were randomly divided into control group, GE group, JEV-infected group, and GE-treated group, with 20 mice in each group. Subsequently, the 14-day survival rate of mice in each group was measured. The Morris water maze experiment was used to test the cognitive memory ability of mice in different groups. Immunofluorescence was used to detect JEV infection in the hippocampus of brain tissue. ELISA was used to detect the level of inflammatory damage and oxidative stress in the hippocampus of brain tissue. Western blotting was used to detect the protein expressions, including autophagy-related protein 5 (Atg5), B-cell lymphoma 2 (Bcl-2), Bcl2-associated X protein (Bax), p62, and microtubule-associated protein light chain 3 (LC3)-Ⅱ/ LC3-Ⅰ. The results showed that GE treatment improved the 14-day survival rate of JEV model mice. Compared to those of the JEV-infected group, the escape latency of GE-treated group was significantly shorter, platform quadrant retention time ratio and the number of platform crossing were significantly increased (P<0.01). Compared to that of the JEV-infected group, the percentage of JEV positive cells of GE-treated group was significantly decreased (P<0.01). Compared to those in the JEV-infected mice, the expressions of IL-1β, TNF-α, reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly decreased in GE-treated mice (P<0.01). The expressions of Atg5, Bax, and LC3-Ⅱ/LC3-Ⅰ in the GE-treated group were significantly lower (P<0.01), while the expressions of p62 and Bcl-2 were significantly higher (P<0.01). Taken together, this study shows that GE treatment may reduce mortality and improve learning and cognitive function in JEV-infected mice. The underlying mechanism may be the reduction of oxidative stress, inflammation, and the improvement of autophagy.
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  Analysis of gene mutation and tumor immune microenvironment characteristics in patients with SMAD4 mutant colorectal cancer

  JI Qing-hua, XU Wei, ZHANG Hu-shan, MA Jun-rui, HUANG Fei, WU Long, GUO Wei, ZHANG Ru-yi, LIANG Zheng-zi

  2024, 44(6): 506-512.

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  The study aims to investigate the mutation and tumor immune microenvironment  characteristics  of the mothers against decapentaplegic homolog 4 (SMAD4) in patients with colorectal cancer in Guizhou province. Next generation sequencing (NGS) technology, multiple immunofluorescence, and immunohistochemical techniques were used to retrospectively analyze the SMAD4 mutation data of 150 colorectal cancer patients in Guizhou and among them the tumor immune microenvironment data of 8 patients were also analyzed. The results showed that 22.00% of the patients had SMAD4 mutations and the majority of the mutations were non-synonymous mutations (66.67%). The main gene co-mutating with SMAD4 was Kirsten rat sarcoma viral oncogene homologue (KRAS, 51.52%). There was no significant difference in the tumor mutational burden (TMB) level and the PD-L1 expression level between patients with and without SMAD4 mutation (P>0.05). The proportion of microsatellite instability-high (MSI-H) in patients with and without SMAD4 mutation showed statistically significant difference (P=0.014), although the total ratio of MSI-H patients was only 6.06% (4/66). Compared to SMAD4 wild-type patients, the SMAD4 mutation patients had more M2 macrophage infiltration in tumor tissues (P=0.038). This study suggests that the increased infiltration of M2 macrophages in the tumor of SMAD4 mutation patients may contribute to worse prognosis and resistance to chemotherapy.
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  Human bone marrow mesenchymal stem cells inhibit neuronal apoptosis in vitro through the BDNF/TrKB pathway

  MA Lin-qing, ZHANG Li-mei, REN Li-yan, SUN Lu, CUI Jun-yi, ZHOU Li-hua

  2024, 44(6): 513-520.

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  This report investigated the mechanism by which human bone marrow mesenchymal stem cells (hBMSC) inhibit neuronal apoptosis in vitro. H2O2 was used as an apoptotic inducer to human neuroblastoma cells (SH-SY5Y cells) or primary cultured mouse nerve cells. The antioxidant effect of hBMSC was evaluated by co-culturing hBMSC with SH-SY5Y cells or primary cultured mouse nerve cells. Specific tyrosine receptor kinase B (TrkB) inhibitors were used to block the brain-derived neurotrophic factor (BDNF)/TrKB pathway, and the changes in the antioxidant effect of hBMSC were observed. The results showed that H2O2 induced apoptosis in SH-SY5Y cells in a concentration and time-dependent manner. Of note, SH-SY5Y cells or primary cultured mouse nerve cells co-cultured with hBMSC showed reduced apoptosis. TrkB inhibitors blocked the BDNF/TrKB pathway and partially impaired the inhibitory effect of hBMSC on apoptosis. Our results show that hBMSC exert their inhibitory effects of neuronal apoptosis through BDNF/TrKB pathway in vitro.
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  Association between 8 single nucleotide polymorphisms of HLA class Ⅱ genes and chronic HBV infection

  KONG Fan-hui, SU Ming-kuan, YANG Hong, WU Shui-qing, GUO Jian-feng, HUANG Jian-cheng

  2024, 44(6): 521-525.

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  In order to verify the association between genetic susceptibility and chronic HBV infection with HLA class Ⅱ gene and to understand the molecular mechanism of chronic HBV infection in different populations, this study screened 8 representative single nucleotide polymorphisms (SNPs) from the HLA class Ⅱ gene region, and re-analyzed the correlation between the selected SNPs and the risk of chronic HBV infection. A hospital-based case-control study was carried out with 183 chronic hepatitis B (CHB) patients as the case group and 196 HBV self-limiting infection patients as the control group. Allele typing of selected SNPs was performed using SNaPshot technology. Dominant model analysis comparing the control group and case group revealed that rs9276370 (GG+GT vs TT: P=0.013), rs7756516 (CC+CT vs TT: P=0.023), rs7453920 (AA+AG vs GG: P=0.003), rs3077 (AA+AG vs GG: P=0.028 ), rs9277535 (AA+AG vs GG: P=0.012), and rs9366816 (TT+TC vs CC: P=0.014) polymorphisms were all positively correlated with HBV self-limiting infection. The rs378352 (AA+AG vs GG: P=0.024) was a risk factor for CHB. Further haplotype analysis showed that rs9276370, rs7756516, and rs7453920 of HLA-DQ gene may exhibit haplotypes of TTG and GCA. Haplotype TTG was positively correlated with the risk of CHB, while haplotype GCA had a strong effect on clearance after HBV infection. Therefore, the results of this study suggest that HLA Ⅱ polymorphism may be associated with chronic HBV infection.
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  Research progress on TREM2-mediated immune responses in Alzheimer's disease

  CAO Jun-zheng, SONG Jun-ying, WANG Rui-fang

  2024, 44(6): 526-529.

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  The incidence of Alzheimer's disease (AD) among the elderly is extremely high. At present, the most well-known pathological changes are hyperphosphorylated tau protein entanglement and amyloid β-protein (Aβ) deposition. Remarkably, the immune inflammatory response also plays an important role in the pathogenesis of AD, and microglia plays the main role in the process of neuroinflammatory response. In brain, triggering receptor expressed on myeloid cells 2 (TREM2) is mainly expressed in microglia, which not only regulates the metabolism, survival, phagocytosis, and proliferation of microglia, but also regulates the  polarization of microglia from M1 type to M2. Over the years, growing evidence has demonstrated that TREM2-mediated  immure response leads to the improvement of  AD pathology. This article reviews the effects of TREM2 and microglia on the central nervous system and AD biogenesis.
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  Research progress of single-chain antibody fragments and their medical application

  JIAO Zi-chen, WANG Zi-hao, KUANG Yu-zhu, ZHANG Lin-bo, ZHANG Wen-hui, FU Lu

  2024, 44(6): 530-535.

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  Genetically engineered modification of antibodies generates various types of new small molecule antibodies, among which single-chain antibody (ScFv) has indisputable advantages including high specificity, high affinity, tissue permeability, excellent stability and low immunogenicity, easy preparation, and large-scale production, etc. ScFv has been widely applied in the fields of drug development and disease treatment. This review summarizes the new research progress and medical application of ScFv.
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  Research progress and trend of universal double negative T cell therapy

  YAN Qiong-yu, LU Li-ming, YANG Li-ming

  2024, 44(6): 536-541.

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  With the development of checkpoint inhibiting antibodies such as PD-1 and chimeric antigen receptor T cells (CAR-T), tumor immunotherapy has become a research hotspot. CAR-T therapy has achieved extraordinary results in antitumor treatments, especially in hematological malignancies and some autoimmune diseases, where it leads to a remarkable complete remission rate with manageable safety profile. Nevertheless, some limitations persist in autologous CAR-T therapy, including high costs, long manufacturing turn-round time, and inconvenient to use. These limitations may be overcome by utilizing allogeneic T cells obtained from healthy donors as a source of universal CAR-T production. Double negative T cells（DNT）have emerged as an allogeneic immunotherapy with no genome editing. DNT are mature T cells that comprise 1% to 10% of peripheral T cells and are defined by the expression of CD3 but not CD4, CD8, or CD56 molecules on their surface. Their TCRs are either TCRαβ or TCRγδ chains and do not bind to invariant natural killer T cells  TCR-CD1d-α-galactosylcenonide (αGalCer) tetramers. A lot of preclinical data showed that DNT possess dual abilities to kill tumor cells while simultaneously inhibiting graft-versus-host disease. DNT can be ex vivo expanded from healthy donors. Therefore, allogeneic DNT can be used as an“off-the-shelf” cellular product that is readily available for clinical applications. Genetically modified CAR-DNT have enhanced target-specific tumoricidal effects against several tumors. In this review, we will summarize the discovery of DNT, its tumoricidal mechanisms and updated clinical applications. DNT may provide a novel perspective immunotherapy for various diseases.
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  Research progress of osteoporosis mediated by intestinal microflora through the immune system

  LEI Pei-pei, MI Tian, YANG Gui-ling, WANG Qiu-ling

  2024, 44(6): 542-547.

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  The gut microbiota consists of commensal bacteria that live in the gut and have important functions including regulating host metabolism and immune homeostasis. Recently, there is evidence that the intestinal flora can regulate bone mass through the interaction of metabolites with host's endocrine and immune systems. This review summarizes the latest references at home and abroad on how intestinal flora affects host bone metabolism through the immune system.
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  Advances in basic and clinical studies of age-associated B cells

  XIAO Yi-qin-wen, CHEN Zi-wei, LI Fei, WANG Shi-xuan

  2024, 44(6): 548-554.

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  Age-associated B cell (ABC) is a new B cell subset discovered in recent years, which accumulates in spleen with age. This type of cell has unique cellular phenotypes and transcriptional features. ABC highly expresses myeloid marker CD11c and transcription factor T-bet, and differentiates and proliferates upon the stimulation of Th1 cytokines and TLR7 and/or TLR9. It can differentiate into plasma cells to produce antibodies, secrete cytokines, and present antigens. ABC plays an important role in immune aging and autoimmune disease. This review aims to introduce the phenotypic characteristics, origin, anatomical distribution, differentiation regulation mechanism, and function of ABC, clarify its role in immune aging and autoimmune diseases, and provide a new target for clinical treatment.