Background:The effect of platelet factor 4(PF4)on bone marrow mesenchymal stem cells(BMMSCs)and osteoporosis is poorly understood.Therefore,this study aimed to evaluate the effects of PF4-triggered bone destruction in...Background:The effect of platelet factor 4(PF4)on bone marrow mesenchymal stem cells(BMMSCs)and osteoporosis is poorly understood.Therefore,this study aimed to evaluate the effects of PF4-triggered bone destruction in mice and determine the underlying mechanism.Methods:First,in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry,respectively.Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S.Next,an osteoporotic mouse model was established by performing bilateral ovariectomy(OVX).Furthermore,the PF4 concentrations were obtained using enzymelinked immunosorbent assay.The bone microarchitecture of the femur was evaluated using microCT and histological analyses.Finally,the key regulators of osteogenesis and pathways were investigated using quantitative real-time polymerase chain reaction and Western blotting.Results:Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs.Furthermore,the levels of PF4 in the serum and bone marrow were generally increased,whereas bone microarchitecture deteriorated due to OVX.Moreover,in vivo mouse PF4 supplementation triggered bone deterioration of the femur.In addition,several key regulators of osteogenesis were downregulated,and the integrinα5-focal adhesion kinase-extracellular signalregulated kinase(ITGA5-FAK-ERK)pathway was inhibited due to PF4 supplementation.Conclusions:PF4 may be attributed to OVX-i nduced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5-FAK-ERK pathway.展开更多
背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
Rab5 is a GTPase protein that is involved in intracellular membrane trafficking. It functions by binding to various effector proteins and regulating cellular responses, including the formation of transport vesicles an...Rab5 is a GTPase protein that is involved in intracellular membrane trafficking. It functions by binding to various effector proteins and regulating cellular responses, including the formation of transport vesicles and their fusion with the cellular membrane. Rab5 has been reported to play an important role in the development of the zebrafish embryo;however, its role in axonal regeneration in the central nervous system remains unclear. In this study, we established a zebrafish Mauthner cell model of axonal injury using single-cell electroporation and two-photon axotomy techniques. We found that overexpression of Rab5 in single Mauthner cells promoted marked axonal regeneration and increased the number of intra-axonal transport vesicles. In contrast, treatment of zebrafish larvae with the Rab kinase inhibitor CID-1067700markedly inhibited axonal regeneration in Mauthner cells. We also found that Rab5 activated phosphatidylinositol 3-kinase(PI3K) during axonal repair of Mauthner cells and promoted the recovery of zebrafish locomotor function. Additionally, rapamycin, an inhibitor of the mechanistic target of rapamycin downstream of PI3K, markedly hindered axonal regeneration. These findings suggest that Rab5 promotes the axonal regeneration of injured zebrafish Mauthner cells by activating the PI3K signaling pathway.展开更多
BACKGROUND C-X-C chemokine receptor type 5(CXCR5)+CD8+T cells represent a unique immune subset with dual roles,functioning as cytotoxic cells in persistent viral infections while promoting B cell responses.Despite the...BACKGROUND C-X-C chemokine receptor type 5(CXCR5)+CD8+T cells represent a unique immune subset with dual roles,functioning as cytotoxic cells in persistent viral infections while promoting B cell responses.Despite their importance,the specific role of CXCR5+CD8+T cells in chronic hepatitis B(CHB),particularly during interferon-alpha(IFN-α)treatment,is not fully understood.This study aims to elucidate the relationship between CXCR5+CD8+T cells and sustained serologic response(SR)in patients undergoing 48 weeks of pegylated IFN-α(peg-IFN-α)treatment for CHB.AIM To elucidate the relationship between CXCR5+CD8+T cells and sustained SR in patients undergoing 48 weeks of peg-IFN-αtreatment for CHB.METHODS This study enrolled 60 patients with hepatitis Be antigen(HBeAg)-positive CHB undergoing 48 weeks of peg-IFN-αtreatment.Participants were assessed for eligibility based on criteria such as persistent HBsAg-positive status for at least six months,HBeAb-negative,hepatitis B virus DNA levels exceeding 2×104 copies/mL,and alanine aminotransferase(ALT)levels between 2 and 10 times the upper limit of normal.Blood samples were collected at baseline and at weeks 12,24,48,and a 24-week treatment-free follow-up(week 72)to measure serum interleukin(IL)-21 concentration via ELISA and to analyze CXCR5 and programmed death-ligand 1(PD-L1)expression on CD8+T cells by flow cytometry,CXCR5 is a chemokine receptor that directs immune cells to specific tissues,while PD-L1 is a protein that regulates immune responses by inhibiting T cell activity.RESULTS Patients with CHB exhibited significantly lower levels of circulating CXCR5+CD8+T cells compared to healthy controls(P<0.01).Notably,CXCR5+CD8+T cells were prominently expressed in patients who achieved sustained SR compared to non-SR(NSR).A significant correlation was observed between CXCR5 and PD-L1 expression(r=-0.189,P=0.002).However,there was no significant correlation between serum IL-21 levels and CXCR5+CD8+lymphocytes(r=-0.03,P=0.625)or serum ALT levels(r=0.026,P=0.678).CONCLUSION The enhanced expression of CXCR5+CD8+T cells in patients achieving HBeAg seroconversion during IFN-αtreatment suggests that these cells play a crucial role in antiviral immune responses against hepatitis B.This study highlights the potential of CXCR5+CD8+T cells as immune regulators in CHB,which may inform future therapeutic strategies to optimize antiviral treatments.展开更多
基金Beijing Natural Science Foundation,Grant/Award Number:L222145CAMS Innovation Fund for Medical Sciences,Grant/Award Number:2019-I2M-5-038+2 种基金Clinical Medicine Plus X-Young Scholars Project,Peking Universitythe Fundamental Research Funds for the Central Universities,Grant/Award Number:PKU2023LCXQ017National Natural Science Foundation of China,Grant/Award Number:81700935。
文摘Background:The effect of platelet factor 4(PF4)on bone marrow mesenchymal stem cells(BMMSCs)and osteoporosis is poorly understood.Therefore,this study aimed to evaluate the effects of PF4-triggered bone destruction in mice and determine the underlying mechanism.Methods:First,in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry,respectively.Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S.Next,an osteoporotic mouse model was established by performing bilateral ovariectomy(OVX).Furthermore,the PF4 concentrations were obtained using enzymelinked immunosorbent assay.The bone microarchitecture of the femur was evaluated using microCT and histological analyses.Finally,the key regulators of osteogenesis and pathways were investigated using quantitative real-time polymerase chain reaction and Western blotting.Results:Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs.Furthermore,the levels of PF4 in the serum and bone marrow were generally increased,whereas bone microarchitecture deteriorated due to OVX.Moreover,in vivo mouse PF4 supplementation triggered bone deterioration of the femur.In addition,several key regulators of osteogenesis were downregulated,and the integrinα5-focal adhesion kinase-extracellular signalregulated kinase(ITGA5-FAK-ERK)pathway was inhibited due to PF4 supplementation.Conclusions:PF4 may be attributed to OVX-i nduced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5-FAK-ERK pathway.
基金supported by the Research Funds of the Center for Advanced Interdisciplinary Science and Biomedicine of IHM,No.QYZD20220002the National Natural Science Foundation of China,No.82071357a grant from the Ministry of Science and Technology of China,No.2019YFA0405600 (all to BH)。
文摘Rab5 is a GTPase protein that is involved in intracellular membrane trafficking. It functions by binding to various effector proteins and regulating cellular responses, including the formation of transport vesicles and their fusion with the cellular membrane. Rab5 has been reported to play an important role in the development of the zebrafish embryo;however, its role in axonal regeneration in the central nervous system remains unclear. In this study, we established a zebrafish Mauthner cell model of axonal injury using single-cell electroporation and two-photon axotomy techniques. We found that overexpression of Rab5 in single Mauthner cells promoted marked axonal regeneration and increased the number of intra-axonal transport vesicles. In contrast, treatment of zebrafish larvae with the Rab kinase inhibitor CID-1067700markedly inhibited axonal regeneration in Mauthner cells. We also found that Rab5 activated phosphatidylinositol 3-kinase(PI3K) during axonal repair of Mauthner cells and promoted the recovery of zebrafish locomotor function. Additionally, rapamycin, an inhibitor of the mechanistic target of rapamycin downstream of PI3K, markedly hindered axonal regeneration. These findings suggest that Rab5 promotes the axonal regeneration of injured zebrafish Mauthner cells by activating the PI3K signaling pathway.
基金Supported by Changsha Science and Technology Program,No.kq2022397Natural Science Foundation of Hunan Province(Departmental Joint Fund),No.2023JJ60440+2 种基金Research Program of Health Commission of Hunan Province,No.202303088786Clinical Medical Research Center for Viral Hepatitis of Hunan Province,No.2023SK4009the Scientific Research Program of FuRong Laboratory,No.2023SK2108.
文摘BACKGROUND C-X-C chemokine receptor type 5(CXCR5)+CD8+T cells represent a unique immune subset with dual roles,functioning as cytotoxic cells in persistent viral infections while promoting B cell responses.Despite their importance,the specific role of CXCR5+CD8+T cells in chronic hepatitis B(CHB),particularly during interferon-alpha(IFN-α)treatment,is not fully understood.This study aims to elucidate the relationship between CXCR5+CD8+T cells and sustained serologic response(SR)in patients undergoing 48 weeks of pegylated IFN-α(peg-IFN-α)treatment for CHB.AIM To elucidate the relationship between CXCR5+CD8+T cells and sustained SR in patients undergoing 48 weeks of peg-IFN-αtreatment for CHB.METHODS This study enrolled 60 patients with hepatitis Be antigen(HBeAg)-positive CHB undergoing 48 weeks of peg-IFN-αtreatment.Participants were assessed for eligibility based on criteria such as persistent HBsAg-positive status for at least six months,HBeAb-negative,hepatitis B virus DNA levels exceeding 2×104 copies/mL,and alanine aminotransferase(ALT)levels between 2 and 10 times the upper limit of normal.Blood samples were collected at baseline and at weeks 12,24,48,and a 24-week treatment-free follow-up(week 72)to measure serum interleukin(IL)-21 concentration via ELISA and to analyze CXCR5 and programmed death-ligand 1(PD-L1)expression on CD8+T cells by flow cytometry,CXCR5 is a chemokine receptor that directs immune cells to specific tissues,while PD-L1 is a protein that regulates immune responses by inhibiting T cell activity.RESULTS Patients with CHB exhibited significantly lower levels of circulating CXCR5+CD8+T cells compared to healthy controls(P<0.01).Notably,CXCR5+CD8+T cells were prominently expressed in patients who achieved sustained SR compared to non-SR(NSR).A significant correlation was observed between CXCR5 and PD-L1 expression(r=-0.189,P=0.002).However,there was no significant correlation between serum IL-21 levels and CXCR5+CD8+lymphocytes(r=-0.03,P=0.625)or serum ALT levels(r=0.026,P=0.678).CONCLUSION The enhanced expression of CXCR5+CD8+T cells in patients achieving HBeAg seroconversion during IFN-αtreatment suggests that these cells play a crucial role in antiviral immune responses against hepatitis B.This study highlights the potential of CXCR5+CD8+T cells as immune regulators in CHB,which may inform future therapeutic strategies to optimize antiviral treatments.
