Transforming liquid flow fuel cells to controllable reactors for highlyefficient oxidation of 5-hydroxymethylfurfural to 2, 5-furandicarboxylic acid at low temperature

Highly-efficient oxidation of 5-hydroxymethylfurtural(HMF) to 2,5-furandicarboxylic acid(FDCA) at low temperature with air as the oxidant is still challenging.Herein,inspired by the respirato ry electron transport chain(ETC) of living cells mediated by electron carriers,we constructed artificial ETCs and transformed liquid flow fuel cells(LFFCs) to flexible reactors for efficient oxidation of HMF to produce FDCA under mild conditions.This LFFC reactor employed an electrodeposition modified nickel foam as an anode to promote HMF oxidation and(VO_(2))_(2)SO_(4) as a cathode electron carrier to facilitate the electron transfer to air.The reaction rate could be easily controlled by selecting the anode catalyst,adjusting the external loading and changing the cathodic electron carrier or oxidants.A maximal power density of 44.9 mW cm^(-2) at room temperature was achieved,while for FDCA production,short-circuit condition was preferred to achieve quick transfer of electrons.For a single batch operation with 0.1 M initial HMF,FDCA yield reached 97.1%.By fed-batch operation,FDCA concentration reached 144.5 g L^(-1) with a total yield of 96%.Ni^(2+)/Ni^(3+) redox couple was the active species mediating the electron transfer,while both experimental and DFT calculation results indicated that HMFCA pathway was the preferred reaction mechanism.

Ghrelin regulates insulin resistance by targeting insulin-like growth factor-1 receptor via miR-455-5p in hepatic cells

Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption, intracellular glycogen content, phosphorylation of PI3K and Akt stimulated by insulin, expression of miR-455-5p, as well as IGF-1R protein level were analyzed. In addition, bioinformatic analysis, dual luciferase reporter assay, miR- 455-5p mimic or inhibitor treatment was conducted to investigate the molecular mechanisms. Results: High glucose treatment upregulated miR-455-5p expression but reduced glucose consumption and glycogen content. DAG reversed the effect of high glucose on glucose metabolism, increased protein level of IGF-1R and phosphorylation of PI3K/Akt stimulated by insulin, as well as downregulated miR-455-5p expression. Bioinformatic analysis indicated IGF-1R was the target of miR-455-5p. Dual luciferase reporter assay, as well as transfection with miR-455-5p mimic/inhibitor confirmed that DAG activated IGF-1R/PI3K/Akt signaling via inhibiting miR-455-5p. Conclusion: DAG improves insulin resistance via miR-455-5p- mediated activation of IGF-1R/PI3K/Akt system, suggesting that suppression of miR-455-5p or activation of DAG may be potential targets for T2DM therapy.

Inhibitory Effect of Flavonoid Glycosides from Chlorophytum comosum on Nasopharyngeal Carcinoma 5-8F Cells and Its Mechanism

[Objectives]To study the inhibitory activity of two flavonoid glycosides isolated from Chlorophytum comosum Laxum R.Br on human nasopharyngeal carcinoma(NPC)cell line 5-8F in vitro and its mechanism.[Methods]The flavonoid glycosides were isolated and purified from the ethanol alcoholic extract of the roots of Liliaceae plant Chlorophytum comosum by silica gel column chromatography,macroporous resin column chromatography,Sephadex LH-20,and reverse column chromatography(ODS).The inhibitory activity of flavonoid glycosides on human nasopharyngeal carcinoma cells was analyzed by CCK-8 method,and the potential mechanism was preliminarily analyzed by molecular docking.[Results]Two flavonoid glycosides were identified as isovitexin 2″-0-rhamnoside and 7-2″-di-O-β-glucopyranosylisovitexin.Two flavonoid glycosides showed promising inhibitory effect on human nasopharyngeal carcinoma cell line 5-8F,with IC_(50) values of 24.8 and 27.5μmol/L,respectively.Molecular docking results showed that the potential targets of two flavonoid glycosides include CyclinD1,Bcl-2β-Catenin,ILK,TGF-β,in addition,two glycosides showed higher predicted binding affinity towards CyclinD1,which verifies the cytotoxicity of the two compounds on human nasopharyngeal carcinoma cell line 5-8F in vitro.[Conclusions]Two flavonoid glycosides are the active molecules in Chlorophytum comosum that can inhibit the proliferation of human nasopharyngeal carcinoma cells,and have the potential to be used in the research and development of anti nasopharyngeal carcinoma drugs.

Gamma-glutamyl transferase 5 overexpression in cerebrovascular endothelial cells improves brain pathology,cognition,and behavior in APP/PS1 mice

In patients with Alzheimer’s disease,gamma-glutamyl transferase 5(GGT5)expression has been observed to be downregulated in cerebrovascular endothelial cells.However,the functional role of GGT5 in the development of Alzheimer’s disease remains unclear.This study aimed to explore the effect of GGT5 on cognitive function and brain pathology in an APP/PS1 mouse model of Alzheimer’s disease,as well as the underlying mechanism.We observed a significant reduction in GGT5 expression in two in vitro models of Alzheimer’s disease(Aβ_(1-42)-treated hCMEC/D3 and bEnd.3 cells),as well as in the APP/PS1 mouse model.Additionally,injection of APP/PS1 mice with an adeno-associated virus encoding GGT5 enhanced hippocampal synaptic plasticity and mitigated cognitive deficits.Interestingly,increasing GGT5 expression in cerebrovascular endothelial cells reduced levels of both soluble and insoluble amyloid-βin the brains of APP/PS1 mice.This effect may be attributable to inhibition of the expression ofβ-site APP cleaving enzyme 1,which is mediated by nuclear factor-kappa B.Our findings demonstrate that GGT5 expression in cerebrovascular endothelial cells is inversely associated with Alzheimer’s disease pathogenesis,and that GGT5 upregulation mitigates cognitive deficits in APP/PS1 mice.These findings suggest that GGT5 expression in cerebrovascular endothelial cells is a potential therapeutic target and biomarker for Alzheimer’s disease.

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

Perilipin 5 regulates hepatic stellate cell activation and high-fat diet-induced non-alcoholic fatty liver disease

Background:Nonalcoholic fatty liver disease(NAFLD)is one of the most common chronic liver diseases globally.Hepatic stellate cells(HSCs)are the major effector cells of liver fibrosis.HSCs contain abundant lipid droplets(LDs)in their cytoplasm during quiescence.Perilipin 5(PLIN 5)is a LD surface-associated protein that plays a crucial role in lipid homeostasis.However,little is known about the role of PLIN 5 in HSC activation.Methods:PLIN 5 was overexpressed in HSCs of Sprague–Dawley rats by lentivirus transfection.At the same time,PLIN 5 gene knockout mice were constructed and fed with a high-fat diet(HFD)for 20 weeks to study the role of PLIN 5 in NAFLD.The corresponding reagent kits were used to measure TG,GSH,Caspase 3 activity,ATP level,and mitochondrial DNA copy number.Metabolomic analysis of mice liver tissue metabolism was performed based on UPLC-MS/MS.AMPK,mitochondrial function,cell proliferation,and apoptosis-related genes and proteins were detected by western blotting and qPCR.Results:Overexpression of PLIN 5 in activated HSCs led to a decrease in ATP levels in mitochondria,inhibition of cell proliferation,and a significant increase in cell apoptosis through AMPK activation.In addition,compared with the HFD-fed C57BL/6J mice,PLIN 5 knockout mice fed with HFD showed reduced liver fat deposition,decreased LD abundance and size,and reduced liver fibrosis.Conclusion:These findings highlight the unique regulatory role of PLIN 5 in HSCs and the role of PLIN 5 in the fibrosis process of NAFLD.

Exosome-Transmitted miR-224-5p Promotes Colorectal Cancer Cell Proliferation via Targeting ULK2 in p53-Dependent Manner

Objective To investigate the role and molecular mechanism of exosomal miR-224-5p in colorectal cancer(CRC).Methods The miR-224-5p expression in CRC patient tissues and cell-derived exosomes was measured by laser capture microdissection and qRT-PCR,respectively.Dual-luciferase reporter gene assay was used to determine the target gene of miR-224-5p.The protein expressions of p53 and unc-51 like kinase 2(ULK2)in CRC cells were detected by western blot.Flow cytometry was used to detect cell cycle and apoptosis.Cell proliferation was measured by CCK8 and EdU assay.Results The miR-224-5p expression was upregulated in CRC tissues and increased progressively with the rise of CRC stage.CRC cells secreted extracellular miR-224-5p mainly in an exosome-dependent manner,and then miR-224-5p could be transferred to surrounding tumor cells to regulate cell proliferation in the form of autocrine or paracrine.Moreover,ULK2 was characterized as a direct target of miR-224-5p and was downregulated in CRC tissues.Interestingly,ULK2 inhibited CRC cell proliferation in a p53-dependent manner.Furthermore,exosome-derived miR-224-5p partially reversed the proliferation regulation of ULK2 on CRC cells.Conclusion Our findings demonstrate that exosome-transmitted miR-224-5p promotes p53-dependent cell proliferation by targeting ULK2 in CRC,which may offer promising targets for CRC prevention and therapy.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

Study on the mechanism of tRNA-ValAAC-5 in promoting the proliferation and migration of hepatocellular carcinoma cells

Objective:To demonstrate the role and mechanism of tRNA-ValAAC-5 expression in hepatocellular carcinoma(HCC)cells.Methods:The expression levels of tRNA-ValAAC-5 in HCC(Hep3B,HuH7,SNU398,Hep3G2)and human hepatocellular carcinoma(THLE2,THLE3)were detected by real-time PCR.HEP3B and Hep3G2 cells were respectively transfected with tRNA-ValAAC-5-inhibitor and tRNA-ValAAC-5-NC as the inhibitor group and the NC group.Then the ability of cell proliferation was detected by CCK-8 assay and the ability of invasion and metastasis was detected by Transwell assay.The protein expression levels of p21,Matrix metalloproteinase 2(MMP2)and Matrix metalloproteinase 9(MMP9)were determined by Western blot.Results:The relative expression of tRNA-ValAAC-5 in Hep3B,HuH7,SNU398 and Hep3G2 cells were significantly higher than THLE2 and THLE3 cells,the differences were statistically significant(P<0.05).After tRNA-ValAAC-5-inhibitor transfection,the expression of tRNA-ValAAC-5 in Hep3B and Hep3G2 cells were reduced than tRNA-ValAAC-NC group.Both of the differences were statistically significant(t=36.52,27.45,P<0.001),which indicated the transfection was successful.The proliferative ability of Hep3B and Hep3G2 cells transfected with tRNA-ValAAC-5-inhibitor after 24,48,72,96 h were inhibited effectively compared with tRNA-ValAAC-5-NC group.All of the differences were statistically significant in Hep3B(t=5.25,8.23,7.33,14.16,P<0.001)and Hep3G2(t=4.25,5.11,9.39,7.59,P<0.001)cells.The number of invasion and metastasis of Hep3B and Hep3G2 cells were reduced in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,there was significant difference(t=14.01,21.85,P<0.001).The protein expression levels of P21 were lower,MMP2 and MMP9 were higher in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,the differences were statistically significant in Hep3B(t=8.96,12.80,4.652,P<0.001)cells and Hep3G2(t=15.17,22.36,12.61,P<0.001)cells.Conclusion:tRNA-ValAAC-5 can effectively promote the proliferation,invasion and metastasis of HCC,and its possible mechanism is related to regulating the expression of p21,MMP2 and MMP9.

MicroRNA-584-5p/RUNX family transcription factor 2 axis mediates hypoxia-induced osteogenic differentiation of periosteal stem cells

BACKGROUND The hypoxic environment during bone healing is important in regulating the differentiation of periosteal stem cells(PSCs)into osteoblasts or chondrocytes;however,the underlying mechanisms remain unclear.AIM To determine the effect of hypoxia on PSCs,and the expression of microRNA-584-5p(miR-584-5p)and RUNX family transcription factor 2(RUNX2)in PSCs was modulated to explore the impact of the miR-584-5p/RUNX2 axis on hypoxiainduced osteogenic differentiation of PSCs.METHODS In this study,we isolated primary mouse PSCs and stimulated them with hypoxia,and the characteristics and functional genes related to PSC osteogenic differentiation were assessed.Constructs expressing miR-584-5p and RUNX2 were established to determine PSC osteogenic differentiation.RESULTS Hypoxic stimulation induced PSC osteogenic differentiation and significantly increased calcified nodules,intracellular calcium ion levels,and alkaline phosphatase(ALP)activity in PSCs.Osteogenic differentiation-related factors such as RUNX2,bone morphogenetic protein 2,hypoxia-inducible factor 1-alpha,and ALP were upregulated;in contrast,miR-584-5p was downregulated in these cells.Furthermore,upregulation of miR-584-5p significantly inhibited RUNX2 expression and hypoxia-induced PSC osteogenic differentiation.RUNX2 was the target gene of miR-584-5p,antagonizing miR-584-5p inhibition in hypoxia-induced PSC osteogenic differentiation.CONCLUSION Our study showed that the interaction of miR-584-5p and RUNX2 could mediate PSC osteogenic differentiation induced by hypoxia.

Establishment of oxygen glucose deprivation reperfusion model of senescent SH-SY5Y cells

Obejective:To explore the establishment of an oxygen glucose deprivation/reperfusion model of senescent SH-SY5Y cells.Methods:SH-SY5Y cells were randomly divided into control(D-galactose 0 mmol/L group),D-galactose(25 mmol/L,50 mmol/L,100 mmol/L,200 mmol/L,400 mmol/L)groups,and treated with corresponding concentrations of D-galactose for 48 h.The changes of cell morphology,β-galactosidase,the cell morphology,β-galactosidase activity by microscopic observation,cell proliferation rate by EdU kit and cell survival rate by CCK-8 assay were used to determine the decaying concentration of D-galactose and to establish the senescence model.The senescent SH-SY5Y cells were randomly divided into control group(oxygen glucose deprivation without treatment group),oxygen glucose deprivation treatment(0.5 h,1 h,1.5 h,2 h)group,followed by re-glucose reoxygenation for 24 h,and CCK-8 assay for the survival rate of senescent SH-SY5Y cells.Results:There were no significant changes in cell morphology and β-gal activity in the 25 mmol/L and 50 mmol/L groups compared with the control group(P>0.05),cytosolic hypertrophy was seen in the cells of the 100 mmol/L group,chromatin fixation in the cells of the 200 mmol/L group,and massive vacuolization in the cells of the 400 mmol/L group;the positive rate ofβ-galactosidase staining in the cells of the(100-400 mmol/L)group was significantly higher compared with the control group(P<0.05),with little difference between the 100 mmol/L and 200 mmol/L groups(P>0.05);the cell proliferation ability of the(100-400 mmol/L)group was significantly decreased in a concentration-dependent manner(P<0.05);the cell survival rate was decreased in a concentration-dependent manner(P<0.05),with IC_(50) between 100 mmol/L and 200 mmol/L.The survival of senescent SH-SY5Y cells showed a time-dependent decrease in oxygen-glucose deprivation(P<0.05),with an IC_(50) close to 1 h.Conclusion:D-gal concentration of 100 mmoL/L and 48 h of cell action could establish a survival rate of about 50%of senescent SH-SY5Y cells,and oxygen glucose deprivation of senescent SH-SY5Y cells for 1 h and reperfusion for 24 h could establish an oxygen glucose deprivation/reperfusion model of senescent SH-SY5Y cells with a survival rate close to 50%.

Exosome-derived miR-146a-5p from decidual macrophages in preeclampsia inhibits the viability and invasive ability of trophoblast cells by targeting HIF1α

Objective:To investigate the effect of exosomes secreted by decidual macrophages on trophoblast cells and their molecular mechanism.Methods:The decidual tissues of patients with preeclampsia(PE)and normal-term pregnant women were collected.Macrophages were obtained by the density gradient method and then flow cell sorting,then the exosomes were extracted.The structure of the exosomes was observed by transmission electron microscope.The expression of CD63,a marker protein of the exocrine body,was detected by western blot,and the exosomes were identified.CCK-8 was used to detect the effect of exosomes on trophoblast cell viability.Transwell migration experiment was used to detect the influence on migration ability.The expression of miR-146a-5p in exosomes was detected by qPCR.The effect of exosomes on the expression of HIF1αprotein in trophoblasts was detected by western blot and detection of the binding site between miR-146a-5p and HIF1αby double luciferase reporter gene was conducted.Results:The exosomes of macrophages present a"cake"structure with a middle depression about 30-130 nm in diameter,and CD63 is highly expressed,which conforms to the characteristics of exosomes.Compared with the normal group,the exosomes of decidual macrophages in the PE group inhibited the activity and migration of trophoblast cells(P<0.001).The expression of miR-146a-5p in the exosomes of decidual macrophages in the PE decreased significantly,and after exosomes of PE decidual macrophages treating trophoblast cells,the protein expression of HIF1αin trophoblast cells was significantly increased.There are targeted binding sites between miR-146a-5p and HIF1α.Conclusion:PE decidual macrophage exosomes can inhibit the viability and migration of trophoblast cells,which may be related to the decreased expression of miR-146a-5p in exosomes,thus promoting HIF1αprotein expression of trophoblast cells.

Quercetin ameliorates oxidative stress-induced senescence in rat nucleus pulposus-derived mesenchymal stem cells via the miR-34a-5p/SIRT1 axis

BACKGROUND Intervertebral disc degeneration(IDD)is a main contributor to low back pain.Oxidative stress,which is highly associated with the progression of IDD,increases senescence of nucleus pulposus-derived mesenchymal stem cells(NPMSCs)and weakens the differentiation ability of NPMSCs in degenerated intervertebral discs(IVDs).Quercetin(Que)has been demonstrated to reduce oxidative stress in diverse degenerative diseases.AIM To investigate the role of Que in oxidative stress-induced NPMSC damage and to elucidate the underlying mechanism.METHODS In vitro,NPMSCs were isolated from rat tails.Senescence-associatedβ-galactosidase(SA-β-Gal)staining,cell cycle,reactive oxygen species(ROS),realtime quantitative polymerase chain reaction(RT-qPCR),immunofluorescence,and western blot analyses were used to evaluated the protective effects of Que.Meanwhile the relationship between miR-34a-5p and Sirtuins 1(SIRT1)was evaluated by dual-luciferase reporter assay.To explore whether Que modulates tert-butyl hydroperoxide(TBHP)-induced senescence of NPMSCs via the miR-34a-5p/SIRT1 pathway,we used adenovirus vectors to overexpress and downregulate the expression of miR-34a-5p and used SIRT1 siRNA to knockdown SIRT1 expression.In vivo,a puncture-induced rat IDD model was constructed,and X rays and histological analysis were used to assess whether Que could alleviate IDD in vivo.RESULTS We found that TBHP can cause NPMSCs senescence changes,such as reduced cell proliferation ability,increased SA-β-Gal activity,cell cycle arrest,the accumulation of ROS,and increased expression of senescence-related proteins.While abovementioned senescence indicators were significantly alleviated by Que treatment.Que decreased the expression levels of senescence-related proteins(p16,p21,and p53)and senescence-associated secreted phenotype(SASP),including IL-1β,IL-6,and MMP-13,and it increased the expression of SIRT1.In addition,the protective effects of Que on cell senescence were partially reversed by miR-34a-5p overexpression and SIRT1 knockdown.In vivo,X-ray,and histological analyses indicated that Que alleviated IDD in a punctureinduced rat model.CONCLUSION In summary,the present study provides evidence that Que reduces oxidative stress-induced senescence of NPMSCs via the miR-34a/SIRT1 signaling pathway,suggesting that Que may be a potential agent for the treatment of IDD.

非小细胞肺癌组织中lncRNA GAS5、LHPP表达与上皮间质化相关性及临床意义

目的探讨非小细胞肺癌(NSCLC)患者癌组织中长链非编码核糖核酸生长抑制特异性基因5(lncRNA GAS5)、磷酸赖氨酸磷酸组氨酸无机焦磷酸盐磷酸酶(LHPP)表达与上皮间质化(EMT)相关性及临床意义。方法收集2018年6月至2020年1月在河北北方学院附属第一医院行手术切除的NSCLC 90例患者的癌组织及癌旁组织,采用实时荧光定量聚合酶链反应检测lncRNA GAS5、LHPP和EMT相关蛋白[E-钙黏蛋白(E-Cad)、N-钙黏蛋白(N-Cad)和波形蛋白(VIM)]表达。分析NSCLC患者癌组织中lncRNA GAS5、LHPP mRNA与临床病理特征的关系,并通过Pearson相关性分析NSCLC患者癌组织中lncRNA GAS5、LHPP mRNA与EMT相关蛋白表达的相关性。采用Kaplan-Meier法绘制不同lncRNA GAS5、LHPP mRNA表达的NSCLC患者生存曲线,多因素Cox回归分析NSCLC患者预后的影响因素。结果NSCLC患者癌组织中lncRNA GAS5、LHPP mRNA、E-Cad mRNA表达低于癌旁组织,N-Cad mRNA、VIM mRNA表达高于癌旁组织,差异有统计学意义(P<0.05)。Pearson相关性分析显示,NSCLC患者癌组织中lncRNA GAS5与E-Cad mRNA表达呈正相关(r=0.724,P<0.001),与N-Cad mRNA、VIM mRNA表达呈负相关(r=-0.699、-0.689,P<0.001);lncRNA GAS5与LHPP mRNA表达呈正相关(r=0.651,P<0.001)。不同分化程度、肿瘤TNM分期、淋巴结转移的NSCLC患者癌组织中lncRNA GAS5、LHPP mRNA表达比较差异有统计学意义(P<0.05)。Kaplan-Meier生存曲线分析显示,lncRNA GAS5高表达组的3年总生存率[68.18%(30/44)]高于lncRNA GAS5低表达组的3年总生存率[36.96%(17/46)];LHPP mRNA高表达组的3年总生存率[67.39%(31/46)]高于LHPP mRNA低表达组的3年总生存率[36.36%(16/44)],差异有统计学意义(χ^(2)=10.274、10.322,P<0.05)。低分化、肿瘤TNM分期Ⅲ期、有淋巴结转移为NSCLC患者死亡的独立危险因素,lncRNA GAS5≥1.32、LHPP mRNA≥1.12为独立保护因素(P<0.05)。结论NSCLC患者癌组织中lncRNA GAS5、LHPP mRNA低表达,与EMT相关蛋白表达、分化程度、肿瘤TNM分期、淋巴结转移和预后有关,可能成为NSCLC诊治的新靶点。

拮抗CC趋化因子受体5信号诱导肿瘤细胞凋亡并调节肿瘤微环境抑制肿瘤生长

目的探索拮抗CC趋化因子受体5(CCR5)信号通路对肿瘤生长和肿瘤微环境的影响。方法采用CCK8细胞毒试验研究CCR5选择性拮抗剂Maraviroc体外对小鼠Lewis肺腺癌细胞增殖的影响,并运用流式细胞术和RT-PCR检测肿瘤细胞凋亡和Caspase 8基因的表达。然后采用免疫荧光组织化学染色法研究了Maraviroc对小鼠体内肿瘤生长和肿瘤微环境中CD4^(+)和CD8^(+)以及Foxp3^(+)细胞比例的影响。结果拮抗CCR5信号在体内外均能够抑制癌细胞的生长。体外研究发现:CCR5拮抗剂可通过增强凋亡基因Caspase 8表达而诱导肿瘤细胞凋亡。在小鼠体内,CCR5拮抗剂可明显增加肿瘤微环境中CD4^(+)和CD8^(+)细胞的浸润而减少Foxp3^(+)细胞的浸润。结论拮抗CCR5信号可能通过诱导肿瘤细胞凋亡,逆转免疫抑制性肿瘤微环境而抑制肿瘤生长。

SLC6A9对结直肠癌细胞生长和对5-FU药物敏感性的影响

目的:研究溶质载体家族6成员9(solute carrier family 6 member 9,SLC6A9)表达对结直肠癌细胞增殖、迁移和5-氟尿嘧啶(5-fluorouracil,5-FU)药物敏感性的影响。方法:TCGA数据库分析、实时荧光定量PCR和Western blot分析检测SLC6A9在结肠癌组织、正常结肠细胞系(NCM460)和结直肠癌细胞系(SW620、HCT116、HT29、Lovo和SW480)中的表达。将SCL6A9过表达质粒及阴性对照(SLC6A9 OE、Vector)转染HT29细胞,将SCL6A9小干扰RNA及阴性对照(SLC6A9 siRNA1#、siRNA2#和Scramble)转染SW620细胞。划痕愈合实验和Transwell实验检测各组细胞的迁移、侵袭能力。Western blot和细胞免疫荧光检测EMT相关蛋白E-cadherin、Vimentin的表达水平。利用CCK-8法和构建裸鼠移植瘤模型检测SLC6A9过表达对结直肠癌细胞5-FU药物敏感性的影响。结果:与正常结肠组织和NCM460细胞相比,SLC6A9在结肠癌组织和结直肠癌细胞系中低表达(均P<0.05)。SLC6A9过表达引起E-cadherin蛋白表达增加,Vimentin蛋白水平降低,抑制结直肠癌细胞的迁移、侵袭(P<0.05)。SLC6A9低表达引起E-cadherin蛋白表达降低,Vimentin蛋白水平增加,促进结直肠癌细胞的迁移、侵袭能力(P<0.05)。SLC6A9过表达提高了5-FU的药物敏感性,并使肿瘤生长缓慢,质量减轻(P<0.05)。而SLC6A9低表达降低了5-FU的药物敏感性(P<0.05)。结论:SLC6A9过表达能够抑制结直肠癌细胞的迁移、侵袭和EMT进程,并增强5-FU对结直肠癌细胞的药物敏感性。

CXCL5通过诱导血管钙化参与颈动脉斑块的形成

背景:CXC基序趋化因子5(CXC-motif chemokine 5,CXCL5)为上皮细胞衍生的中性粒细胞激活肽,研究发现其可能参与动脉病变。然而,CXCL5在血管钙化中的作用未见报道。目的:探讨CXCL5在颈动脉粥样硬化的血管钙化中的作用。方法:①细胞实验:将小鼠血管平滑肌细胞分成以下各组:成骨培养基组,Vector组(空白质粒转染到细胞中),CXCL5组(CXCL5质粒转染到细胞中),si-NC组(CXCL5阴性对照siRNA转染到细胞中),si-CXCL5组(CXCL5 siRNA转染到细胞中),Vector+LY2157299组和CXCL5+LY2157299组(细胞转染24 h后,将转化生长因子β受体1激酶抑制剂LY2157299加入细胞中)。进行茜素红染色、碱性磷酸酶染色和钙含量测定以评估血管平滑肌细胞成骨分化水平。②动物实验:48只ApoE-/-小鼠随机分成4组:Con+si-NC组、Con+si-CXCL5组、CAS+si-NC组和CAS+si-CXCL5组,前2组不造模,尾静脉注射si-NC或si-CXCL5慢病毒;后2组制备颈动脉粥样硬化模型,尾静脉注射si-NC或si-CXCL5慢病毒。采用Von Kossa染色和免疫组织化学染色评估小鼠颈动脉血管钙化以及CXCL5、转化生长因子β受体1表达情况。结果与结论:①CXCL5组细胞Runt相关转录因子2蛋白水平上调、α-平滑肌肌动蛋白水平下调,si-CXCL5组中的发现与其相反;CXCL5过表达上调了转化生长因子β受体1水平,而CXCL5敲低抑制了转化生长因子β受体1水平。②与Vector组相比,CXCL5组细胞茜素红染色的强度、碱性磷酸酶活性和钙含量显著增加(P<0.05);与si-NC组相比,si-CXCL5组上述2项指标显著降低(P<0.05);当用LY2157299抑制转化生长因子β受体1表达时,CXCL5对平滑肌细胞的成骨转化作用减弱。③与Con+si-NC组相比,CAS+si-NC组大鼠颈动脉中CXCL5蛋白表达和血管钙化面积显著增加(P<0.05);与CAS+si-NC组相比,CAS+si-CXCL5组颈动脉中上述2项指标显著降低(P<0.05)。④与Con+si-NC组相比,CAS+si-NC组大鼠颈动脉中Runt相关转录因子2蛋白表达显著增加(P<0.05)和α-平滑肌肌动蛋白表达显著降低(P<0.05);与CAS+si-NC组相比,CAS+si-CXCL5组颈动脉中上述2项指标呈相反变化(P<0.05)。⑤结果说明,CXCL5通过激活转化生长因子β受体1通路诱导血管平滑肌细胞成骨样转化,抑制CXCL5表达对于改善颈动脉粥样硬化小鼠颈动脉血管钙化是有效的。

DCLK1和ITGA5在非小细胞肺癌患者中的表达及其与临床病理特征及预后的关系研究

目的 检测非小细胞肺癌(non-small cell lung cancer,NSCLC)患者血清双皮质素样激酶1(doublecortin-like kinase 1,DCLK1)和整合素α5(integrin alpha5,ITGA5)水平并讨论二者与临床病理特征及预后的关系。方法 选取2017年6月1日至2020年7月1日在我院收治的116例NSCLC患者(NSCLC组)作为研究对象,收集同期100例健康受试者作为对照组,采用酶联免疫吸附法检测NSCLC组和对照组受试者血清中DCLK1和ITGA5的水平;根据预后情况将患者分为生存组和死亡组,ROC曲线分析血清DCLK1、ITGA5的水平对NSCLC患者3年发生死亡的预测价值,多因素Cox回归分析影响NSCLC患者发生死亡的因素。结果 NSCLC组患者血清DCLK1、ITGA5的水平高于对照组(P<0.05);不同吸烟史、TNM分期、分化程度、肿瘤直径以及淋巴结转移的NSCLC患者血清DCLK1、ITGA5的水平比较,差异有统计学意义(P<0.05);死亡组患者血清DCLK1、ITGA5的水平均高于生存组(P<0.05);血清DCLK1、ITGA5单独及联合预测患者发生死亡的AUC分别为0.895(95%CI=0.824~0.944)、0.828(95%CI=0.747~0.892)、0.926(95%CI=0.862~0.966);DCLK1、ITGA5及淋巴结转移是影响NSCLC患者发生死亡的独立危险因素(P<0.05)。结论 NSCLC患者血清DCLK1、ITGA5的水平异常升高,二者的表达水平与NSCLC患者临床病理特征和不良预后有关。

BCG-CpG-DNA佐剂人用狂犬病疫苗(MRC-5细胞)在食蟹猴体内的免疫原性

目的 评价BCG-CpG-DNA佐剂人用狂犬病疫苗(MRC-5细胞)在食蟹猴体内的免疫原性。方法 将食蟹猴随机分为对照组、3针组及4针组,经后肢肌肉免疫,1.0 mL/剂。3针组及4针组均接种BCG-CpG-DNA佐剂人用狂犬病疫苗,3针组于0 d、7 d、21 d各接种1剂,4针组于0 d、3 d、7 d、14 d各接种1剂;对照组接种市售国产同类疫苗,于0 d注射2剂、7 d及21 d各1剂。初免前(0 d)及初免后7 d、14 d、28 d、35 d、49 d、63 d后肢隐静脉采血,分离血清及外周血淋巴细胞。快速荧光灶抑制实验(Rapid Fluorescent focus Inhibition Test, RFFIT)及酶联免疫斑点检测技术(Enzyme-linked Immunospot Assay, ELISPOT)分别检测血清中抗狂犬病病毒中和抗体水平和外周血淋巴细胞中细胞因子IFN-γ、IL-2的表达水平。结果 食蟹猴血清中和抗体水平于免疫前均呈阴性(抗体效价<0.5 IU/mL),3针组和对照组在初免后7~28 d呈上升趋势,于28 d达最高,4针组在14 d达最高;在初免后28 d,对照组、3针组中和抗体水平显著高于4针组;初免后35 d,3针组中和抗体水平显著高于4针组,其余时间点同时间各组间差异均无统计学意义(P均>0.05)。3针组及4针组外周血淋巴细胞中IL-2及IFN-γ水平在7~14 d呈上升趋势,于14 d达最高,且3针组IFN-γ水平显著高于对照组,其余时间点同时间各组间差异均无统计学意义。结论 BCG-CpG-DNA佐剂人用狂犬病疫苗(MRC-5细胞)具有良好的免疫原性,具有优化狂犬病免疫接种程序的应用价值。

山柰酚对博来霉素诱导小鼠肺纤维化、MRC-5细胞胶原蛋白生成的影响

目的探讨山柰酚对博来霉素诱导小鼠肺纤维化的预防作用和对MRC-5细胞胶原蛋白生成的影响。方法将C57BL/6小鼠随机分为假手术组、模型组、吡非尼酮组(100 mg/kg)、山柰酚组(20 mg/kg),每组10只。在假手术组气管内注射生理盐水,模型组气管内注射博来霉素诱导肺纤维化模型。术后第2天开始给药,连续28 d,检测小鼠呼吸功能、肺组织羟脯氨酸水平,Massson染色观察肺组织胶原活性,免疫组化法检测肺组织TGF-β1、ɑ-SMA、Ⅰ型胶原、Ⅲ型胶原表达量。培养MRC-5细胞株,设置空白组、TGF-β1组和山柰酚20、40、80μmol/L组,分别干预48 h后,CCK-8检测山柰酚对MRC-5细胞抑制率,ELISA检测培养基中Ⅰ型胶原和Ⅲ型胶原水平。结果与模型组比较,山柰酚能改善博来霉素诱导小鼠肺纤维化后的呼吸功能,减轻肺组织胶原沉积,降低肺组织羟脯氨酸水平和TGF-β1、ɑ-SMA、Ⅰ型和Ⅲ型胶原蛋白表达(P<005,P<001)。细胞实验表明,山柰酚能抑制TGF-β1对MRC-5细胞的增殖和Ⅰ型、Ⅲ型胶原生成(P<005,P<001)。结论山柰酚能预防博来霉素诱导小鼠肺纤维化,抑制体外MRC-5细胞株增殖、胶原生成。