Astrocyte-neuron communication mediated by the Notch signaling pathway:focusing on glutamate transport and synaptic plasticity

Maintaining glutamate homeostasis after hypoxic ischemia is important for synaptic function and neural cell activity,and regulation of glutamate transport between astrocyte and neuron is one of the important modalities for reducing glutamate accumulation.However,further research is needed to investigate the dynamic changes in and molecular mechanisms of glutamate transport and the effects of glutamate transport on synapses.The aim of this study was to investigate the regulatory mechanisms underlying Notch pathway mediation of glutamate transport and synaptic plasticity.In this study,Yorkshire neonatal pigs(male,age 3 days,weight 1.0–1.5 kg,n=48)were randomly divided into control(sham surgery group)and five hypoxic ischemia subgroups,according to different recovery time,which were then further subdivided into subgroups treated with dimethyl sulfoxide or a Notch pathway inhibitor(N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester).Once the model was established,immunohistochemistry,immunofluorescence staining,and western blot analyses of Notch pathway-related proteins,synaptophysin,and glutamate transporter were performed.Moreover,synapse microstructure was observed by transmission electron microscopy.At the early stage(6–12 hours after hypoxic ischemia)of hypoxic ischemic injury,expression of glutamate transporter excitatory amino acid transporter-2 and synaptophysin was downregulated,the number of synaptic vesicles was reduced,and synaptic swelling was observed;at 12–24 hours after hypoxic ischemia,the Notch pathway was activated,excitatory amino acid transporter-2 and synaptophysin expression was increased,and the number of synaptic vesicles was slightly increased.Excitatory amino acid transporter-2 and synaptophysin expression decreased after treatment with the Notch pathway inhibitor.This suggests that glutamate transport in astrocytes-neurons after hypoxic ischemic injury is regulated by the Notch pathway and affects vesicle release and synaptic plasticity through the expression of synaptophysin.

Activation of the Notch signaling pathway promotes neurovascular repair after traumatic brain injury

The Notch signaling pathway plays a key role in angiogenesis and endothelial cell formation, but it remains unclear whether it is involved in vascular repair by endothelial progenitor cells after traumatic brain injury. Therefore, in the present study, we controlled the Notch signaling pathway using overexpression and knockdown constructs. Activation of the Notch signaling pathway by Notch1 or Jagged1 overexpression enhanced the migration, invasiveness and angiogenic ability of endothelial progenitor cells. Suppression of the Notch signaling pathway with Notch1 or Jagged1 si RNAs reduced the migratory capacity, invasiveness and angiogenic ability of endothelial progenitor cells. Activation of the Notch signaling pathway in vivo in a rat model of mild traumatic brain injury promoted neurovascular repair. These findings suggest that the activation of the Notch signaling pathway promotes blood vessel formation and tissue repair after brain trauma.

Effect of the Notch signaling pathway on retinal ganglion cells and its neuroprotection in rats with acute ocular hypertension

AIM： To explore the effect of the Notch signaling pathway on retinal ganglion cells（RGCs） and optic nerve in rats with acute ocular hypertension（OH）.METHODS： Totally 48 Sprague-Dawley（SD） rats were included, among which 36 rats were selected to establish acute OH models. OH rats received a single intravitreal injection of 2 μL phosphate buffered solution（PBS） and another group of OH rats received a single intravitreal injection of 10 μmol/L γ-secretase inhibitor（DAPT）. Quantitative real-time polymerase chain reaction（qPCR） and Western blot assay were adopted to determine the mRNA level of Notch and the protein levels of Notch, Bcl-2, Bax, caspase-3, and growth-associated protein 43（GAP-43）. The RGC apoptosis conditions were assessed by TUNEL staining.RESULTS： The OH rats and PBS-injected rats had increased expression levels of Notch1, Bax, caspase-3, and GAP-43, decreased expression levels of Bcl-2, and increased RGC apoptosis, with severer macular edema and RGCs more loosely aligned, when compared with the normal rats. The DAPT-treated rats displayed increased expression levels of Notch1, Bax, caspase-3, and GAP-43, decreased expression levels of Bcl-2, and increased RGC apoptosis, in comparison with the OH rats and PBSinjected rats. RGCs were hardly observed and macular edema became severe in the DAPT-treated rat.CONCLUSION： The Notch signaling pathway may suppress the apoptosis of retinal ganglion cells and enhances the regeneration of the damaged optic nerves in rats with acute OH.

Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via Angiogenesis through Endothelial Progenitor Cells and Notch1 Signaling Pathway

Protective effect of catalpol on myocardium was studied in relation to endothelial progenitor cells, Notch1 signaling pathway and angiogenesis in rats with isoprenaline (INN)-induced acute myocardial infarcts. To analyze the pathological status and impact of catalpol on the rats, 3 weeks after intragastric gavage, the animals were verified for myocardial infarcts with electrocardiogram and measured for enzyme activity of lactate dehydrogenase (LDH), malondialdehyde (MDA), creatine kinase (CK) and superoxide dismutase (SOD) in myocardium, and further analyzed using HE and TTC staining, as well as visual examination of infarct area. Flow cytometry study of endothelial progenitor cells (EPCs) indicated that the EPCs were mobilized during infarction. The roles of Notch1 signaling pathway in angiogenesis of the infracted animals were studied using immunohistochemistry analysis of RBPjκ and Western blot analysis of Notch1 and Jagged1. Our results obtained from the rats treated with catalpol, positive drug and control showed that catalpol could protect rats from infarction probably by mobilization of EPCs and activation of Notch1 signaling pathway.

Role of the Notch Signaling Pathway in Fibrosis of Denervated Skeletal Muscle

In order to investigate the role of the Notch signaling pathway in skeletal muscle fibrosis after nerve injury, 60 Sprague-Dawley rats were selected and divided randomly into a control and two experimental groups. Group A served as controls without any treatment. Rats in groups B were injected intraperitoneally with 0.2 mL PBS and those in group C were injected intraperitoneally with 0.2 mL PBS+100 ymol/L, 0.2 mL N-[N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT, a gamma-secretase inhibitor that suppresses Notch signaling) respectively, on postoperative days 1, 3, 7, 10, and 14 in a model of denervation-induced skeletal muscle fibrosis by right sciatic nerve transection. Five rats from each group were euthanized on postoperative days 1, 7, 14, and 28 to collect the right gastrocnemii, and hematoxylin and eosin (HE) staining, immunohistochemistry test, real-time PCR, and Western blotting were performed to assess connective tissue hyperplasia and fibroblast density as well as expression of Notch 1, Jagged 1, and Notch downstream molecules Hes 1 and collagen I (COL I) on day 28. There was no significant difference in HE-stained fibroblast density between group B and C on postoperative day 1. However, fibroblast density was significantly higher in group B than in group C on postoperative days 7, 14, and 28. Notch 1, Jagged 1, Hes 1, and COL I proteins in the gastrocnemius were expressed at very low levels in group A but at high levels in group B. Expression levels of these proteins were significantly lower in group C than in group B (P<0.05), but they were higher in group C than in group A (P<0.05) on postoperative day 28. We are led to conclude that locking the Notch signaling pathway inhibits fibrosis progression of denervated skeletal muscle. Thus, it may be a new approach for treatment of fibrosis of denervated skeletal muscle.

Acupuncture and moxibustion for the prevention of contrast induced nephropathy in diabetic rats by inhibiting Notch signaling pathway

Objective Apoptosis is recognized as an important mechanism in contrast-induced nephropathy(CIN).Acupuncture and moxibustion,the auxiliary treatment in China,are effective interventions for cell apoptosis in many ischemic diseases.In our previous study,we found acupuncture and moxibustion could prevent CIN.The objective of this research is to study the mechanism of acupuncture and moxibustion on tubular epithelial cell apoptosis in diabetic CIN rats.

Effect of Tripterine on Notch Signaling Pathway in IgA Nephropathy Rats

The objective of the study was to investigate the effect of tripterine on the Notch signaling pathway in renal tissue of IgA nephropathy rats.SD male rats were divided into the control group,IgA nephropathy model group,benazepril group,1 mg/kg/day tripterine intervention group,and 10 mg/kg/day tripterine intervention group according to the random number table method,with 10 rats in each group.The urinary sediment and 24-h urinary protein quantity were detected by conventional methods.The expressions of Notch1,Jagged1,Hes1,and Hey1 in renal tissue of rats were detected by real-time fluorescent quantitative polymerase chain reaction.IgA nephropathy model was successfully established.The hematuria and proteinuria in model group were higher than those of control group(P<0.05).The expressions of Notch1,Jagged1,Hes1,and hey1 in kidney tissue of IgA nephropathy rats were significantly increased(P<0.05).Compared with the model group,hematuria and proteinuria in the tripterine intervention group were alleviated.The expressions of Notch1,Jagged1,Hes1,and Hey1 in rat renal tissue were decreased(P<0.05).Moreover,the expressions of Notch1,Jagged1,Hes1,and Hey1 in renal tissue of rats in 10 mg/kg/day tripterine intervention group were decreased(P<0.05).Tripterine can decrease the levels of hematuria and proteinuria in IgA nephropathy rats.The expression of the Notch signaling pathway in IgA nephropathy rats is increased by the down-regulation of tripterine,suggesting that tripterine has a certain therapeutic effect on IgA nephropathy rat.Moreover,its role may be realized through this signal pathway so as to provide a new mentality for the diagnosis and treatment of IgA nephropathy.

Research progress of Notch signaling pathway and biological behavior of tumor

As an important signal transduction pathway between cells, Notch signaling pathway plays a very important role in cell recognition, proliferation, differentiation ,and apoptosis. At the same time, more and more related studies show that abnormal activation of Notch signaling pathway plays an important role in the occurrence and development of a variety of malignant tumors, and has become a hot topic in the field of tumor research. Instead of focusing on the relationship between Notch signaling pathway and various organ tumors or the relationship between Notch signaling pathway and tumor single regulatory factors, this paper focuses on the role of Notch signaling pathway by summing up and summarizing the role of the signal pathway. A series of biological behaviors of the tumor, such as angiogenesis, invasion, and metastasis, involved in Notch signaling pathway, are reviewed in this paper, as well as the recent advances in the regulation of tumor biological behavior, such as angiogenesis, invasion, metastasis and so on.

Notch1/Jagged1信号对人肝癌SMMC7721细胞增殖和侵袭的影响及其作用机制

目的研究激活的Notch 1信号系统对人肝癌SMMC7721细胞增殖和侵袭力的调控,并初步探讨其可能机制。方法体外培养人肝癌SMMC 7721细胞,RT-PCR技术检测细胞中Notch信号系统的表达情况,向人SMMC 7721细胞中分别加入Notch信号通路的激活剂Jagged 1蛋白(激活剂组)、抑制剂γ-分泌酶抑制剂DAPT(抑制剂组),空白对照组加PBS缓冲液。MTT法和软琼脂集落形成试验检测各组细胞增殖的情况,Transwell小室法观察细胞侵袭的能力,RT-PCR检测各组细胞中Hes-1、Bcl-2及Snail基因的表达。结果SMMC 7721细胞中存在Notch 1/Jagged 1信号系统的表达,培养12、24、36、48 h后,激活剂组、抑制剂组与空白对照组的吸光度值比较,差异均有统计学意义(P<0.01)。激活剂组、空白对照组和抑制剂组的细胞侵袭数分别为43.8±6.8、64.6±5.6和90.0±6.9(P<0.05)。激活剂组细胞中Hes-1基因的表达增强,Bcl-2及Snail基因的表达显著降低;相反,抑制剂组细胞的Hes-1基因表达降低,Bcl-2及Snail基因的表达显著增强(P<0.05)。结论 Notch 1/Jagged 1信号通路在人肝癌SMMC 7721细胞的增殖和侵袭中发挥重要的调控作用,其机制初步认为可能与下调抗凋亡基因Bcl-2以及肿瘤转移相关基因Snail的表达有关。

Notch配体Delta-like-1和Jagged-1 mRNA在骨髓增生异常综合征患者骨髓间充质干细胞中表达研究

本研究检测骨髓增生异常综合征(MDS)患者骨髓间充质干细胞Notch信号通路配体Delta-like-1和Jagged-1 mRNA表达水平,探讨其与M DS发病的关系。采用实时荧光定量聚合酶链反应检测38例M DS患者和16例正常对照者骨髓间充质干细胞中Delta-like-1和Jagged-1的基因表达水平。结果表明,MDS患者的Delta-like-1和Jagged-1 mRNA表达水平较正常对照组均明显升高(P<0.05)。在WHO分组中,RA/RARS、RCMD及RAEB组Delta-like-1的表达量均高于正常对照组(P<0.05),RARB组Jagged-1与正常对照组差异有显著性(P<0.05)。Delta-like-1表达与骨髓原始细胞比例有显著相关性(r=0.502,P<0.05)。伴染色体异常M DS组Deltalike-1和Jagged-1 mRNA表达水平较无染色体异常M DS组明显升高(P<0.05)。在IPSS分组中,较高危组Deltalike-1表达显著高于较低危组(P<0.01),而Jagged-1表达在两组间无统计学差异(P>0.05)。结论:M SC中Delta-like-1和Jagged-1高表达可能参与了M DS的发病过程。

Jagged/Notch信号通路在铜负荷大鼠肝纤维化发生过程中的作用

目的探讨Jagged/Notch信号通路在铜负荷大鼠肝纤维化发生发展中的作用。方法雄性SD大鼠36只随机分为对照组、模型组,模型组按造模后不同时间分为8、12、16、20、24周5个亚组,每组6只。模型组喂饲含1.5g/kg硫酸铜的粉状饲料和0.185%硫酸铜去离子水,观察大鼠一般情况,全自动生化分析仪测定各组大鼠肝组织血清丙氨酸氨基转移酶(ALT)、血清天冬氨酸氨基转移酶(AST)、透明质酸(HA)、层粘蛋白(LN)、Ⅲ型前胶原(PCⅢ)水平;比色法测定大鼠肝脏羟脯氨酸(Hyp)水平;HE、Masson染色法观察大鼠肝脏病理学改变;实时荧光定量PCR法检测TGF-β1、Notch1、Notch3、Hes1、Hes5mRNA表达情况;Western blot检测Jagged1、Notch3、Hes1蛋白表达情况,免疫组化方法检测Hes1、a-SMA表达情况。结果与正常组相比,模型组大鼠肝组织ALT、AST、HA水平自造模第8周开始升高,PCⅢ、LN、Hyp水平自第12周开始升高,差异有统计学意义(P<0.05),肝组织炎症及纤维化程度进行性加重,TGF-β1、Notch3、Hes1mRNA及Jagged1、Notch3、Hes1蛋白表达显著升高,差异有统计学意义(P<0.05);Hes1蛋白在模型组纤维化区域大量表达,与肌成纤维细胞标志蛋白a-SMA表达区域高度重合,两者表达量呈同步增加关系(r=0.952,P<0.05)。结论 Jagged1/Notch3/Hes1信号通路可能参与铜负荷大鼠肝纤维化的发生与发展,并与肝纤维化的严重程度密切相关。

Effect of spinal cord extracts after spinal cord injury on proliferation of rat embryonic neural stem cells and Notch signal pathway in vitro

Objective:To investigate the effect of the spinal cord extracts(SCE)after spinal cord injuries(SCIs)on the proliferation of rat embryonic neural stem cells(NSCs)and the expressions of mRNA of Notch1 as well as of Hes1 in this process in vitro.Methods:The experiment was conducted in 4 different mediums:NSCs+PBS(Group A-blank control group),NSCs+SCE with healthy SD rats(Croup B-normal control group),NSCs+SCE with SD rats receiving sham-operation treatment(Croup C-sham-operation group)and NSCs+SCE with SCIs rats(Group D-paraplegic group).Proliferative abilities of 4 different groups were analyzed by MTT chromatometry after co-culture for 1,2,3,4 and 5 d,respectively.The expressions of Notch 1 and Hes1 mRNA were also detected with RT-PCR after co-culture for 24 and 48 h,respectively.Results:After co-culture for 1,2,3,4 and 5 d respectively,the MTT values of group D were significantly higher than those of group A,group B and group C(P<0.05).However,there were no significantly differences regarding MTT values between group A,group B and group C after co-culture for 1,2,3,4 and 5 d,respectively(P>0.05).Both the expressions of Notch1 and Hes1 mRNA of group D were significantly higher than those of other 3 groups after co-culture for 24 h and 48 h as well(P<0.05).But there was no difference oin expressions of Notch1 and Hes1 mRNA among group A,group B and group C after co-culture for 24 h and 48 h(P>0.05).There was no difference in expressions of Notch1and Hes1 mRNA between 24 h and 48 h treatment in group D.Conclusions:SCE could promote the proliferation of NSCs.It is demonstrated that the microenvironment of SCI may promote the proliferation of NSCs.Besides,SCE could increase the expression of Notch1 and Hes1 mRNA of NSC.It can be concluded that the Notch signaling pathway activation is one of the mechanisms that locally injured microenvironment contributes to the proliferation of ENSC after SCIs.This process may be performed by up-regulating the expressions of Notch1 and Hes1 gene.

Notch信号通路——对健康和疾病的机械论观点

The Notch signaling pathway is evolutionarily conserved across metazoan species and plays key roles in many physiological processes.The Notch receptor is activated by two families of canonical ligands(Deltalike and Serrate/Jagged)where both ligands and receptors are single-pass transmembrane proteins usually with large extracellular domains,relative to their intracellular portions.Upon interaction of the core binding regions,presented on opposing cell surfaces,formation of the receptor/ligand complex initiates force-mediated proteolysis,ultimately releasing the transcriptionally-active Notch intracellular domain.This review focuses on structural features of the extracellular receptor/ligand complex,the role of posttranslational modifications in tuning this complex,the contribution of the cell membrane to ligand function,and insights from acquired and genetic diseases.

Notch signalling pathway in development of cholangiocarcinoma

Cholangiocarcinoma(CCA)comprises of extra-hepatic cholangiocarcinoma and intrahepatic cholangiocarcinoma cancers as a result of inflammation of epithelium cell lining of the bile duct.The incidence rate is increasing dramatically worldwide with highest rates in Eastern and South Asian regions.Major risk factors involve chronic damage and inflammation of bile duct epithelium from primary sclerosing cholangitis,chronic hepatitis virus infection,gallstones and liver fluke infection.Various genetic variants have also been identified and as CCA develops on the background of biliary inflammation,diverse range of molecular mechanisms are involved in its progression.Among these,the Notch signalling pathway acts as a major driver of cholangiocarcinogenesis and its components(receptors,ligands and downstream signalling molecules)represent a promising therapeutic targets.Gamma-Secretase Inhibitors have been recognized in inhibiting the Notch pathway efficiently.A comprehensive knowledge of the molecular pathways activated by the Notch signalling cascade as well as its functional crosstalk with other signalling pathways provide better approach in developing innovative therapies against CCA.

Effects of the Nocth signaling pathway on expression of inflammatory factors and transforming growth factors in diabetic foot ulcers

Objective:persistent hyperinflammation is an important reason for the development of diabetic foot ulcer.Notch signaling is an important signaling pathway involved in the inflammatory response and cell proliferation in diabetic foot ulcer rats.This paper aims to explore the effect of Notch signaling on inflammatory factors,chemokines and growth factors through the intervention of Notch signaling in diabetic foot ulcer rats.Methods:the experimental model was made by using high-fat feed combined with streptozotocin(STZ)to cause diabetes,and the experimental model of diabetic foot ulcer was established by constant temperature and constant pressure scald apparatus.The normal ulcer model was used as a control.The intervention controls of the experimental model included normal saline,western medicine growth factor,Notch agonist Jagged1,Notch signaling inhibitor ly-411575,and the intervention of traditional Chinese medicine Zizhu ointment for 7 days.Serum il-1,il-6,TNF-radiation,and il-17 were detected by ELISA.Real-time PCR was used to detect the inflammatory factors,chemokines,and growth factors associated with Notch signaling in wound tissues:tnf-uum,il-1,il-6,il-17,interleukin-8,ip-10,McP-1,TGF-uum,TGF-livelihood.Results:serum levels of il-1,il-6,TNF-radiation and il-17 in diabetic foot ulcer rats were significantly higher than that in normal ulcer rats.The contents of il-1,il-6,TNF-radiation and il-17a in ly-411575 group and Zizhu ointment group were significantly reduced.Real-time PCR results of wound tissue showed that the levels of inflammatory cytokines il-1,il-6,TNF-radiation,il-17 and chemokines ip-10,il-8 and McP-1 in the wound tissue of diabetic foot ulcer rat model were significantly higher than that of normal ulcer model,and the levels of growth factor TGF-exposure were lower than that of normal ulcer model.LY-411575 significantly reduced il-1,il-6,TNF-maxima,il-17,and the chemokines ip-10,il-8,and McP-1 in diabetic foot ulcer rats,and reduced the expression of TGF-,TGF-earth.Jagged1 can increase the expression of TGF--,TGF---,suggesting that inhibition of the Notch signaling pathway can reduce the expression of the inflammatory factors il-1,il-6,TNF--,il-17a,il-8,and the growth factors TGF--,TGF---.Zizhu ointment can reduce the levels of il-1,il-6,TNF-benand,il-17,and the chemokines ip-10,il-8,and McP-1 on the wound surface of diabetic foot rats,and improve the expression of TGF-benand TGF-SUNS.Ly-411575 inhibited the expression of TGF-bento and TGF-promoting of Zizhu ointment.Conclusion:the expression of inflammatory cytokines and chemokines was higher and the expression of growth factors was lower in diabetic foot ulcer rats than in normal ulcer rats.Inhibition of Notch signaling pathway can reduce the expression of inflammatory factors,chemokines and growth factors in experimental model rats,and Notch signaling pathway can promote inflammation and cell proliferation.Zizhu ointment can reduce the levels of inflammatory cytokines and chemokines in diabetic foot ulcer rats,improve the expression of growth factors,and reduce wound inflammation,which may be related to the inhibition of Nocth signal expression.

Regulation of osteoprotegerin expression by Notch signaling in human oral squamous cell carcinoma cell line

Objective: To investigate the influence of Notch signaling on osteoprotegerin(OPG)expression in a human oral squamous cell carcinoma cell line.Methods: Activation of Notch signaling was performed by seeding cells on Jagged1 immobilized surfaces. In other experiments, a g-secretase inhibitor was added to the culture medium to inhibit intracellular Notch signaling. OPG m RNA and protein were determined by real-time PCR and ELISA, respectively. Finally, publicly available microarray database analysis was performed using connection up- or down-regulation expression analysis of microarrays software.Results: Jagged1-treatment of HSC-4 cells enhanced HES1 and HEY1 m RNA expression, confirming the intracellular activation of Notch signaling. OPG m RNA and protein levels were significantly suppressed upon Jagged1 treatment. Correspondingly, HSC-4 cells treated with a g-secretase inhibitor resulted in a significant reduction of HES1 and HEY1 m RNA levels, and a marked increase in OPG protein expression was observed.These results implied that Notch signaling regulated OPG expression in HSC-4 cells.However, Jagged1 did not alter OPG expression in another human oral squamous cell carcinoma cell line(HSC-5) or a human head and neck squamous cell carcinoma cell line(HN22).Conclusions: Notch signaling regulated OPG expression in an HSC-4 cell line and this mechanism could be cell line specific.

可溶性Jagged1对大鼠肺动脉高压的抑制作用

目的 Notch在肺动脉高压形成过程中起到重要作用,本文拟探讨可溶性Jagged1(sJag1)对MCT诱导的大鼠肺动脉高压的影响。方法用MCT诱导SD大鼠形成肺动脉高压,分别用载体和sJag1复合体在肺动脉高压诱发开始时加以治疗,测量治疗后肺动脉压和血管中层厚度比例,以及notch1和Jagged1蛋白的表达,并检测血管平滑肌细胞的增生和凋亡情况。结果与载体治疗组相比,sJag1治疗可以通过抑制血管平滑肌细胞增生并促进其凋亡,从而明显缓解肺动脉高压和减轻肺血管中层厚度。结论 sJag1对MCT诱导的大鼠肺动脉高压有明显的抑制作用。

苦参碱含药血清对WB-F344细胞形态、增殖和Jagged1信号分子表达的影响

目的研究苦参碱含药血清对大鼠肝卵圆细胞系WB-F344细胞形态、增殖及Jagged1信号分子表达的影响,探讨苦参碱对肝干细胞诱导分化的作用机制。方法用不同浓度苦参碱含药血清(5%、10%、20%)作用于WB-F344细胞,以未用苦参碱含药血清处理的WB-F344细胞为空白对照组,观察细胞形态学改变,MTT法检测细胞增殖情况,免疫细胞化学法观察ALB、Jagged1蛋白表达变化,RT-PCR观察Jagged1、Hes1mRNA表达的变化。结果经苦参碱含药血清干预后,WB-F344细胞形态发生明显改变:药物组细胞较对照组细胞体积增大,核缩小,核浆比减小,培养48 h大部分细胞呈多边形。MTT结果显示,5%、10%、20%浓度的苦参碱含药血清24、48、72 h均能不同程度抑制WB-F344增殖,且存在时间、剂量依赖性。免疫细胞化学结果显示,空白对照组WB-F344细胞表达Jagged1蛋白,不表达ALB蛋白,经苦参碱含药血清处理后Jagged1蛋白表达显著下降(P<0.01),ALB蛋白表达显著升高(P<0.01)。RT-PCR结果显示,经苦参碱含药血清处理后Jagged1mRNA、Hes1mRNA表达显著下降(P<0.01)。结论苦参碱含药血清可诱导WB-F344细胞向肝细胞特性方向分化,其作用机制与调节Notch信号通路的关键分子有关。

Brucine Inhibits Bone Metastasis of Breast Cancer Cells by Suppressing Jagged1/Notch1 Signaling Pathways

Objective： To examine the effects of brucine on the invasion, migration and bone resorption of receptor activator of nuclear factor-kappa B ligand（RANKL）-induced osteoclastogenesis. Methods： The osteoclastogenesis model was builded by co-culturing human breast tumor MDA-MB-231 and mouse RAW264.7 macrophages cells. RANKL（50 ng/m L） and macrophage-colony stimulating factor（50 ng/m L） were added to this system, followed by treatment with brucine（0.02, 0.04 and 0.08 mmol/L）, or 10 μmol/L zoledronic acid as positive control. The migration and bone resorption were measured by transwell assay and in vitro bone resorption assay. The protein expressions of Jagged1 and Notch1 were investigated by Western blot. The expressions of transforming growth factor-β1（TGF-β1）, nuclear factor-kappa B（NF-κB） and Hes1 were determined by enzyme-linked immunosorbent assay. Results： Compared with the model group, brucine led to a dose-dependent decrease on migration of MDA-MB-231 cells, inhibited RANKL-induced osteoclastogenesis and bone resorption of RAW264.7 cells（P 〈0.01）. Furthermore, brucine decreased the protein levels of Jagged1 and Notch1 in MDA-MB-231 cells and RAW264.7 cells co-cultured system as well as the expressions of TGF-β1, NF-κB and Hes1（P〈0.05 or P〈0.01）. Conclusion： Brucine may inhibit osteoclastogenesis by suppressing Jagged1/Notch1 signaling pathways.

人Jagged-1蛋白在真核细胞中的表达及稳定株的建立

Jagged-1蛋白属于Notch信号通路的配体之一,而Notch信号通路是介导细胞和细胞之间接触的主要信号通路之一,在造血微环境中调节造血细胞增殖与分化的过程中起重要作用。为研究Notch信号通路中受体与配体结合后的生物学功能及Notch信号通路在骨髓基质细胞影响细胞耐药的作用机制,构建过表达Jagged-1蛋白的NIH-3T3细胞株。构建含Jagged-1全编码区基因的pEGFP-IRES2-Jagged-1真核表达载体。结果表明,重组质粒转染哺乳动物细胞NIH-3T3后,Western blot分析证实转染后NIH-3T3细胞高效表达Jagged-1蛋白;经过选择性培养基筛选及有限稀释法挑取单克隆细胞后,流式细胞术(FCM)分析证实建立起过表达Jagged-1蛋白的单克隆细胞模型,即NIH-3T3-pEGFP-IRES2-Jagged-1细胞株。结论:本研究成功构建了Jagged-1真核表达载体,并建立了稳定转染的单克隆细胞株。过表达Jagged-1蛋白的NIH-3T3单克隆细胞株的构建为我们研究骨髓基质细胞相关的细胞耐药的作用机制并为发现新的药物作用靶点提供条件。