Increasing Accumulation Level of Foreign Protein in Transgenic Plants Through Protein Targeting

Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.

Osteopontin promotes gastric cancer progression via phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

BACKGROUND Gastric cancer(GC)is one of the most common malignant tumors.Osteopontin(OPN)is thought to be closely related to the occurrence,metastasis and prognosis of many types of tumors.AIM To investigate the effects of OPN on the proliferation,invasion and migration of GC cells and its possible mechanism.METHODS The mRNA and protein expression of OPN in the GC cells were analyzed by realtime quantitative-reverse transcription polymerase chain reaction and western blotting,and observe the effect of varying degree expression OPN on the proliferation and other behaviors of GC.Next,the effects of OPN knockdown on GC cells migration and invasion were examined.The short hairpin RNA(shRNA)and negative control shRNA targeting OPN-shRNA were transfected into the cells according to the manufacturer’s instructions.Non transfected cells were classified as control in the identical transfecting process.24 h after RNA transfection cell proliferation activity was detected by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay,and cell invasiveness and migration were detected by Trans well assay.Meanwhile,the expression of protein kinase B(AKT),matrix metalloproteinase 2(MMP-2)and vascular endothelial growth factor(VEGF)in the human GC cell lines was detected by reverse transcription polymerase chain reaction and western blotting.RESULTS The results of this study revealed that OPN mRNA and protein expression levels were highly expressed in SGC-7901 cells.OPN knockdown by specific shRNA noticeably reduced the capabilities of proliferation,invasion and migration of SGC-7901 cells.Moreover,in the experiments of investigating the underlying mechanism,results showed that OPN knockdown could down-regulated the expression of MMP-2 and VEGF,it also decreased the phosphorylation of AKT.Meanwhile,the protein expression levels of MMP-2,VEGF and phosphorylated AKT was noticeable lower than that in control group in the GC cells after they were added to phosphatidylinositol-3-kinase(PI3K)inhibitor(LY294002).CONCLUSION These results suggested that OPN though PI3K/AKT/mammalian target of rapamycin signal pathway to upregulate MMP-2 and VEGF expression,which contribute SGC-7901 cells to proliferation,invasion and migration.Thus,our results demonstrate that OPN may serve as a novel prognostic biomarkers as well as a potential therapeutic targets for GC.

Identification of Prognosis-Related Genes and Key Target Genes for Pancreatic Cancer: A Bioinformatics Analysis

Objective: The mortality and morbidity rates associated with pancreatic cancer (PaCa) are extremely high. Various studies have demonstrated that pancreatic cancer will be the fourth cancer-related death by 2030, raising more concern for scholars to find effective methods to prevent and treat in order to improve the pancreatic cancer outcome. Using bioinformatic analysis, this study aims to pinpoint key genes that could impact PaCa patients’ prognosis and could be used as therapeutic targets. Methods: The TCGA and GEO datasets were integratively analyzed to identify prognosis-related differentially expressed genes. Next, the STRING database was used to develop PPI networks, and the MCODE and CytoNCA Cytoscape in Cytoscape were used to screen for critical genes. Through CytoNCA, three kinds of topology analysis were considered (degree, betweenness, and eigenvector). Essential genes were confirmed as potential target treatment through Go function and pathways enrichment analysis, a developed predictive risk model based on multivariate analysis, and the establishment of nomograms using the clinical information. Results: Overall, the GSE183795 and TCGA datasets associated 1311 and 2244 genes with pancreatic cancer prognosis, respectively. We identified 132 genes that were present in both datasets. The PPI network analysis using, the centrality analysis approach with the CytoNCA plug-in, showed that CDK2, PLK1, CCNB1, and TOP2A ranked in the top 5% across all three metrics. The independent analysis of a risk model revealed that the four key genes had a Hazard Ratio (HR) > 1. The monogram showed the predictive risk model and individual patient survival predictions were accurate. The results indicate that the effect of the selected vital genes was significant and that they could be used as biomarkers to predict a patient’s outcome and as possible target therapy in patients with pancreatic cancer. GO function and pathway analysis demonstrated that crucial genes might affect the P53 signaling pathway and FoxO signaling pathway, through which Meiotic nuclear division and cell cycle may have a significant function in essential genes affecting the outcome of patients who have pancreatic cancer. Conclusions: This study suggests that CDK2, CCNB1, PLK1 and TOP2A are four key genes that have a significant influence on PaCa migration and proliferation. CDK2, CCNB1, PLK1, and TOP2A can be used as potential PaCa prognostic biomarkers and therapeutic targets. However, experimental validation is necessary to confirm these predictions. Our study comes into contributions to the development of personalized target therapy for pancreatic cancer patients.

Mitochondrial uncoupling protein 2 and pancreatic cancer:A new potential target therapy

Overall 5-years survival of pancreatic cancer patients is nearly 5%,making this cancer type one of the most lethal neoplasia.Furthermore,the incidence rate of pancreatic cancer has a growing trend that determines a constant increase in the number of deceases caused by this pathology.The poor prognosis of pancreatic cancer is mainly caused by delayed diagnosis,early metastasis of tumor,and resistance to almost all tested cytotoxic drugs.In this respect,the identification of novel potential targets for new and efficient therapies should be strongly encouraged in order to improve the clinical management of pancreatic cancer.Some studies have shown that the mitochondrial uncoupling protein 2(UCP2) is over-expressed in pancreatic cancer as compared to adjacent normal tissues.In addition,recent discoveries established a key role of UCP2 in protecting cancer cells from an excessive production of mitochondrial superoxide ions and in the promotion of cancer cell metabolic reprogramming,including aerobic glycolysis stimulation,promotion of cancer progression.These observations together with the demonstration that UCP2 repression can synergize with standard chemotherapy to inhibit pancreatic cancer cell growth provide the molecular rationale to consider UCP2 as a potential therapeutic target for pancreatic cancer.In this editorial,recent advances describing the relationship between cancer development and mitochondrial UCP2 activity are critically provided.

Xihuang pills induce apoptosis in hepatocellular carcinoma by suppressing phosphoinositide 3-kinase/protein kinase- B/mechanistic target of rapamycin pathway

BACKGROUND The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin(PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills(XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma(HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHPassociated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway.AIM To confirm the effect of XHP on HCC and the possible mechanisms involved.METHODS The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS). Cellbased experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP(0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay.Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction(RT-qPCR), respectively.Third, Western blotting and RT–qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway.Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed.RESULTS The following 12 compounds were identified in XHP using high-resolution mass spectrometry:Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose-and timedependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract(0.625mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins(e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights.CONCLUSION XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3.Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

Host-targeting-motif Harbored Secretary Proteins in Genome of Plant Pathogenic Fungus Botrytis cinerea

According to our previous study, saprophytic fungi Botrytis cinerea contained 579 predicted secretary proteins. Among them, we found that 122 of these proteins contained the highly conserved pathogenic-related host-targeting-motif RxLx within 100 residues adjacent to the signal peptide cleavage site. According to PEDNAT and COG of the GenBank database, the functions of this motif containing proteins included metabolism modification and cell secretion. We blasted them in GenBank and found 47.54% had highly conserved homologues in other species, among them 74.1% had putative functional domains. This suggests these proteins are presumably ancient and vertically transmitted within the species. Many of these domains belonged to proteins which played roles in the pathogenic process of other kinds of pathogens and some had already been proved to be pathogenic secretary proteins of Botrytis cinerea. So we postulated that proteins contained host-targeting-motif RxLx were candidates participating in the pathogenesis of Botrytis cinerea.

Targeting amyloid precursor protein shuttling and processing-long before amyloid beta formation

Targeting early steps in amyloid-beta production：Alzheimer’s disease（AD）has a long history as the＂amyloid deposit＂disorder.Many disorders are now known to be caused by proteinβ-sheet misfolding and aggregation（e.g.,Parkinson’s disease：α-synuclein;Huntington’s disease：Huntingtin;

Bone morphogenetic protein signaling：a promising target for white matter protection in perinatal brain injury

Prematurely born newborns,as well as those born at term,may suffer from several types of brain injury including hypoxic-ischemic injury,intracranial hemorrhage,both intraventricular and parenchymal,and injury that is the consequence of intrauterine growth restriction（IUGR）.Injury of all types can impact the motor and cognitive abilities of survivors.The mechanisms leading to disability are not completely understood.

Searching for Novel Targets to Control Wheat Head Blight Disease—I-Protein Identification, 3D Modeling and Virtual Screening

Fusarium head blight (FHB) is a destructive disease of wheat and other cereals. FHB occurs in Europe, North America and around the world causing significant losses in production and endangers human and animal health. In this article, we provide the strategic steps for the specific target selection for the phytopathogen system wheat-Fusarium graminearum. The economic impact of FHB leads to the need for innovation. Currently used fungicides have been shown to be effective over the years, but recently cereal infecting Fusaria have developed resistance. Our work presents a new perspective on target selection to allow the development of new fungicides. We developed an innovative approach combining both genomic analysis and molecular modeling to increase the discovery for new chemical compounds with both safety and low environmental impact. Our protein targets selection revealed 13 candidates with high specificity, essentiality and potentially assayable with a favorable accessibility to drug activity. Among them, three proteins: trichodiene synthase, endoglucanase-5 and ERG6 were selected for deeper structural analyses to identify new putative fungicides. Overall, the bioinformatics filtering for novel protein targets applied for agricultural purposes is a response to the demand for chemical crop protection. The availability of the genome, secretome and PHI-base allowed the enrichment of the search that combined experimental data in planta. The homology modeling and molecular dynamics simulations allowed the acquisition of three robust and stable conformers. From this step, approximately ten thousand compounds have been virtually screened against three candidates. Forty-five top-ranked compounds were selected from docking results as presenting better interactions and energy at the binding pockets and no toxicity. These compounds may act as inhibitors and lead to the development of new fungicides.

Effects of Dietary Soy Protein Concentrate on Growth, Digestive Enzymes Activities and Target of Rapamycin Signaling Pathway Regulation in Juvenile Soft-Shelled Turtle, <i>Pelodiscus sinensis</i>

Soft-shelled turtle, Pelodiscus sinensis is important aquatic species in China, and searching for alternatives protein resources to fish meal (FM)-based feeds in feed has become urgent and important for its sustainability development. The present study was conducted to assess the effects of dietary soy protein concentrate (SPC) on growth, digestive enzymes and target of rapamycin (TOR) signaling pathway of juvenile P. sinensis (4.56 ± 0.09 g). SPC was applied to replace FM protein at 0%, 15%, 30% and 60% (designated as T0, T15, T30 and T60, respectively), and each diet was fed to triplicate groups. The results showed that there was no significant difference in growth performance and feed utilization except of the turtles fed with T60 diet, of which showed poorer daily weight gain and feed conversion rate. The pepsin/trypsin and Na+-K+ ATP-ase activities decreased dramatically when SPC level increased, and lipase activities in liver and intestinal tract also showed decline tendency. However, amylase activities were unaffected. No significant differences were observed in TOR, S6K1 and 4E-BP1 genes mRNA expression level of TOR signaling pathway among the treatments. However, the relative phosphorylated level of these proteins decreased significantly when SPC level increased. The present study indicated that high SPC substitution level would suppress digestive enzymes and TOR signaling pathway proteins phosphorylated level and eventually result in growth reduction of P. sinensis.

鳖甲煎丸对肝癌大鼠黏着斑激酶/雷帕霉素靶蛋白通路的影响

目的:探讨鳖甲煎丸对肝癌模型大鼠肝组织病理特征及FAK/mTORC1通路的影响。方法:HepG2(1×10^(7))细胞注射于大鼠右腋皮下,建立肝癌模型,分为模型组、5-氟尿嘧啶组、鳖甲煎丸低剂量组、鳖甲煎丸高剂量组,另设正常组。各药物组给予相应药物灌胃,持续给药4周,正常组、模型组给予等体积的0.9%氯化钠溶液。实验结束后,测定肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、肝癌FAK、mTORC1水平、肝癌VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白水平。结果:与正常组比较,模型组肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白、肝癌FAK、mTORC1 mRNA和蛋白表达升高(均P<0.05)。与模型组比较,5-氟尿嘧啶组、鳖甲煎丸低剂量组、鳖甲煎丸高剂量组肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白、肝癌FAK、mTORC1 mRNA和蛋白表达降低(均P<0.05)。鳖甲煎丸高剂量组肝癌组织重量、肝癌组织体积、肝癌组织抑瘤率、VEGFR-2、VEGF、IL-4、IL-8、TNF-α蛋白、肝癌FAK、mTORC1 mRNA和蛋白表达低于鳖甲煎丸低剂量组(均P<0.05)。结论:鳖甲煎丸对大鼠肝癌具有明显抑制作用,能减轻大鼠肝癌所致的病理损伤,其机制可能与鳖甲煎丸抑制肝癌FAK、mTORC1表达,诱导肝癌细胞凋亡,降低炎症反应有关。

基于NOD2介导的AMPK/mTOR信号通路探讨宫颈癌细胞恶性行为的机制

目的 基于核苷酸结合寡聚化结构域受体2(NOD2)介导的AMP活化蛋白激酶(AMPK)/雷帕霉素靶蛋白(mTOR)信号通路探讨宫颈癌(CC)细胞恶性行为的机制。方法 生物信息学分析确定NOD2在CC组织中的表达。将靶向NOD2(shNOD2)、shRNAs阴性对照(shNC)以及NOD2过表达(NOD2)质粒和载体(Vec)转染CC细胞。通过CCK-8测定、集落形成和Transwell细胞侵袭测定来确定NOD2对CC细胞生长的影响。通过高通量RNA测序(RNA-Seq)进行转录组分析。Western blot试验检测细胞系中NOD2、AMPK/mTOR信号通路和自噬蛋白的表达。24只雌性BALB/c裸鼠随机分为4组,每组6只:载体组(Vec组)、NOD2过表达组(NOD2组)、shNC组和shNOD2组。构建小鼠远处转移模型,监测肺转移的荧光强度,计数肺转移结节的数量。结果 在线数据库分析显示,NOD2在CC组织中表达明显高于正常组织,并且不同分期的CC中NOD2的mRNA表达差异有统计学意义(P<0.05)。此外,NOD2的高表达与较差的总生存期和无病生存期相关(P<0.05)。NOD2过表达对CC细胞增殖、集落形成、迁移和侵袭具有促进作用,而NOD2敲低则相反。与体外结果一致,在转移的小鼠尾静脉注射模型中,NOD2组CC细胞的肺定殖、肺转移灶较Vec组增加(P<0.05),而shNOD2组CC细胞的肺定殖、肺转移灶较shNC组减少(P<0.05)。RNA-Seq结果显示NOD2表达与AMPK信号激活、mTOR信号抑制、自噬调节途径激活和自噬体形成显著相关。与shNC组相比,shNOD2组磷酸化AMPK、LC3蛋白表达水平减少(P<0.05),磷酸化mTOR、p62蛋白表达水平增加(P<0.05);与Vec组相比,NOD2组LC3、AMPK蛋白表达水平增加(P<0.05),磷酸化mTOR、p62蛋白表达水平减少(P<0.05)。与shNC组相比,shNOD2组GFP-mRFP-LC3的点积累减少(P<0.05);与Vec组相比,GFP-mRFP-LC3的点积累增加(P<0.05)。结论 NOD2可能通过AMPK/mTOR信号促进CC增殖、迁移和侵袭,其作用机制部分涉及自噬激活。

Predicted essential proteins of Plasmodium falciparum for potential drug targets

Objective:To identify novel drug targets for treatment of Plasmodium falciparum.Methods: Local BT.ASTP were used to find the proteins non-homologous to human essential proteins as novel drug targets.Functional domains of novel drug targets were identified by InterPro and Pfam.3D structures of potential drug targets were predicated by the SWISS-MODEL workspace. Ligands and ligand-binding sites of the proteins were searched by Ef-seek.Results:Three essential proteins were identified that might be considered as potential drug targets.AAN37254.1 belonged to 1-deoxy-D-xylulose 5-phosphate reductoisomerase,CAD50499.1 belonged to chorismale synthase,CAD51220.1 belonged to FAD binging 3 family,but the function of CAD51220.1 was unknown.The 3D structures,ligands and ligand-binding sites of AAM37254.1 and CAD50499.1 were successfully predicated.Conclusions:Two of these potential drug targets are key enzymes in 2-C-methyl-d-erythritol 4-phosphate pathway and shikimate pathway, which are absent in humans,so these two essential proteins are good potential drug targets.The function and 3D structures of CAD50499.1 is still unknown,it still need further study.

下调HMGB2表达对肝癌LM3细胞上皮-间质转化的抑制作用及其AKT/mTOR信号通路机制

目的:探讨下调肝癌细胞中高迁移率族框蛋白2 (HMGB2)表达对肝癌细胞生物学行为及上皮-间质转化(EMT)进程的影响,并阐明其作用机制。方法:对数生长期的人肝癌LM3细胞分为阴性对照组和HMGB2 RNA干扰组(HMGB2 siRNA组),分别以Lipofectamin 2000为载体转染无关序列的RNA寡核苷酸(RNA oligo)和敲除HMGB2序列的RNA oligo。采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测2组细胞中HMGB2 mRNA和蛋白表达水平,分别采用细胞划痕实验和Transwell小室实验检测2组细胞的迁移和侵袭能力,采用Western blotting法检测2组细胞中E-钙黏蛋白(E-cadherin)、 N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达水平。结果:与阴性对照组比较,HMGB2 siRNA组细胞中HMGB2 mRNA和蛋白表达水平均明显降低(P<0.05),HMGB2 siRNA组细胞划痕愈合率明显降低(P<0.01),侵袭细胞数明显减少(P<0.01),细胞中E-cadherin蛋白表达水平明显升高(P<0.01),N-cadherin、Vimentin、mTOR、AKT和磷酸化AKT (p-AKT)蛋白表达水平明显降低(P<0.05或P<0.01)。结论:下调HMGB2的表达可降低肝癌LM3细胞迁移和侵袭能力并抑制EMT,其作用机制可能与参与调节AKT/mTOR通路相关蛋白表达有关。

淫羊藿苷调控mTOR/Akt/CREB通路对高糖诱导的足细胞自噬及凋亡的影响

目的 探讨淫羊藿苷对高糖诱导的足细胞自噬、凋亡及哺乳动物雷帕霉素靶蛋白(mTOR)/丝氨酸苏氨酸蛋白激酶(Akt)/环磷酸腺苷反应元件结合蛋白(CREB)通路的影响。方法 将小鼠足细胞MPC5分为5组:正常对照组(5.5 mmol·L^(-1)葡萄糖)、高糖组(30 mmol·L^(-1)葡萄糖)、淫羊藿苷组(30 mmol·L^(-1)葡萄糖+5μmol·L^(-1)淫羊藿苷)、GDC-0349组(30 mmol·L^(-1)葡萄糖+50μmol·L^(-1)GDC-0349)、淫羊藿苷+GDC-0349组(30 mmol·L^(-1)葡萄糖+5μmol·L^(-1)淫羊藿苷+50μmol·L^(-1)GDC-0349)。培养48 h后,噻唑蓝法检测MPC5细胞活力;吖啶橙染色观察MPC5细胞自噬情况;流式细胞术检测MPC5细胞凋亡;蛋白印迹法检测MPC5细胞自噬[微管相关蛋白1轻链3(LC3)Ⅱ、LC3Ⅰ、自噬相关蛋白(Beclin-1)]、凋亡[Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)]和mTOR/Akt/CREB通路相关蛋白的表达。结果 与正常对照组比较,高糖组MPC5细胞活力、Bcl-2、磷酸化mTOR(p-mTOR)/mTOR、磷酸化Akt(p-Akt)/Akt、磷酸化CREB(p-CREB)/CREB蛋白表达水平显著降低(P<0.05),自噬能力增强,自噬体表现出橙色荧光,细胞凋亡率、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bax蛋白表达水平显著升高(P<0.05)。与高糖组比较,淫羊藿苷组MPC5细胞活力、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bcl-2、p-mTOR/mTOR、p-Akt/Akt、p-CREB/CREB蛋白表达水平显著升高,自噬能力进一步增强,自噬体数量增多,自噬体呈现出砖红色荧光(P<0.05),细胞凋亡率、Bax蛋白表达水平显著降低(P<0.05);GDC-0349组MPC5细胞活力、LC3Ⅱ/LC3Ⅰ、Beclin-1、Bcl-2、p-mTOR/mTOR、p-Akt/Akt、p-CREB/CREB蛋白表达水平显著降低,自噬能力减弱,自噬体数量减少,自噬体表现出橙色荧光(P<0.05),细胞凋亡率、Bax蛋白表达水平显著升高(P<0.05);淫羊藿苷+GDC-0349可逆转淫羊藿苷对高糖诱导MPC5细胞的作用效果(P<0.05)。结论 淫羊藿苷通过激活mTOR/Akt/CREB通路促进高糖诱导的足细胞自噬抑制细胞凋亡。

间变性淋巴瘤激酶在治疗非小细胞肺癌中的作用及潜在医学应用

在非小细胞肺癌(non-small cell lung cancer,NSCLC)的治疗过程中,随着靶向表皮生长因子受体剂(epidermal growth factor receptor,EGFR)抑制剂的使用,不少患者出现了耐药,耐药的主要原因有EGFR位点的突变,旁路激活等原因。在旁路激活中主要有间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)等通路的激活。最近有权威研究发现ALK与免疫治疗有密切的关系。该文从ALK的结构和生理功能,ALK的小分子抑制剂,ALK的生物学功能及其与NSCLC相关的应用现状进行综述,并展望了未来的发展方向,从而使ALK更好地应用于NSCLC的治疗。

蛋白激酶mTOR调控心肌细胞衰老机制的研究进展

人口老龄化导致心血管疾病的发病率持续增高,机体衰老和心肌细胞衰老常相伴而行。心肌细胞衰老的特征主要表现为氧化应激增加、细胞周期活动停滞、代谢紊乱等,其主要与细胞自噬、线粒体功能障碍、端粒缩短以及慢性炎症等途径有关。在这些导致心肌细胞衰老的途径中,常伴有蛋白激酶的异常表达和功能障碍。蛋白激酶mTOR可通过激活一系列级联反应参与衰老相关的心血管疾病的发生和发展。现就mTOR调节心肌细胞衰老的机制进行讨论并总结,提示mTOR未来可能成为预防或治疗心肌细胞衰老的重要靶点。

橙皮苷对慢性应激抑郁模型大鼠海马组织Akt/mTOR信号通路调节机制的研究

目的:通过构建慢性应激抑郁模型大鼠模型,探究橙皮苷是否通过调控蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白(mTOR),参与影响慢性应激抑郁模型大鼠病情。方法:将72只大鼠随机分为6组:健康组、慢性应激抑郁组、橙皮苷高剂量组、橙皮苷低剂量组、氟西汀组、橙皮苷^(+)Akt抑制组,每组12只。构建慢性应激抑郁模型大鼠模型,糖水偏好实验、强迫游泳实验、旷场实验观察各组大鼠行为学情况;HE染色、Tunel染色观察各组大鼠海马组织病理学变化及细胞凋亡情况;检测各组大鼠海马组织皮质酮(CORT)、5-羟色胺(5-HT)、IL-6含量;Western blot检测海马组织Akt、mTOR、p-Akt、p-mTOR表达水平。结果:健康组大鼠海马组织完整;与健康组相比,慢性应激抑郁组大鼠海马组织损伤明显,强迫游泳不动时间、凋亡指数、CORT、IL-6含量显著升高,糖水偏好度、跨格次数、站立次数、存活神经元数、5-HT含量、p-Akt/Akt、p-mTOR/mTOR显著降低(P<0.05);与慢性应激抑郁组相比,氟西汀组、橙皮苷高剂量组、橙皮苷低剂量组大鼠海马组织逐渐恢复,强迫游泳不动时间、凋亡指数、CORT、IL-6含量显著降低,糖水偏好度、跨格次数、站立次数、存活神经元数、5-HT含量、p-Akt/Akt、p-mTOR/mTOR显著升高(P<0.05);与橙皮苷高剂量组相比,橙皮苷低剂量组、橙皮苷^(+)Akt抑制组大鼠海马组织恢复缓慢,强迫游泳不动时间、凋亡指数、CORT、IL-6含量显著升高,糖水偏好度、跨格次数、站立次数、存活神经元数、5-HT含量、p-Akt/Akt、p-mTOR/mTOR显著降低(P<0.05);橙皮苷高剂量组与氟西汀组相比,各项指标差异无统计学意义(P>0.05)。结论:橙皮苷通过调控Akt/mTOR通路,激活Akt、mTOR,缓解神经细胞凋亡,改善抑郁大鼠的神经功能和行为学功能,从而治疗慢性应激抑郁。

基于“肾脑相济”理论探讨艾灸对阿尔茨海默病大鼠海马AMPK/mTOR信号通路的影响

目的观察艾灸对阿尔茨海默病(Alzheimer’s disease,AD)大鼠腺苷酸活化蛋白激酶(adenosine 5-monophosphate activated protein kinase,AMPK)/雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路相关递质表达的影响,探讨艾灸治疗AD的作用机制。方法将SD大鼠按照随机数字表法分为正常组8只、模型组32只,采取侧脑室注射β淀粉样蛋白(amyloidβ-protein,Aβ)_(25-35)的方法建立大鼠AD模型。将模型复制成功的大鼠随机分为模型组、药物组、艾灸组,每组8只。艾灸组大鼠用艾条灸“百会”“肾俞”“三阴交”,每次15 min,同时按3 mg/kg灌胃蒸馏水;药物组大鼠按3 mg/kg灌胃盐酸多奈哌齐;对照组及模型组大鼠按3 mg/kg灌胃蒸馏水。采用Morris水迷宫法检测大鼠行为学表现,苏木精—伊红染色法观察大鼠海马病理组织改变,Western blot法检测大鼠海马磷酸化雷帕霉素靶蛋白(phosphorylated mammalian target of rapamycin,p-mTOR)、核糖体蛋白S6激酶(ribosomal protein S6 kinase p70,P70S6K)、自噬相关基因5(autophagy-related gene 5,ATG5)、磷酸化腺苷酸活化蛋白激酶(phosphorylated adenosine 5-monophosphate activated protein kinase,p-AMPK)、微管相关蛋白1轻链3B(microtubule associated protein light chain 3B,LC3B)-Ⅱ/LC3B-Ⅰ的表达水平。结果苏木精—伊红染色结果表明,模型组海马神经元萎缩明显,与模型组比较,药物组和艾灸组海马神经元形态及分化程度均有明显改善。与正常组比较,模型组大鼠的逃避潜伏期显著延长(P<0.05),p-mTOR及P70S6K表达水平均显著升高(P<0.05),ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著降低(P<0.05)。与模型组比较,药物组和艾灸组大鼠的逃避潜伏期均显著缩短(P<0.05),p-mTOR及P70S6K表达水平均显著下降(P<0.05),ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著上升(P<0.05)。与药物组比较,艾灸组大鼠逃避潜伏期显著缩短(P<0.05);p-mTOR及P70S6K表达水平显著降低(P<0.05),ATG5、LC3B-Ⅱ/LC3B-Ⅰ、p-AMPK表达水平均显著上升(P<0.05)。结论艾灸能够调控AMPK/mTOR信号通路,诱导细胞自噬,阻断脑内Aβ表达,从而改善认知功能。

牛乳和鸡蛋中致敏原物质的检测

牛乳、鸡蛋及乳、蛋制品富含蛋白质、维生素和矿物质等,是人们饮食结构中的常见食物,在为人类提供丰富营养素的同时,也容易引起部分人群的过敏。针对目前过敏发生情况和过敏原检测技术的发展现状,该文简述了牛乳和鸡蛋中的主要过敏蛋白、酶等,并介绍了过敏原常见检测方法,比较各检测方法的优缺点,为牛乳和鸡蛋过敏原的检测提供理论依据和指导。