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Sample preparation and separation is a very crucial and complicated process in proteomics research.Based on the actual condition of university and students,this article explored the way to improve the teaching quality of sample preparation and separation in proteomics course for medical post-graduates aiming at cultivating and enhancing the students' scientific thought,making them have a better understanding of the rules,technologies,strategies in sample preparation and separation procedure.
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Urine provides an alternative to blood plasma as a potential source of disease biomarkers. Exosomes was separated by ultracentrifuge at 200000 g in normal persons and non-small cell lung cancer (NSCLC) patients′ urine. For proteomic analysis of urinary exosome, 1D sodium dodecylsulfonate-polyacrylate gel electrophoresis(SDS-PAGE) was carries out and cut the gel 31 kDa-20 kDa bands in normal group and disease group′s. These gel blocks were subjected to in-gel trypsinization, and the extracted peptides were analyzed HPLC-CHIP-MS/MS. Approximately 24 unique proteins were identified in the UniProtKB/SWISS-PORT. The difference expression proteins were found in urinary exosome from NSCLC patients, including three fragment of the immunoglobulin kappa, two kinds of Ras related proteins, glutathione S-transferase A2, serum amyloid P-component precursor and phosphatidylethanolamine-binding protein 1.
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Aim To screen the express-altered proteins before or after effect of Ecdysterone on HepG_2 cell model of insulin resistance by the strategy of comparative proteomics, which may approach new proves exploring the target of sensitizer.Methods HepG_2 cells were incubated with 5×10~(-7) mol·L~(-1) insulin for 16 h, and glucose consumption was determined. After treatment, the insulin resistant cells were incubated with 10~(-5) mol·L~(-1) Ecdysterone for 24 h.Then glucose consumption contents were determined. The proteins of two groups before and after treatment with Ecdysterone were extracted by lysis buffers. The express-altered proteins were screened by 2-DE technique.Some of them were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Results 53 express-altered protein spots of insulin resistant HepG_2 cells before and after treated by Ecdysterone were screened by 2-DE technique,in which 35 ones were up-regulated and the others down-regulated, 6 spots of which were analyzed by MALDI-TOF-MS mass spectrometry and MS-Fit database.Conclusion The target of Ecdysterone as a sensitizer involves many proteins and kinases which correlate insulin resistant. These results lay a foundation for further studies on the function of these target proteins.
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HEK293 or HeLa cells were transfected by NIRF and, or P53, whole cell extracts andimmunoprecipitates were subjected to SDS-PAGE followed by Western blotting. GST pull-down was carried outto identify the interactions between NIRF and P53. In vitro ubiquitination reaction was carried out to identify P53ubquitinate by NIRF. The results suggested that NIRF could interact with P53 in vivo and in vitro. The results alsoshowed that NIRF could ubiquitinate P53 in vivo and in vitro. The results indicated that NIRF would be a newnegative regulator of P53.
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Proteomic technology has been widely used in the research of differently-expressed proteins under different physiology and pathologic conditions.In the aspect of the human spermatozoa proteomics,many reports have demonstrated the differently expressed proteins and the roles of sperm under different physiology and pathologic state from proteome level,and these results provided reliable theoretic base for well understanding of sperm molecule mechanism in physiology and pathologic conditions.
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The Caco-2 cell model established as a tool for in vitro investigations of intestinal drug transport processes has been widely used because of its growth characteristics, i.e., it forms polarized monolayers in cultures and differentiates into cells with high homology to human intestinal epithelial absorptive cells. Caco-2 cell cultures have provided a major conceptual advance in our understanding of intestinal drug absorption, biotransformation and bioavailability at the cellular level. Caco-2 cells have received considerable attention from the pharmaceutical industry because they have been widely accepted as a potent in vitro model membrane to screen for potential absorption problems in drug discovery programs. However, the Caco-2 monolayers model is still not perfect. The tightness of the monolayers resembles more colonic than small intestinal tissue, resulting in poor permeabilities for hydrophilic compounds traversing the epithelium via the aqueous paracellular pathway. Caco-2 cells have no mucus layer that is a potential barrier to drug absorption and display low expression of cytochrome P450 which are drug metabolizing enzymes. Further refinements of the Caco-2 cell culture model are needed to better predict human intestinal drug transport. To optimize Caco-2 model, the following technics have been used: modifying the condition of the cell culture, using molecular cloning strategies and inducing the expression of relevant enzymes. They are described in this review.
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Humans , Biological Availability , Biological Transport , Caco-2 Cells , Cell Biology , Colon , Physiology , Epithelial Cells , Cell Biology , Intestinal Absorption , Models, BiologicalABSTRACT
Objective To investigate some points in evaluating the effects of insulin sensitizer on obesityassociated non-insulin-dependent diabetes mellitus Zucker fa/fa rat model. Methods Total 20 male Zucker fa/fa rats were divided into model group and rosiglitazone groups. Another 10 male Zucker fa/? rats served as normal control. Rosiglitazone at 6 mg/kg was given intragastrically once per day for 4 weeks,and the rats of the other 2 groups were fed with same solvent at same volume. Results Rosiglitazone reduced the levels of fasting insulin,triglyceride ( TG) and free fatty acid ( FFA) of Zucker fa/fa rats ( P
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Objective To explore the difference of male and female rats in establishing diabetic model by feeding lardy diet and intraperitoneally injecting a low dose streptozotocin(STZ).Methods Totally 184 male and female Wistar rats(each 92 rats) were induced to diabetes mellitus by feeding lardy diet and intraperitoneally injecting 25 mg/kg STZ for 5 weeks.For the 114 left living rats(70 females and 44 males),they were randomized into female high rosiglitazone group(n=23),male high rosiglitazone group(n=15),female low rosiglitazone group(n=23),male low rosiglitazone group(n=15),female model group(n=24),and male model group(n=14).Rosiglitazone at 2 or 0.5 mg/kg were intragastrically administered to corresponding rats once a day for 4 weeks.Another 8 health female and 8 health male rats receiving same volume solvent served as normal control.The body weight,taken food amount,fasting blood glucose,plasma insulin content and the morphology of the spleen were measured and examined to validate the animal models.Results Blood glucose,total plasma lipids and cholesterol of model rats were markedly increased after STZ injection.And there were some other symptoms of model rats,such as polyuria,polydipsia and polyphagia,which indicated that diabetes had been induced in the models.The male model rats had higher mortality,body weight,triglyceride level and lower plasma insulin than in female(P
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Aim To establish a model of focal cerebral ischemic tissue of rat brain and explore the injury mechanism of focal cerebral ischemia reperfusion in whole level of proteins.Method The model was established with suture method by reperfusion 24 h after ischemic 2 h according to Koizumi′s method,total brain tissue proteins were extracted with Lysis buffer,proteins were separated by two-dimensional electrophoresis(2-DE),stained by Coomassie brilliant blue,the patterns were gotten,differential proteins were found out,PMS was obtained by MALDI-TOF-MS,and related information of proteins was gained by MS-Fit database.Results A comparative proteomic study of model and normal group was performed.Compared with model group,the normal group gained 23 differential protein spots,13 spots expressed lowly,and 10 spots high,6 protein spots were identified,the relative cerebral ischemic proteins such as Leukotriene A-4 hydrolase,Thyrotropin-releasing hormone-degrading ectoenzyme etc were found out.Conclusions Establishing a 2-DE technology is applied to protein analysis of brain tissue,and the relative proteins of cerebral ischemia are found from proteome aspect.This will contribute to the research on the injury mechanism of cerebral ischemic tissues.
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Aim The potentiality as a differentiation inducer of the new steroidal drug(NSC67657) had been studied.Then the expression differences of protein between treated and untreated HL60 cell line could be analysed.Method Firstly,the expression patterns of C/EBP alpha gene and protein were observed between treated and untreated HL60 cell line.Then the proliferation of HL60 cell line could be investigated by MTT.At the same time cellular chemical staining could be employed to investigate which direction HL60 cell line would be induced by NSC67657.Then the flow cytometry(FCM) could be employed to detect the profile of differentiation of HL60 cell line induced in different time and at different drug concentrations,by which the most suitable drug concentration and inducing time could be found. Following that,the information of cellular cycle and ultramicrostructure could be analysed by FCM and electronmicro scope,by which whether the apoptosis had happened or not under the drug treatment could also be found. Finally,the protein of these two group HL60 cell lines could be separated by modified two-dimensional electrophoresis (2-DE).Results The expression of C/EBP alpha gene and protein could be promoted under the treatment of NSC67657.Then the proliferation of HL60 cell line was inhibited significantly.From cellular chemical staining,the monocytic differentiation could be easily found and the perfect inducing time and drug concentration were defined as 10 ?mol?L-1NSC67657 and constantly inducing HL60 cell line within 5 days.The cellular number of G0~G1 was increased and hardly any apoptosis which might be happened during drug inducing ability could be seen.The protein of HL60 cell lines were separated by modified 2-DE technology.Then there were 14 protein spots which could only be found in the differentiated gels,on the other hand,20 protein spots could only be found in the undifferentiated gels,which would have been analyzed by MALDI-TOF.Conclusions HL60 cell line could be induced to monocyte by NSC67657 which could also stimulate the C/EBP? in the early stage.2-DE could separate the protein directly which expressed differentially,from which some proteins essential in cellular differentiation might be found.
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Aim To study the effect of the total saponins of Rubus parvifolius(TSRP)on the intracellular free calcium levels in ischemic hippocampus neurons.Methods The cytosolic free calcium concentration in ischemic hippocampal neurons was measured by using Ca2+-sensitive fluorescent probe fluo-2/AM with a laser scanning confocal microscope.Results Application of TSRP inhibited free intracellular calcium in ischemic hippocampus neurons in a concentration-dependent manner.Conclusions These results suggest that TSRP might protect hippocampus neurons via attenuating calcium overload induced by ischemia.
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Aim To study the effect of tubeimosides on human rectal cancer cell line SW480 in vitro and the mechanisms of its antitumor effect.Method The morphological changes were determined by means of light microscopy and electron microscopy respectively.Cell apoptosis was detected by flow cytometry through Annexin V assay and PI fluorescent staining methods.The protein levels of Bc1-2,p53 and Fas were determined using immunohistochemical technique.Results Apparent morphological characteristic of apoptosis was detected under the optical and electric microscope.The percentage of apoptotic cell increased when comparing the drug groups with control after treatment with tubeimoside Ⅰand Ⅲ.Immunohistochemical analysis revealed apoptosis was and induced by tubeimosides through inhibiting Bcl-2 and p53,and upregulating Fas.Conclusion Tubeimosides can induce apoptosis of SW480 cells,which may be one reasons of its antitumor effects.
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Aim To investigate whether ecdysterone is able to exert glucose-lowering effect on hepatocytes or stimulate the secretion of insulin.Methods For glucose consumption studies,the amounts of glucose disappeared from the culture medium of HepG2 cells within 24 h were determined.?TC3 cells were also used to monitor insulin secretion.Results The glucose concentrations decreased significantly by ecdysterone 1?10~(-6)～10~(-4) mol?L~(-1),while glucose consumption increased by 44%～77% with ecdysterone.Glucose consumption declined as its concentrations increased.Insulin had no effect on glucose-lowering of ecdysterone.?TC3 cells were not stimulated by ecdysterone.Conclusion Ecdysterone is able to exert glucose-lowering effect on hepatocytes which is insulin independent,but has no effect on insulin secretion.
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This article reviewed the appli ca tion of molecular biology technology, such as genomics, DNA labeling technology, and DNA microarray. The aim is to provide the reference for the modernization o f Chinese material medica.
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Objective: To make out effects of different absorbents, mositure and alcohol amount on the absorption of lipophilic extract of Rhizoma Alismatis in Jinzhe Guanxin Capsules in order to solve its preparation procedure problem. Methods: The absorbent activity and absorbed dose were determined. Results: The moisture of lipophilic extract of Rhizoma Alismatis was lower than 25%, 95% alcohol amount for every gram was 0.25～ 0.5 ml when aluminum hydroxide gel was used as an absorbent. This was the best procedure condition.Conclusion: Jinzhe Guanxin Capsule preparad by aluminum hydroxide gel conforms with the preparation quality requirement.
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OBJECTIVE:To study the content change regularities and the dynamic characteristics of paeonol,the major active ingredients in Cortex moutan from Chongqing Dianjang county under different influencing variables.METHODS:The determination of paeonol in moutan cortex samples was conducted by HPLC,and dynamic analysis on content change regularities was conducted.RESULTS:The content of paeonol took a para-curve pattern at an altitude from 400m to 800m,with that from 600m in altitude has the highest content;The content increased year by year from growing life of 3 to 5 years;The content took a para-curve pattern when harvested from 8mo to 11mo,with that harvested in 10mo was the highest.CONCLUSION:The optimal altitude for the growing of Cortex moutan was from 400m to 800m,in particular at an altitude of 600m,and it is preferable to harvest Cortex moutan with 4 or 5 years growing life in the first 20 days of 10mo.
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Aim To screen alteration in protein level in brain tissue between middle cererbral artery occlusion(MCAO) and the total saponins of Rubus Parviflolius L(TSRP) treated rats and to learn the mechanism of nerve-protecting effects of TSRP after focal cerebral ischemia-reperfusion.Methods The MCAO 2 h reperfusion of 24 h model was established in rats,2-DE was applied to preparein step 1 for the separation of protein,and imaging analysis of gels was done with Image-Master 2D software.Different protein spots expressing between cerebral ischemic reperfusion(CIR) model and TSRP group were cut from the gels for the MS analysis,Different protein spots expressing between cerebral ischemic tissue administered with or without TSRP were also cut from the gels for the MS analysis.MALDI-TOF-MS and database search: protein spots from step 2 were subject to in-gel digestion and MALDI-TOF-MS analysis,which resulted in the peptide mass fingerprint spectra(PMF).PMF were search against swiss-prot database using search program of Ms-Fit.Results By comparative proteome analysis,29 expression-altered spots were excised for MS and informatics analysis,comparing to the CIR model,18 proteins up-regulated in the TSRP group,11 proteins down-regulated,7 spots of them were identified and characterized.Conclusion The protective effect of TSRP on cerebral ischemia-reperfusion is the result of the participation of a number of proteins.
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Aim To explore the mechanism of neuro-protecting effects of TGRP on ischemic cerebral injury.Methods The middle cerebral artery was occluded for 2 h to produce focal ischemic followed by 24 h reperfusion.HE staining,TUNEL labeling and immunohistochemical methods were used to investigate changes of neuronal apoptosis and expression of the apoptosis-related proteins after administration of TGRP.Result TGRP decreased the pathologic changes significantly.The apoptosis of neural cells and the expression of Bax positive cells in the TGRP groups significantly decreased compared with the control group(P
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Aim To establish insulin resistant HepG2 cell model induced by high concentration insulin in vitro culture and evaluate the effects of pioglitazone on insulin sensitivity and glucose metabolism in the cell model.Methods HepG2 cells were incubated with 5?10~(-7)mol?L~(-1) insulin for 16 h and insulin-stimulated()~3H-D glucose incorporation was determined.Then,insulin-stimulated glucose incorporation rate which was used to estimate insulin sensitivity of HepG2 cell,and glucose consumption,the amountsof glucose disappeared from the culture medium of HepG2 cells within 24 hours was determined.The insulin resistance cells were incubated with pioglitazone 1?10~(-5) mol?L~(-1).Results It was demonstrated that the incorporation rate of()~3H-D-glucose in insulin-resistant HepG2 cells was decreased significantly than that in the control cells.Insulin-resistant HepG2 cells were free from stimulation for 48 h by insulin,but the incorporation rate of()~3H-D-glucose was lower than that in control cells.Incubation of insulin resistance cells with pioglitazone significantly increased glucose uptake and glucose consumption by the cells compared with that of the control cells(P
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Aim This study examined the uptake and transport of ecdysterone(EDS) using Caco-2 cell monolayers as a model of human intestinal mucosa.Methods Two kinds of Caco-2 cell monolayer model(Caco-2 cell monolayers;CYP3A4 expressing Caco-2 cell monolayers) were set up to study the uptake and transport of EDS.Results Compared bi-directional charaterizaton of Caco-2 cell,the apparent permeability coefficients(Papp) values of EDS were between 0.1?10-6 cm?s-1 and 1?10-6 cm?s-1.EDS absorption was 1%~10% for two kinds of Caco-2 models.The RPapp values were all less than 1.5 for 4 h.Conclusions The uptake and transport of EDS was a passive transcellular diffusion mechanism.Ecdysterone was not influenced by CYP3A4 mediate mechanism.