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.

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.

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.

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.

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.

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.
Biological Availability ; Biological Transport ; Caco-2 Cells ; cytology ; Colon ; physiology ; Epithelial Cells ; cytology ; Humans ; Intestinal Absorption ; Models, Biological

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 its 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.

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.

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.

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.