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.

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.

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.

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 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.

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

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

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.