ABSTRACT
Primary hepatocytes are widely used in drug metabolism and toxicity assessment. As the culture of primary hepatocytes in vitro is a process of dedifferentiation, hepatocytes lose normal metabolic detoxification function gradually. The mechanism of hepatocyte dedifferentiation has been not clear so far. TFs play an important role in the dedifferentiation and non-parenchymal cells can maintain the function of hepatocytes in vitro. However, the current methods cannot be used in effective identification and quantitative analysis of a large number of TFs. In this paper, the mo-culture system (only primary hepatocytes) and co-culture system (primary hepatocytes and non-parenchymal cells) were established. The cells were cultured for 24 h, 48 h, 72 h as monolayer. The changes of TFs during the culture were obtained by TOT (Transcription factor response elements on tip) transcription factor enrichment method and mass spectrometry. A total of 219 TFs were identified in three individual replicates. The result revealed that up-regulated TFs were enriched in cell proliferation, death and immune response pathways, and down-regulated TFs were involved in metabolism pathway. The establishment of such culture-TFs identification system is of great significance to reveal the mechanism of primary hepatocyte dedifferentiation and crosstalk between hepatocytes and non-parenchymal cells.
ABSTRACT
Steady improvement in mass spectrometers technology has transformed the targeted proteome analysis into a new stage. Parallel reaction monitoring (PRM) technology has evolved from the basic multiple reaction monitoring (MRM) targeted proteomics methods in recent years. PRM performs with a higher sensitivity, throughput and reproducibility in targeted quantification, however its limitations in effectiveness and accurate quantification of samples with higher complexity still remain unsolved. In this study through improving the chromatographic conditions of PRM we established a simple and robust platform for targeted proteomic quantification. The newly established PRM system is equipped with columns with increased inner diameter (150 μm) and decreased total length (8 cm); faster liquid phase elution rate (800 nL/min) and shortened elution gradient (35 min). These modifications enable PRM platform to combine with dual reverse phase chromatography, to quantify up to 400 low abundance peptides in human 293T cells whole cell extract. Our findings would benefit the promotion of PRM technology, especially providing a technical option for accurate quantification of low abundance proteins.
ABSTRACT
Objective To investigate the cognition and care needs of the patients with acute stage fracture thus providing reference for the construction of intermediate care model. Methods The data of 504 patients with acute stage fracture from December 2014 to April 2015 were investigated through questionnaire, and analyzed. The issues included gender, age, marital status, cultural level, degree of understanding of intermediate care, care needs, etc. Results 63.29%(319/504) of the patients didn′t know intermediate care before they filled in the questionnaire,only 3.97%(20/504) of the patients understood the conception of intermediate care; different stage of age had different cognition towards intermediate care, the differences were statistically significant( χ2=148.771, P<0.01); different stage of age, sex, marital status, cultural level had different care needs, the differences were statistically significant (H/u=9.692-129.097, P<0.05). Conclusions At present, the majority of patients have no conception about intermediate care, so it is necessary to build the model of intermediate care. As nursing staff we should provide different help according to different type people, thus we can promote patients' physical and mental health, improve patient satisfaction to the greatest extent.