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Study on the Release Characteristics of HMGB1 from Liver Cells and Its Purification Methods

Author: DaiXiaHong
Tutor: FanXueGong
School: Central South University
Course: Internal Medicine
Keywords: High mobility Group Box-1 Protein Hepatic Failure Chromatography release Two-dimensional gel electrophoresis
CLC: R575.1
Type: PhD thesis
Year: 2011
Downloads: 28
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Abstract


Background and aims:Hepatic Failure is a potentially devastating syndrome with a high mortality rate. Accumulating evidence has established that extracellular high mobility group protein -1 (HMGB1) plays a critical role in the pathophysiology of hepatic Failure. As a nucleoprotein, HMGB1 binds to the chromatin in non-necrotic parenchymal cells commonly. However, our previous studies showed that liver cells could also release HMGB1 in the case of non-necrotic, remaining an unclear process. In the first part of this study, we take the clear processing characteristics of HMGB1 secretion from immune cells as controls, to investigate the release characteristics of HMGB1 from liver cells.On the other hand, purification of HMGB1 protein secreted by liver cells is the prerequisite to study its biological activity. However, the purification technique of natural proteins still remains in an exploratory stage at present. In the second part of this study, the purification methods and conditions of secreted HMGB1 were explored preliminarily.Methods:(1) Different concentrations of LPS were added to the liver cell line L02 in vitro. At different time point after treated, cells and supernatants were collected to detect cell viability by MTT assay and level of HMGB1 by western blot to determine the optimal LPS stimulating concentration.(2) 400ng/mL of LPS was added to the liver cell lines (L02 and HepG2) and immune cell line (U937) in vitro. At different time point (0h,4h,8h,12h,16h,20h,24h) after treated, cells and supernatants were collected. Cell viability was determined by MTT assay and level of LDH in the supernatant. The apoptotic rats were determined by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay and flow cytometry. The cell HMGB1mRNA level was determined by RT-PCR and supernatant HMGB1 level was determined by western blot. The morphological changes were detected by HE staining and DAPI staining of nuclear. blotting. The cytoplasmic translocation of HMGB1 was observed by immunofluorescence. Equal volume of saline was added as control.(3) The supernatant secreted HMGB1 protein was purified using protein precipitation, ultrafiltration, chromatography (ion chromatography, gel filter ration) et al, while verified by Western blot, Coomassie brilliant blue and silver staining.Results:(1) LPS stimulation in vitro induced release of HMGB1 from L02 cells, and the level of HMGB1 in the supernatant increased as dose dependent way within a certain dose range of 0-400ng/mL. However, when the stimulation concentration of LPS increased to 800ng/mL, the level of HMGB1 in the supernatant was lower than 400ng/mL of LPS was added. MTT showed that the cell survival rate was negatively correlated with the concentration of LPS. When the LPS concentration ranged from 0 to 400ng/mL, more than 95% of L02 cells would be survival.(2) MTT, TUNEL assay and level of LDH in the supernatant demonstrated that no significant necrosis or apoptosis was observed when 400ng/mL of LPS was added to the three cell lines within 0-24h (P>0.05). Besides, no significant morphological changes were observed by HE and DAPI staining in the three cell lines within 0-24 h after LPS stimulation.(3) Western blot showed that the levels of HMGB1 in U937 cell supernatants were significantly higher than the corresponding controls at 8-24h after LPS treated (P<0.05). While in L02 and HepG2 cell lines, the supernatant HMGB1 levels increased at 16-24h after LPS treated, much later than in U937 cells (P<0.05). The supernatant HMGB1 levels in the three cell lines reached to a peak at 20h after LPS treated. Further analysis to compare the amount of HMGB1 in the three types of cells at a certain time point showed an absolute advantage in U937 cells (P<0.05).(4) The results of RT-PCR showed that HMGB1mRNA expression level in L02 and HepG2 cells increased at 20h after LPS treated. While in the U937 cell line, it increased ever since 12h after LPS treated, much earlier than that in the liver cells (P<0.05). Besides, at a certain LPS stimulating time point, the HMGB1mRNA expression level in U937 cells was significantly higher than that in L02 and HepG2 cells (P<0.05).(5) Significant cytoplasmic translocations of HMGB1 in L02 and HepG2 cells were observed at 12-24h after LPS treated by immunofluorescence. However, in U937 cells, the translocation occurred much earlier at about 4h after LPS was added (P<0.05).(6) Among the conventional purification methods of natural proteins, the protein precipitation and ultrafiltration separation are not suitable for HMGB1 purification. While the sequential application of molecular sieve and ion exchange chromatography could purify the secreted HMGB1 protein to a greater degree. Conclusions:(1) The activated liver cells could secrete the late inflammatory mediator HMGB1 after LPS stimulating as the immune cells did. However, they secrete HMGB1 in a later phase and much less amount when compared to the immune cells. And the secretion was related to the displacing of nucleus HMGB1 to the endochylema.(2) Sequential chromatography is suitable method as an initial purification of the secreted HMGB1 protein, which lays the foundation for the further purification and biological activity study of the secreted HMGB1.

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CLC: > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Hepatitis
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