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Role of Heat Shock Protein 27 in Regulating Mouse Embryonic Stem Cells Differentiation Induced by Icariin in Vitro

Author: CuiRong
Tutor: ZhuDanYan
School: Zhejiang University
Course: Pharmacology
Keywords: heat shock protein 27 ES cells cardiomyocytes ICA
CLC: R285.5
Type: Master's thesis
Year: 2012
Downloads: 46
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Abstract


Embryonic stem cells (ES cells) are derived from the inner cell mass of the blastocyst and they have special character described as "pluripotency" which means they can differentiate into many different cell lines. The models of ES cells differentiation are applied in the drug discovery, test of embryonic toxicological compounds and cell therapy. There are many methods of mouse ES cells directional differentiation into cardiomyocytes. The classic method is "hanging drop culture-suspension culture- adherent culture". Certain compounds and growth factors can promote ES cells differentiation into cardiomyocytes. Our research group found icraiin (ICA), which is active ingredient in Herba Epimedii, can observably induce mouse embryonic stem cells differentiation into cardiomyocytes in 10-7 mol/L by increasing the expression of cardiac-specific proteins and heart development related genes. Presently many signalling pathways are found participant in cardiomyocytes differentiation. However, the mechanisms of ES cells differentiation into cardiomyocytes are too complicated to be understood clearly. The mechanisms of ICA promoting cardiac cells differentiation involve the generation of reactive oxygen species which activating the p38 pathway and NF-κB signaling pathway, the activation of p53 dependent apoptosis in the early differentiation, and so on. However, the regulating mechanisms need further in-depth exploration since they are not yet fully revealed.Heat shock proteins also called "stress proteins" express comprehensively in many tissues. Their molecular chaperones activity can help many proteins fold or unfold, which expression will increase when cells are heated or injured. Someone make a hypothesis that heat shock proteins can not only protect cells but also participant in cell differentiation by translocation and transient expression in cells. Heat shock proteins named by molecular weight include hsp100, hsp90, hsp70, hsp60, small heat shock proteins (sHsps). Main function of sHsps involves molecular chaperones, thermotolerance, inhibition of apoptosis and cell differentiation. As a member of sHsps family, Hsp27 also has the above mentioned function. Hsp27 can help non-natural proteins fold by forming complex with proteins in case they assemble nonspecifically. Compared with other family members, molecular chaperone of Hsp27 is ATP independent. Hsp27 can combine with Akt to active it, and glutathione is induced by Hsp27 in order to inhibit apoptosis of cells injured by ROS. Hsp27 also can combine with actin to maintain cell structure. Hsp27 can combine with Ub-conjunctive protein to facilitate the degradation of proteins. Moreover, Hsp27 promotes degradation of IκB and actives NF-κB pathway. Hsp27 not only protects cells from apoptosis but also plays an important role in cell differentiation. Hsp27 is a predifferentiation marker of leukemia cells and C2C12 cells. The mechanism is related with the overexpression of Hsp27 making cell cycle of the cells stay in G0/G1. The differentiation of mouse CGR8 cells is inhibited followed by apoptosis because of Hsp27 expressing deficiency. Hsp27 may be a switch between apoptosis and differentiation. The role of Hsp27 in regulating ES cells differentiation is not understood. It is very important to explore the role of Hsp27 in ES cells differentiation for revealing the mechanism of heart development.Aim:To explore the roles of Hsp27 in regulating the mouse ES cells differentiation during the course of startup and differentiation into cardiomyocytes. To investigate the effects of ICA on the expression and function of Hsp27 when inducing the mouse ES cells differentiation.Methods:Using the hanging drop culture method, ES cells were differentiated into cardiomyocytes by ICA at the concentration of 10-7mol/L in vitro. RA (10-8 mol/L) and DMSO are used as positive control and negative control respectively. Beating of cardiomyocytes was observed under the light microscope. The formation of cardiac-specific sarcomeric structure was observed using immunofluorescence. Samples of EBs at d 5+3 (DMSO and ICA) were collected and protein expression profiles were got using two-dimensional electrophoresis and mass spectrometry (MS). Hsp27 expression was ramarkable different in ICA group compared to that in control gruop. Expression of Hsp27 in different differentiation phase was identified by western blot analysis. Co-expression of Hsp27 and a-actinin was observed by immunofluorescence. In order to evaluate the function of Hsp27 on cardiac differentiation, we silenced the Hsp27 gene in ES cells. When Hsp27 was silenced, the percentage of the beating EBs, expression of a-actinin and expression of NF-κB in nucleus were detected. On the other hand, in order to explore role of Hsp27 in regulating differentiation start of ES cells induced by ICA. ICA was added when LIF was withdrawn. ES cells clones were observed at 6 h,12 h,24 h respectively after LIF was withdrawn. Expression of Hsp27, p-Hsp27 and p53 was detected by western blot and translocation of Hsp27, p53 was detected using immunofluorescence. Location of p53 was also detected in ES cells after Hsp27 silencing.Results:Embryoid bodys (EBs) were formed after hanging drop culture of ES cells. EBs were transferred to the 6-well plates and ICA was added to induce ES cells differentiation into cardiomyocytes. Automatic rhythmic contraction center appeared and sarcomere was observed by immunofluorescence which suggested that ES cells differentiated into myocardial cells. Protein expression profiles were obtained by two-dimensional electrophoresis and Hsp27 was identified as a member of proteins expressing differently. Expression of Hsp27 increased from d 5+2 to d 5+4 and decreased at d 5+7. Expression of p-Hsp27 increased from d 5+2 to d 5+7. Compared with control, ICA and RA could increase expression of Hsp27 and decrease p-Hsp27 expression at d 5+7. Hsp27 protein was co-expressed withα-actinin. After Hsp27 siliencing, the percentage of beating EBs was 30%, which was lower than that in si-con group at d 5+7. ICA could reverse the percentage of beating EBs inhibited by Hsp27 siliencing and reach 50%. After Hsp27 silencing, expression of NF-κB in the nucleus was decreased in response of the decrease of Ub-conjunctive protein and the increase of IκBα, however p-IκBαwas not affected. ICA could raise the expression of IκBαbecause of increasing the Ub-conjunctive protein and activating NF-κB pathway to induce ES cells differentiation into cardiomyocytes.On the other hand, the expression of Hsp27 and p53 increased significantly when ICA was added in ES cells to start differentiation from 6 h to 12 h, although expression of p-hsp27 decreased. Hsp27 transform from nucleus to cytoplasm and expression of p53 increased in the nucleus when ES cells differentiation started. ICA could facilitate translocation of Hsp27 and expression of p53 in the nucleus. Siliencing of Hsp27 inhibited expression of p53 in the nucleus. ICA can reverse the inhibition of p53 translation because of siliencing Hsp27 gene. Our data suggested that Hsp27 expressed transiently and transform from nucleus to cytoplasm during the initial differentiation of ES cells, meanwhile, the expression of p53 increased in the nucleus which facilitated by Hsp27. ICA induced ES cells differentiation by regulating Hsp27 pathway. Conclusion:1. At the early stage of ES cells differentiation into cardiomyocytes, Hsp27 induced ubiquitin combining with IκBαand activated NF-κB pathway to regulate ES cells differentiation into cardiomyocytes. ICA could induce expression and chaperone activity of Hsp27, additionally promote ubiquitin combining with IκBαand activate NF-κB pathway to regulate ES cells differentiation into cardiomyocytes.2. At the startup stage of ES cells differentiation, Hsp27/p-Hsp27 and p53 expression in nucleus occurred transient increase. Hsp27 transferred into cytoplasm from nucleus. ICA could regulate the start of ES cells differentiation by inducing Hsp27 expression and translocation into cytoplasm, furthermore increasing p53 expression in nucleus to induce apoptosis.

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