The Experimental Study Of The Dedifferentiation Mechanism Of Apicidin On The Mature Adipocyte

With the obesity problem has been more and more attention. Especially in relatively good living conditions in developed countries and some developing countries. Obesity increases the incidence of some diseases, particularly heart disease, type2diabetes, obstructive sleep apnea, and some cancer diseases. So research on fat cells has been more widespread attention. Except in the problem of obesity, research on fat cells can also be applied in the fields such as stem cell research, tissue engineering, tissue augmentation and animal husbandry.Although there are many types of cells in the adipose tissue or fat, but the fat in adipose tissue cells are always major cell types, and is responsible for an important function of that lipid droplets metabolism. Recently, the phenomenon of dedifferentiation of mature fat in vitro has been found in many species. Such cells are known dedifferentiate fat (DFAT) cell. In addition, several studies have found to be redifferentiation into fat cells filled with lipid droplets, or under suitable conditions in transdifferentiate into chondrocytes, osteoblasts, myoblasts stock or other possible cell types. Differentiation of pluripotent stem cells to differentiate fat cells to make this become a new stem cell research, in particular, it is relatively easier to extract isolated from potential patients. However, the mechanism of adipocyte dedifferentiation still unclear.Chromatin remodeling resulting from the reversible acetylation of core histones has been suggested to be a critical component of transcriptional regulation. The turnover of histone acetylation is tightly regulated by the balance of opposing activities between histone acetyltransferases (HATs) and histone deacetylases (HDACs). The inhibition of HDACs causes an accμMulation of acetylated histones in the nucleus and a subsequent activation of transcription for target genes. Indeed, it has repeatedly been demonstrated that treatment with HDAC inhibitors such as Apicidin, trichostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA) upregulate the transcription of p21WAF1/Cip1, cyclin E, and telomerase reverse transcriptase via histone hyperacetylation. Thus, HDAC inhibitors are emerging as a promising class of chemotherapeutic agents against cancer. However, in addition to the anticancer effect of HDAC inhibitors, recent work indicates that cellular differentiation processes can be regulated by HDAC inhibitors. This notion is supported by work demonstrating that the differentiation process requires dynamic changes in gene expression through chromatin remodeling. Indeed, in the course of adipocyte differentiation, sodiμM butyrate (NaB) will stimulate a fat gene expression after treatment.Apicidin is a fungal metabolite that has been identified as an antiprotozoal agent that inhibits parasite histone deacetylase more research is now being used for anti-cancer drugs. Apicidin has been reported as an HDAC inhibitor can influence the differentiation of adipocyte. And there is a certain phenomenon of dedifferentiation. So can Apicidin induce the adipocyte dedifferentiation? What is the mechanism? And select a more representative experiment DFAT cell model to study.Thus, the present study mainly consists of three parts:1, From the use of mature adipose tissue by enzymatic digestion and ceiling adherent to get the DAFT cells.2, Apicidin processing DFAT cells was observed morphological changes and quantitative changes in lipid droplets in adipocytes. And selected its safe and effective concentrations.3, After Apicidin treament, transcription and protein expression were determined quantitatively in adipose differentiation selected key genes. Analysis of the mechanism dedifferentiation.Methods 1. DFAT cells Obtain and identificationAdipose tissue obtained from Nanfang Hospital, washed with PBS three times, was cut with scissors into a paste, digestion, with0.075%Ⅰ collagenase digestion shaker at37℃45min, to be repeated shocks during mixing, the same amount of volμMe complete medium (high glucose DMEM+10%FBS+1%double anti) after,200μM nylon mesh filter, filtrate200g/min centrifuge5min, cells were collected with a second layer of fat and150μM200μM nylon mesh filtered to remove the maximμM contamination of other cells. The fat cells were seeded on culture dish, with4%paraformaldehyde mature adipocytes10min, then cells were washed with PBS, first with1μg/ml Nile red staining after30min with10μg/ml of Hoechst33342staining30min. Use stained cells were observed under fluorescence microscope. Nile red fluorescence was observed with598nm filter, Hoechst33342fluorescence with460nm filter observation.2. DFAT ceiling adherent cell culture methodFat cells were collected25cm2flasks, on average0.5-1×105cells per bottle, the bottle filled with complete medium (high glucose DMEM+10%FBS+1%double antibody), the bottle is inverted upside down at37℃,5%CO2incubator, so that contact with the floating fat cells culture bottle.48h after righting flasks, carefully suck out the floating fat cells were inoculated in new25cm2flasks, a new round ceiling adherent culture. After the second48h, repeat the previous procedure to remove adherent fibroblast-like cells. If after culture bottles still observed fibroblast-like cells exist, the above process is repeated a third time. When the microscope when seen as fibroblast-like cell contamination, suck out the fat cells were inoculated in an inverted, sealed and filled with complete medium in25cm2culture flask into the incubator so that fat cells cultured10d firmly adherent.10d, the righting flasks, each replacement2d complete medium changes were observed daily adherent fat cells. Frozen or passaged to the fifth generation for subsequent experiments.3. Morphological observation of Apicidin affect DFAT differentiation and dedifferentiation. The paired cell P5DFAT generation cell suspension5x104/ml, and seeded into96-well culture plates. Each hole200μl ordinary medium. Placed in37℃,5%CO2incubator. Until the cells reached80-90%confluence. Were observed:1, the effects of different concentrations of Apicidin after adipogenic DFAT cells.2, in the adipogenic environment, the effects of different concentrations of Apicidin on adipogenic DFAT cells.3, in the adipogenic environment, the effects of different concentrations of Apicidin on DFAT cells.4, Apicidin treatment, DFAT cells into the ability of fat.4. Oil Red O stainingUntil the cells reached80to90%confluence. The P5generation DFAT cells dubbed5x104/ml of cell suspension was inoculated into two six-well plates (9holes), the complete medium3ml/hole into37℃,5%CO2incubator culture. Adipogenic differentiation induction step:When the cells reached to be80%-90%confluence, taking nine holes to absorb the old medium, add3ml/hole adipogenic DMEM medium (containing10%fetal bovine serum,1%double anti-,1μmol/L dexamethasone,10μmol/L insμlin,200μmol/L indomethacin,0.5mmol/L IBMX); leaving a hole made blank. Each3d replace1medium directed differentiation-inducing first eight days. Take8-hole induced holes were replaced with1,2,4,8,16,32,64,128μM/L Apicidin common medium, leaving a hole induced hole ordinary medium was changed to maintain the control group did. Join Apicidin the first three days, oil red O staining and quantification. Each doing three wells. Microplate reader using a wavelength of490nm, measure OD values.5. MTT assayThe medium,1x PBS and Trypsin-EDTA to the pre-equilibrated to room temperature. The cells were washed with1x PBS2-3times to remove residual serum. Aspirate PBS. Adding an appropriate amount Trypsin-EDTA, gently rotate, so Trypsin-EDTA culture vessel surface evenly covered, for1-2minutes digestion, cell gap increases seen under the microscope, the cells became round, hand pat culture vessel wall, adding2ml immediate complete medium to terminate digestion. Pipette the liquid surface of the culture vessel by pipetting gently, repeated3-5times, the cells were completely detached from the wall of the bottle bottom of the plate, the cells were shifted into a centrifuge tube, again flask was added1×PBS washed1-2times, and washings were also transferred to the centrifuge tube. Centrifugation (244g,4min), the supernatant was aspirated. Add5ml of cell pellet resuspended in complete medium with a pipette gently pipetting the cell suspension collected in the sampling count. Take two96-well plates, each plate containing complete medium previously added8concentration0.1,1,2,4,8,16,32,64μM/L Apicidin of,200μl/hole,5wells for each concentration and set up a blank control, each plate45holes. Take2×103cells/hole (96well plate) were seeded to a96well plate add two holes in the medium. Cultured for3days, take a plate measuring MTT. Cultured for7days, take another piece of plate measured MTT.6. RT-PCRDetection of PPARγ, C/EBPα, C/EBPβ, SREBP1C, GATA4, C-MYC, OCT4, SOX2, TBX1, KLF4, NANOG, SOX17gene expression changes. When the cells reached100%confluence, BO group, B1group, B3group, A1group, A3group to absorb the old medium, add2ml/hole adipogenic induction of complete medium started, the control group not treated. Adipogenic induction of complete medium7d, cells appear more large lipid droplets. After induction7d, B0, and the control group, for PCR testing. Meanwhile, B1group, B3group replaced added between1μmol/L Apicidin normal medium, each well2ml. The group A1, A3group convertible into normal medium, each well2ml. Group B1dosing1d, A1group of normal culture medium1d, two groups for PCR testing. B3group dosing effect3d, A3group of ordinary culture medium3d, two groups for PCR testing.7. Western-blottingDetection of PPARγ, C/EBPα, C/EBPβ, SREBP1C, GATA4, C-MYC, OCT4, SOX2, TBX1, KLF4, NANOG, SOX17protein level. When the cells reached100%confluence, B0group, B1group, Al group set to absorb the old culture medium, complete medium was added to start adipogenic induction. After induction7d, B0extracted proteins. Meanwhile, B1group replaced added1μmol/L Apicidin common medium, each well2ml. The Al group convertible into normal medium, each well2ml. Group B1dosing role1d, A1group Normal culture medium1d, two cell extract proteins.Results1. DFAT cells Obtain and identificationFreshly isolated mature adipocytes in culture flasks filled with medium float up and because of the buoyancy reason, too close to the train on the bottle wall under the ceiling.2-3days prior to culture, is still not mature adipocytes stay close to the ceiling of the bottle. Within these fat cells and there is still a large lipid droplets and in the light exhibits high light reflectivity, showing translucent yellow color. Did not find any other cell contamination. Hoechst33342by Nile Red staining and can be found in mature adipocytes lipid droplets are dyed red, nuclei were stained blue, and often located in the extracellular side of the body, the annular ring into the fat. Such results show that this method can be isolated adipocytes having high purity and activity.2. DFAT ceiling adherent cell culture methodProbably from about the beginning of the fourth day began to mature fat cells of snapping the ceiling, and there are already some dedifferentiation performance. But this time the cells are still not very firmly adherent, while shaking flasks adherent cells would have come off, so upside down within10days of culture, the bottle placed in the incubator is more stable, uninterrupted or move location Jia. Not all fats can all be de-differentiation, as well as a considerable number of fat cells rupture and release of free lipid droplets. Dedifferentiation process is the first one or both sides of fat cells form small prominent, and intracellular lipid droplets in a single room and more space gradually formed small lipid droplets. Fusiform cells and eventually form which contains many small lipid droplets. Then take a very long process, small lipid droplets within the spindle cells gradually disappear, and the cells gradually become fibroblast-like cells. Finally start clonal proliferation of fibroblasts.3. Apicidin affect morphological differentiation and dedifferentiation of DFAT1) The effect of the different concentrations Apicidin on the adipogenic induced DFAT cells in normal culture medium. After the observation that DFAT cells into fat, after adding Apicidin and normal medium. Under the effect of different concentrations of Apicidin. While the first three days of lipid droplet formation continues than observed on day1.But in the fifth day observation. It has been found to be inhibited fat and has a lipid droplets decrease. Apicidin and this phenomenon is related to the concentration varies.2) The effect of the different concentrations Apicidin on the adipogenic induced DFAT cells in adipogenesis culture medium.After the observation that DFAT cells into fat, after adding Apicidin and adipogenesis culture medium. Under the effect of different concentrations of Apicidin. While the first three days of lipid droplet formation continues than observed on day1.But in the fifth day observation. It has been found to be inhibited fat and has a lipid droplets decrease. Apicidin and this phenomenon is related to the concentration varies.3) Adipogenic effects of Apicidin on DFAT cellsWill replace the normal medium into adipogenic medium containing different concentrations Apicidin continue culture. In the third and fifth day found, DFAT kept the fibroblast-like morphology characteristics.4) After Apicidin treatmenteffect on the ability of DFAT cells into fatReplace normal medium containing1μM/L Apicidin normal medium.3days after the observation DFAT remained a fibrous morphology found no lipid droplet formation, replaced after adipogenic liquid does not contain Apicidin continues to culture, for the first three days after adipogenic cells observed deformation. Appeared in the cytoplasm of some small lipid droplets and transparent and gradually gathered, six days after the induction of differentiation, DFAT cell morphology changed significantly, the formation of fibroblasts appearance from long shuttle gradually turned round and began to appear more lipid droplets in cells.4. Oil Red OAdded Apicidin the first three days, were observed with increasing Apicidin concentration, degree of lipid droplets are destroyed gradually increased.4,8μM/L is a cut-off point. More than8μM/L concentration of lipid droplets in addition to being severely damaged, the morphology of the cells have been destroyed Greater than8 concentration of lipid droplets in addition to being severely damaged, the morphology of the cells have been destroyed. Concentrations of less than4μM/L under cell morphology has affected but not severely. Lipid droplets to maintain a good shape and have lipid droplets shrink phenomenon. By Oil Red O quantitative detection method, the measured and OD of1,2,4,8,16,32,64,128μM/L Apicidin treatment DFAT cells and the control group. Oil Red0OD were:0.16±0.00,0.16±0.01,0.17±0.03,0.15±0.09,0.15±0.07,0.18±0.06,0.14±0.03,0.14±0.09,0.19±0.16. Measured in each group there was a significant difference (F=8.627, P=0.000) between the OD values of intracellular lipid droplets content.5. MTT assayEffects of different concentrations Apicidin on DFAT cell activity,0.25,0.5,1,2,4,8,16,32,64μM/L obtained in comparison with the blank inhibition rate was0.33±0.10,0.38±0.09,0.44±0.09,0.53±0.07,0.61±0.08,0.68±0.05,0.66±0.08,0.67±0.06,0.67±0.07. There were significant differences (F=14.232, P=0.000) between the groups. Inter8,16,32,64μM/L four groups was not statistically significant for multiple comparisons. Using0.25,0.5,1,2,4μM/L with blank inhibition rate comparison was carried regression measured:Apicidin50%DFAT cell proliferation inhibition concentration1.475μM/L.6. RT-PCR and Western-blotting test resultsPPARy compared after adipogenic induction group (B0) is not induced group (A0group) gene expression. In the use of normal medium containing Apicidin (A1, A2) group and the general media does not contain Apicidin (B1, B2group) whose expression levels have declined. But in the first three days and training (Al, B1group) compared to the first seven days culture (B1,B2group) expressing a slight increase. Protein levels also showed a drop phenomenon. But the difference is that the gene expression and protein levels of B1group decreased more significantly. C/EBPα mRNA expression, B0group is highly expressed. A1and B1expression levels decreased, B1decline more apparent. A2and B2levels continue to decline. B2decline more apparent. Gene expression and protein levels in line. mRNA expression of C/EBPβ show, B0group expression, but showed a high expression of B1group and B2continues to increase. However, the low expression group A1, but not statistically different from the BO group, while A2group increased performance. Similar gene expression and protein levels. SREBP-1C gene expression and protein levels similar to C/EBPβ. Gene expression is also no significant difference in Al and B1group. NANOG gene expression is highly expressed in the group AO, BO group decreased, while Al and B1group have declined, and the decline is more evident in group A1, A1and A2groups has declined. Protein levels and gene match. BO group of highly expressed genes TBX1, B1expression and no significant difference BO,A1group declined, A2and B2groups were continuing to decline. Similar gene expression and protein levels. SOX17gene expression, the expression of both groups AO and BO, but not statistically significant. B1expression was significantly increased, but decreased expression Al. A2and B2expression decreased. Similar gene expression and protein levels. GATA4in gene expression. BO has expression, B1expression was increased, A1expression decreased. There was no statistical difference comparing A2and A1, B2expression decreased. Similar gene expression and protein levels. Gene expression of OCT4, AO and BO high expression group, but I saw no statistically significant difference. A1and B1group decreased expression, A1group decreased significantly. A2and B2are continuing to decline. Similar gene expression and protein levels. KLF4gene expression, BO lower than the expression A0, A1slightly lower than B0, B1, but increased significantly over B0. A3and B3were increased. Similar gene expression and protein levels.C-MYC gene expression increased in BO, B1expression increased, B2expression continues to increase, while the decrease in the expression of A1, A2expression continued to decline. Gene expression and protein levels in line. In AO and BO SOX2expression of genes, A1and B1were decreased significantly reduced A1. A2and B2significantly decreased.ConclusionMature adipocytes by digestion centrifugation manner, high purity can be obtained free of fat cells. Through the ceiling culture method can get DFAT cells. And can be passaged and cryopreservation process. Research can be carried out as a cell model.Through the cell morphology, different concentrations of Apicidin for DFAT adipogenic process had an impact.1, in normal culture medium environment, different concentrations of Apicidin inhibits adipogenesis of DFAT continues into fat, and have a certain de-differentiation effects.2, in the adipogenic-based culture, different concentrations of Apicidin inhibits adipogenesis of DFAT continues into fat, and have a certain de-differentiation effects.3, in the adipogenic environment, different concentrations of Apicidin DFAT were completely inhibited fibroblast-like cells into fat. After4Apicidin treatment. Adipogenic-based culture with DFAT cells, DFAT cells can occur adipogenic process. These conclusions indicate Apicidin completely inhibited adipogenesis process DFAT, and there dedifferentiation effect. And this phenomenon is reversible.Quantified by Oil Red O found that, with the increase of concentration Apicidin, which causes the effect of the more significant dedifferentiation, but found that the higher the concentration which causes a decrease in cell activity phenomenon is more obvious. By MTT assay for cell activity also confirms previous observations. With higher concentrations Apicidin, the lower the cell activity. Especially in the detection of several high-concentration group, the inhibition rate was not statistically significant. High concentrations were observed phenomenon is more obvious necrosis. By linear regression analysis to determine Apicidin DFAT cells for50%inhibition rate1.475μM/L. And Oil Red O and morphological observation, election1μM/L concentration for subsequent studies.Gene and protein by the key test results before and after treatment in the Apicidin. Expression of C-MYC gene causing our high interest. Because the replacement of normal medium containing Apicidin increased expression of C-MYC, and after replacing the medium does not contain a Normal expression of C-MYC Apicidin reduced. Gene expression and protein levels were also consistent. Gene expression and increases with increasing time. Already there is evidence that the C/EBPβ and C/EBPα expression sites between C-MYC expression sites. In adipogenic process feedback mechanism exists between C/EBPβ and C/EBPα. This experiment also proved that the gene expression in downstream C/EBPβ down, and that the elevated expression of the gene upstream of the phenomenon. Therefore, Apicidin inhibiting the adipocyte differentiation and induce the dedifferentiation maybe cause by the expression of C-MYC. According to the available information indicates that, C-MYC is likely to be achieved through the WNT path.