OBJECTIVE: To establish an in vitro cell model simulating acute graft-versus-host disease (aGVHD) bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells (MSCs). METHODS: The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model, respectively. Bone marrow transplantation (BMT) mouse model (n=20) was established by being injected with bone marrow cells (1×10⁷ per mouse) from donor mice within 4-6 hours after receiving a lethal dose (8.0 Gy, 72.76 cGy/min) of γ ray general irradiation. A mouse model of aGVHD (n=20) was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells (1×10⁷ per mouse) and spleen lymphocytes (2×10⁶ per mouse). The blood was removed from the eyeballs and the mouse serum was aspirated on the 7^th day after modeling. Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2%, 5% and 10% BMT mouse serum and aGVHD mouse serum in the medium, respectively. The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining. The expression levels of related proteins PPARγ and CEBPα were detected by Western blot. The expression differences of key adipogenic transcription factors including PPARγ, CEBPα, FABP4 and LPL were determined by real-time quantitative PCR (RT-qPCR). RESULTS: An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established. Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10% compared with BMT serum. Western blot experiments showed that adipogenesis-related proteins PPARγ and CEBPα expressed in MSCs were down-regulated. Further RT-qPCR assay showed that the production of PPARγ, CEBPα, FABP4 and LPL, the key transcription factors for adipogenic differentiation of MSC, were significantly reduced. CONCLUSION The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.
Animals ; Mesenchymal Stem Cells/cytology* ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Adipogenesis ; Female ; Cell Differentiation ; Graft vs Host Disease/blood* ; Bone Marrow Cells/cytology* ; PPAR gamma/metabolism* ; Disease Models, Animal ; CCAAT-Enhancer-Binding Protein-alpha/metabolism*

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPβ and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.
3T3-L1 Cells ; Activating Transcription Factor 3/pharmacology* ; Adipocytes ; Adipogenesis/genetics* ; Animals ; CCAAT-Enhancer-Binding Protein-alpha/pharmacology* ; Cell Differentiation ; Goats ; Mice ; PPAR gamma/metabolism*

OBJECTIVE: To investigate the expression of C/EBPα gene in elderly patients with multiple myeloma （MM） and its prognostic significance. METHODS: Sixty-nine olderly patients with multiple myeloma (MM) treated in our hospital from February 2015 to October 2017 were selected and enrolled in the MM group, 38 healthy persons received physical examination were selected and enrolled in the control group. The bone marrow of 2 groups was collected and the mononuclear cells were isolated.The mRNA expression level of C/EBPα gene in mononuclear cells was determined by RT-PCR, the Western blot was used to detect the protin expression level of PBMNC C/EBPα, and the protein level of C/EBPα in bone marrow was detected by immunohistochemistry. The correlations of C/EBPα gene expression with the clinical characteristics and survival time in MM patients were analyzed. RESULTS: The expression level of mRNA and protein of C/EBPα in MM patients was significantly lower than that in the control group (P<0.05). The expression level of C/EBPα gene significantly correlated with the ISS stage, CRP, Calcium, β2-MG, LDH and the percentage of myeloma cells in MM patients (P<0.05). The expression of C/EBPα gene was not correlate with sex, age, immunoglobulin typing, Hb in MM patients (P>0.05).Immunohistochemical staining showed that the bone marrow samples of the control group were stained more deeply, and the staining intensity in bone marrow samples of MM patients with CR, PR and relapse was successively descended. The protein level of C/EBPα in CR patients with MM was significantly higher than that in PR and relapsed patients by Western blot (P<0.05). Kaplan-Meier survival analysis showed that OS and DFS in the patients with high expression of C/EBPα gene were higher than those in low expression group (P<0.05). Multivariate Cox regression analysis showed that CRP,ratio of myeloma cells and C/EBPα gene were independent factors affecting OS and PFS (P<0.05). CONCLUSION The expression level of C/EBPα gene in MM patients is low that may stimulate the genesis of MM, and the expression of C/EBPα gene closely relates with the development of MM disease.
Aged ; Bone Marrow ; CCAAT-Enhancer-Binding Protein-alpha ; Humans ; Multiple Myeloma ; genetics ; Neoplasm Recurrence, Local ; Prognosis

OBJECTIVETo study the expression of SUMO-modified CCAAT enhancer binding protein &alpha; (C/EBP&alpha;) in preterm rat model of bronchopulmonary dysplasisa (BPD) induced by hyperoxia exposure and its role.

METHODSEighteen preterm rats were randomly divided into an air group and a hyperoxia group (n=9 each). The model of BPD was prepared in preterm rats exposed to hyperoxia. The rats from the two groups were sacrificed on postnatal days 4, 7 and 14 respectively (3 rats at each time) and lung tissues were harvested. Periodic acid-Schiff (PAS) staining was used to observe the differentiation of rat lung tissues. Ki67 expression was detected by immunohistochemistry. Western blot was used to measure the protein expression of small ubiquitin-related modifier-1(SUMO1) and C/EBP&alpha;. A co-immunoprecipitation assay was performed to measure the protein expression of SUMO-modified C/EBP&alpha;.

RESULTSCompared with the air group, the hyperoxia group showed a decreased glycogen content in the lung tissue on postnatal day 4, and an increased content on postnatal days 7 and 14. Over the time of hyperoxia exposure, the hyperoxia group showed an increased expression of Ki67 in the lung tissue compared with the air group at all time points. Compared with the air group, the protein expression of C/EBP&alpha; increased on postnatal day 4 and decreased on postnatal days 7 and 14 in the hyperoxia group (P<0.05). The hyperoxia group had significantly upregulated expression of SUMO1 and SUMO-modified C/EBP&alpha; compared with the air group at all time points (P<0.05). In the hyperoxia group, the protein expression of SUMO-modified C/EBP&alpha; was positively correlated with the glycogen content (r=0.529, P<0.05) and the expression of Ki67 (r=0.671, P<0.05).

CONCLUSIONSHyperoxia may induce over-proliferation and differentiation disorders of alveolar epithelial cells in preterm rat model of BPD, possibly through an increased expression of SUMO-modified C/EBP&alpha.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; etiology ; metabolism ; pathology ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Cell Proliferation ; Disease Models, Animal ; Hyperoxia ; complications ; pathology ; Ki-67 Antigen ; analysis ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Sumoylation

The CCAAT enhancer binding protein α (C/EBP α:p42 and p30),which encoded by CCAAT enhancer binding protein α () gene,plays a pretty crucial role in the regulation of myeloid hematopoiesis.The disorder of CEBPA gene expression is an pivotal mechanism of acute myeloid leukemia (AML). The result of uncontrolled expression of C/EBP α gene is the over-expression of p30 and the incomplete loss of p42, both of which contribute to the occurrence of AML. Restoring the expression ratio of C/EBP α such as over-expression of p42 or blocking the carcinogenic pathway of p30 seems to be important for the treatment of AML caused by such causes. In order to better guide medical decision-making, this article reviews research progress on C/EBPα in the pathogenesis of AML.
CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Gene Expression Regulation, Neoplastic ; Hematopoiesis ; genetics ; Humans ; Leukemia, Myeloid, Acute ; physiopathology

OBJECTIVETo investigate the expressions of inflammation- and fibrosis-related genes in perinephric and subcutaneous adipose tissues in patients with adrenocorticotropic hormone (ACTH)-independent Cushing's syndrome.

METHODSThe perinephric and subcutaneous adipose tissues adipose tissues were obtained from 8 patients with ACTH-independent Cushing's syndrome undergoing laparoscopic retroperitoneal adrenalectomy. Real-time PCR was used to detect the mRNA expression levels of interleukin 6 (IL-6), tumor necrosis factor-&alpha; (TNF-&alpha;), matrix metallopeptidase 2 (MMP-2), TIMP metallopeptidase inhibitor 1 (TIMP-1), early growth response 1 (EGR1), CCAAT/enhancer binding protein β(CEBPβ), uncoupling protein 1(UCP-1), PPARγ coactivator 1 alpha (PGC1&alpha;) and cell death-inducing DFFA-like effector a (CIDEA).

RESULTSThe mRNA level of CIDEA was significantly higher in the perinephric adipose tissue (peri-N) than in the subcutaneous adipose tissue (subQ) (P<0.05). The expressions of CEBPβ, UCP-1, and PGC1&alpha; mRNA in the peri-N were similar with those in the subQ. The expressions of IL-6, TIMP1 and EGR1 mRNA in the subQ were significantly higher than those in the peri-N (P<0.05). No significant difference in TNF-&alpha; and MMP-2 mRNA levels was found between peri-N and subQ.

CONCLUSIONThe expression levels of the inflammation- and fibrosis-related genes are higher in the subQ than in the peri-N of patients with ACTH-independent Cushing's syndrome, suggesting that chronic exposure to endogenous hypercortisolism may cause adipose tissue dysfunction.

Adrenalectomy ; Adrenocorticotropic Hormone ; CCAAT-Enhancer-Binding Protein-beta ; metabolism ; Cushing Syndrome ; metabolism ; surgery ; Early Growth Response Protein 1 ; metabolism ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; metabolism ; Real-Time Polymerase Chain Reaction ; Subcutaneous Fat ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; Uncoupling Protein 1 ; metabolism

Obesity is associated with a number of metabolic abnormalities such as type 2 diabetes and has become a major health problem worldwide. In the present study, we investigated the effects of Epimedium koreanum Nakai (Herba Epimedii, HE) and its main constituent icariin on the adipocyte differentiation in 3T3-L1 preadipocytes. HE extract and icariin significantly reduced lipid accumulation and suppressed the expressions of PPARγ, C/EBPα, and SREBP-1c in 3T3-L1 adipocytes. They also inhibited fatty acid synthase (FAS), acyl-Co A synthase (ACS1), and perilipin. Moreover, HE extract and icariin markedly increased the phosphorylation of AMPK. These results indicated that HE extract and icariin can inhibit the adipocyte differentiation through downregulation of the adipogenic transcription factors, suggesting that HE containing icariin may be used as a potential therapeutic agent in the treatment and prevention of obesity.
3T3-L1 Cells ; Adipocytes ; cytology ; drug effects ; metabolism ; Adipogenesis ; drug effects ; Animals ; CCAAT-Enhancer-Binding Protein-alpha ; genetics ; metabolism ; Epimedium ; chemistry ; Flavonoids ; pharmacology ; Lipid Metabolism ; drug effects ; Mice ; PPAR gamma ; genetics ; metabolism ; Plant Extracts ; pharmacology ; Sterol Regulatory Element Binding Protein 1 ; genetics ; metabolism

OBJECTIVE: To explore the effect of H. pylori on the expression of CCAAT enhancer binding protein α (C/EBPα) and connexin 43 (Cx43) in gastric carcinogenesis.
 METHODS: Different gastric mucosal epithelial cell lines (GES-1 cells, AGS cells and SGC-7901 cells) were cultured. A total of 6 cases of chronic non-atrophic gastritis tissues, 12 cases of gastric precancerous lesions tissues and 12 cases of gastric cancer tissues were collected under endoscopy. The expression of C/EBPα and Cx43 mRNA in the above-mentioned cells and tissues was detected by real-time PCR. The GES-1 cells and East Asian CagA+ H. pylori strains were co-cultured for 24 and 48 h as an experimental group, and those cell lines without H. pylori infection were cultured for 24 and 48 h as a control group. Real-time PCR and Western bolt were used to detect the expression of mRNA and proteins of C/EBPα and Cx43 in GES-1 cells, respectively.
 RESULTS: The expressions of C/EBPα and Cx43 mRNA in the AGS and SGC-7901 cells were lower than those in GES-1 cells (all P<0.05), and both of them decreased more profoundly in the SGC-7901 cells than those in the AGS cells (both P<0.05). The expressions of C/EBPα and Cx43 mRNA were lower in the gastric cancer tissues than those in the chronic non-atrophic gastritis tissues (both P<0.05) and gastric precancerous lesion tissues (both P<0.05). The C/EBPα expression was positively correlated with Cx43 expression (gastric precancerous lesion tissues: r=0.679, gastric cancer tissues: r=0.792; both P<0.05). Compared with the control group, the expressions of C/EBPα and Cx43 mRNA and protein in the experimental group were decreased at 24 and 48 h after culture (both P<0.05), which were lower at 48 h than those at 24 h (both P<0.05).
 CONCLUSION H. pylori infection can down-regulate the expressions of C/EBPα and Cx43 on gastric epithelial cells, which may be associated with gastric carcinogenesis.
CCAAT-Enhancer-Binding Protein-alpha ; Cell Line ; Cell Transformation, Neoplastic ; Connexin 43 ; Down-Regulation ; Gastric Mucosa ; Helicobacter Infections ; Helicobacter pylori ; Humans ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Stomach Neoplasms

OBJECTIVETo compare the adipogenesis and the adipocyte function between 3T3-L1 cells and human bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODSBy density gradient centrifugation and adherent culture, the MSCs were isolated from human bone marrow and purified. The cell morphology was observed under an inverted microscope. After the induction of adipogenic differentiation, the differentiation level was detected by oil red O staining and OD values. The expression of PPARγ, FABP4 and C/EBP&alpha; mRNA was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Adipocytes and THP-1 cells were co-cultured by adding 1 µg/ml cytarabine. The ability of chemotherapy resistance was measured after 48 h.

RESULTSThe Oil Red O staining and measuring the absorbance showed that the lipid content in 3T3-L1 cells group was more than that in MSCs group, and the OD value was higher than that in MSCs group (P < 0.05). The results of qRT-PCR showed that the relative expression of PPARγ, FABP4 and C/EBP&alpha; mRNA of 3T3-L1-derived adipocytes was higher than that of human bone marrow MSCs-derived adipocytes (P < 0.05). Coculture experiments showed that the number of viable THP-1 cells in the group containing adipocytes was more than that in the control group (P < 0.05). The difference between 3T3-L1 cell group and MSC group was statistically significant (P < 0.05).

CONCLUSIONThe ability of adipogenesis of 3T3-L1 cells is higher than that of human bone marrow MSCs in vitro. Adipocytes can protect THP-1 cell line against cytarabine, and the effect of adipocytes from 3T3-L1 cell group is greater than that from the human bone marrow MSC group.

3T3-L1 Cells ; Adipocytes ; Adipogenesis ; Animals ; Bone Marrow Cells ; CCAAT-Enhancer-Binding Protein-alpha ; Cell Differentiation ; Fatty Acid-Binding Proteins ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; Mice ; PPAR gamma ; RNA, Messenger

OBJECTIVEOur research was aim to investigate the effect of microRNA-328 (miR-328) on proliferation of chronic myeloid leukemia(CML) cell K562 and the mediated effect of C/EBP&alpha;.

METHODSThe eukaryotic expression vectors of miR-328 targeting gene and suppressor gene (hsa-miR-328 and hsa-miR-328-inhibitor) were constructed, and transfected into K562 cells respectively. The mRNA expression levels of miR-328 and C/EBP &alpha; were detected by real-time fluorescence quantitative RT-PCR; C/EBP &alpha; protein expression was detected by Western blot; CCK-8 was used to estimate the cell viability.

RESULTSThe recombinant genes of hsa-miR-328 and hsa-miR-328-inhibitor were successfully constructed and transfected into K562 cells. Fluorescent cells were observed after 24 h, and the visible fluorescence cells were gradually increased after 48 h or 72 h, the miR-328 showed no effect on the mRNA expression of C/EBP&alpha; detected by RT-PCR. Meanwhile, miR-328 showed recovering effect on C/EBP&alpha; translation and inhibition of K562 cells proliferation.

CONCLUSIONmiR-328 has been successfully constructed and transfected into K562 cells, miR-328 inhibits the proliferation of K562 cells by up-regulation of C/EBP&alpha;.

CCAAT-Enhancer-Binding Protein-alpha ; Cell Proliferation ; Cell Survival ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; MicroRNAs ; Transfection ; Up-Regulation