Knockdown of long non-coding RNA MIR4697 host gene inhibits adipogenic differentiation in bone marrow mesenchymal stem cells / 北京大学学报(医学版)

OBJECTIVE@#To preliminarily investigate the role of long non-coding RNA (lncRNA) MIR4697 host gene (MIR4697HG) in regulating the adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).@*METHODS@#For adipogenic differentiation, BMSCs were induced in adipogenic media for 10 days. The mRNA expression levels of lncRNA MIR4697HG and adipogenic marker genes including peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhanced binding protein α (CEBP/α) and adiponectin (ADIPQ) were detected by quantitative real-time polymerase chain reaction (qRT-PCR) at different time points (0, 1, 2, 3, 5, 7, 10 days). The MIR4697HG stable knockdown-BMSC cell line was generated by infection of MIR4697HG shRNA-containing lentiviruses. To avoid off-target effect, two target sequences (shMIR4697HG-1, shMIR4697HG-2) were designed. And then cells were induced to differentiate in adipogenic medium. Oil red O staining, Western blot and qRT-PCR were used to detect the effect of MIR4697HG knockdown on adipogenic differentiation of BMSCs.@*RESULTS@#The mRNA expression level of MIR4697HG was significantly increased during adipogenic differentiation (P < 0.01), and adipogenic differentiation of BMSCs was evidenced by upregulated mRNA levels of specific adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ. Observed by fluorescence microscopy, more than 90% transfected target cells expressed green fluorescent protein successfully after shMIR4697HG-1 group, shMIR4697HG-2 group and shNC group transfection for 72 h. And the transfection efficiency of MIR4697HG examined by qRT-PCR was above 60%. Then the BMSCs were treated with adipogenic media for 7 days and showed that the mRNA expression levels of adipogenesis-related genes including PPARγ, CEBP/α and ADIPQ were significantly decreased in the MIR4697HG knockdown group (P < 0.01), while the expression levels of PPARγ and CEBP/α proteins were decreased remarkably as well (P < 0.01). Consistently, MIR4697HG knockdown BMSCs formed less lipid droplets compared with the control BMSCs, which further demonstrated that MIR4697HG knockdown inhibited adipogenic differentiation of BMSCs.@*CONCLUSION@#lncRNA MIR4697HG played a crucial role in regulating the adipogenic differentiation of BMSCs, and MIR4697HG knockdown significantly inhibited the adipogenic differentiation of BMSCs. These data may suggest that lncRNA MIR4697HG could serve as a therapeutic potential target for the aberrant adipogenic differentiation-associated disorders including osteoporosis.

Mechanism of Bushen Huatan Prescription in Regulation of Osteogenesis and Adipogenic Differentiation and Improvement of Postmenopausal Bone Loss in Ovariectomized Rats with Osteoporosis / 中国实验方剂学杂志

ObjectiveTo explore the underlying mechanism of Bushen Huatan prescription in alleviating postmenopausal osteoporosis （PMOP） by maintaining the balance of osteogenesis and adipogenic differentiation in ovariectomized rats with osteoporosis. MethodSeventy-five 6-month-old non-pregnant female SD rats were randomly divided into sham-operation group， model group， atorvastatin group， liviol group， and Bushen Huatan prescription group. Bilateral ovaries were removed in the four groups except the sham-operation group， while only the same mass of adipose tissue around the ovaries was removed in the sham-operation group. On the 5th week after surgery， drugs were consecutively administrated for 8 weeks. Rats in the Bushen Huatan prescription group received 9.4 mg·kg-1 of the prescription， rats in the atorvastatin group received 0.92 mg·kg-1 of atorvastatin， rats in the Liviol group received 0.23 mg·kg-1 of liviol， and rats in the model group and the sham-operation group received saline once a day. Micro-computed tomography （Micro CT） was used to detect bone mineral density （BMD） of rat tibia in each group. Hematoxylin-eosin （HE） staining was used to detect the relative area of rat bone marrow adipose tissue （BMAT） in each group. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the relative expression levels of Runt-related transcription factor 2 （Runx2）， peroxisome proliferator-activated receptor （PPARγ）， leptin （LPN）， and leptin receptor （OBR） in bone tissues. ResultAs compared with the sham operation group， the BMD of rats in the model group decreased （P<0.05）， while the relative area of BMAT increased （P<0.05）. In addition， the expression levels of LPN， OBR， and Runx2 decreased in the model group （P<0.05）， while the level of PPARγ increased （P<0.05）. As compared with the model group， the BMD of rats in the atorvastatin group， the Livial group， and the Bushen Huatan prescription group increased （P<0.05）， and the relative area of BMAT decreased （P<0.05）. The expression levels of LPN， OBR， and Runx2 in these groups increased （P<0.05）， while the expression level of PPARγ decreased （P<0.05）. ConclusionBushen Huatan prescription plays the anti-osteoporosis role in the rat model of PMOP through up-regulating LPN and OBR in bone tissues and maintaining the balance of osteogenesis and adipogenic differentiation， thereby reducing postmenopausal bone loss and playing a role in the prevention and treatment of PMOP.

C/EBPb converts bovine fibroblasts to adipocytes without hormone cocktail induction

BACKGROUND: Adipogenesis and fibrogenesis can be considered as a competitive process in muscle, which may affect the intramuscular fat deposition. The CCAAT/enhancer-binding protein beta (C/EBPb) plays an important role in adipogenesis, which is well-characterized in mice, but little known in bovine so far. RESULTS: In this study, real-time qPCR revealed that the level of C/EBPb was increased during the developmental stages of bovine and adipogenesis process of preadipocytes. Overexpression of C/EBPb promoted bovine fibroblast proliferation through mitotic clonal expansion (MCE), a necessary process for initiating adipogenesis, by significantly downregulating levels of p21 and p27 (p < 0.01). Also, the PPARc expression was inhibited during the MCE stage (p < 0.01). 31.28% of transfected fibroblasts adopted lipid-laden adipocyte morphology after 8 d. Real-time qPCR showed that C/EBPb activated the transcription of early stage adipogenesis markers C/EBPa and PPARc. Expression of ACCa, FASN, FABP4 and LPL was also significantly upregulated, while the expression of LEPR was weakened. CONCLUSIONS: It was concluded C/EBPb can convert bovine fibroblasts into adipocytes without hormone induction by initiating the MCE process and promoting adipogenic genes expression, which may provide new insights into the potential functions of C/EBPb in regulating intramuscular fat deposition in beef cattle.

Effect of platelet-derived growth factor (PDGF-BB) and bone morphogenic protein 2 (BMP-2) transfection of rBMSCs compounded with platelet-rich plasma on adipogenic differentiation

The aim of this study was to inhibit adipogenic differentiation by transfecting two growth factors, platelet-derived growth factor (PDGF-BB) and bone morphogenic protein 2 (BMP-2), into modified rat bone marrow mesenchymal stem cells (rBMSCs) and then compounded with platelet-rich plasma (PRP). To achieve rBMSCs, the osteoporosis model of rats was established, and then the rBMSCs from the rats were isolated and identified. Co-transfection of rBMSCs with PDGF-BB-GFP and BMP-2 and detection of PDGF-BB/BMP-2 expression in transfected BMSCs was assessed by qRT-PCR and western blot, respectively. Moreover, the effect of the two growth factors transfection of rBMSCs on adipogenic differentiation was evaluated by oil red O staining and western blot, respectively. Finally, construction of the two growth factors transfection of rBMSCs compounded with PRP and detection of adipogenic differentiation were assessed by oil red O staining, CCK-8, and western blot, respectively. In vitro studies revealed that the two growth factors transfection of rBMSCs compounded with PRP promoted cell viability and inhibited adipogenic differentiation and could be promising for inhibiting adipogenic differentiation.

Effects of astragalus polysaccharide on radiation-induced adipogenic differentiation of bonemarrow mesenchymal stem cells / 西安交通大学学报(医学版)

Objective: To study the effects of astragalus polysaccharide (APS) on adipogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) irradiated by X-ray. Methods: Human bone marrow mesenchymal stem cells irradiated by 2 Gy X-ray were intervened with 50 μg/mL of APS. The number and size of lipid droplets of stem cells were observed by oil red O staining and the expressions of CEBPα and PPAR-γ were detected by Western blot after induction with special medium. Results: The number of adipocytes differentiated from bone marrow mesenchymal stem cells decreased, and the area of lipid droplets in adipocytes decreased significantly after irradiation (P<0.05). The area of lipid droplets in the bone marrow mesenchymal stem cells treated with APS in advance increased compared with that in radiation alone (P<0.05). Similarly, the expressions of PPAR-γ and recombinant human CCAAT enhancer binding protein (CEBPα ) decreased after X-ray irradiation, while the expressions of PPAR-γ and CEBPα increased after the intervention of APS (P<0.05). Conclusion: X-ray can damage the directional adipogenic differentiation of human bone marrow mesenchymal stem cells, and APS has a protective effect on the adipogenic differentiation of human bone marrow mesenchymal stem cells after X-ray irradiation.

Acellular adipose tissue preparation: A soft-tissue filler allowing for allogeneic injection or in situ adipogenesis? / 中国组织工程研究

BACKGROUND: Construction of seedless tissue-engineered adipose tissue from acellular adipose tissue is a hot research topic in soft tissue filling. OBJECTIVE: To investigate the effects of preparation methods of acellular adipose tissue on the induction of adipose regeneration after transplantation in recent years, and to look forward to its clinical application prospects. METHODS: A computer-based online search of PubMed and Elsevier databases was performed to retrieve papers regarding acellular adipose tissue preparation and transplantation published between January 1971 and December 2018 with the search terms “adipose tissue engineering; adipose tissue extracellular matrix; soft tissue repair; angiogenesis; adipogenic induction”. The retrieved papers were summarized from the perspectives of improvement in preparation methods of acellular adipose tissue, cross-linking cytokines and biomaterials. RESULTS AND CONCLUSION: Retrieved studies have shown that extracellular matrix of adipose tissue can act as an ideal scaffold material for soft tissue filling. Subcutaneous implantation of extracellular matrix of adipose tissue can recruit host stem cells and induce their proliferation and adipogenesis. However, existing acellular schemes can lead to the loss of extracellular matrix proteins and structures. This greatly affects the fat regeneration ability of acellular adipose tissue implanted in vivo. However, supercritical carbon dioxide deoiling, mechanical pretreatment, cross-linking cytokines or biomaterials can reduce the loss of extracellular matrix proteins and supplement the proteins that promote tissue regeneration during the preparation of acellular adipose tissue. This can ultimately enhance the angiogenesis and adipogenesis of acellular adipose tissue after transplantation. Acellular adipose tissue has strong application prospects in adipose tissue engineering because of its natural adipogenic induction ability. If the loss of extracellular matrix protein can be overcome during preparation of acellular adipose tissue or under the premise of safety and controllability, acellular adipose tissue is expected to become a suitable soft tissue filler that allows allogeneic injection and in situ adipogenesis.

Regulatory effect of uncarboxylated osteocalcin on osteogenic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions / 中国组织工程研究

BACKGROUND: The method of promoting osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions to inhibit adipogenic differentiation can provide prevention and treatment ideas for the treatment of bone metabolic diseases such as diabetic osteoporosis. OBJECTIVE: To explore the effects of uncarboxylated osteocalcin on adipogenic and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions so as to reveal the action mechanism of uncarboxylated osteocalcin on the differentiation of bone marrow mesenchymal stem cells. METHODS: Mouse bone marrow mesenchymal stem cells were cultured by whole bone marrow culture and adherent purification. Cells were treated with uncarboxylated osteocalcin at different concentrations (0, 1, 3, 10, and 30 μg/L). Cell proliferation was detected by cell counting kit-8 to determine the best mass concentration. Passage 3 bone marrow mesenchymal stem cells were incubated with adipogenic (or osteogenic) differentiation medium, and assigned to four groups: control group, high glucose group, uncarboxylated osteocalci n group, and high glucose + uncarboxylated osteocalcin group. Corresponding groups received the addition of 25.5 mmol/L exogenous glucose and 3 μg/L uncarboxylated osteocalcin. Lipid droplets and calcium nodules were detected by oil red and alizarin red staining. Quantitati ve reverse transcription-polymerase chain reaction was used to detect the relative expression levels of adipogenic marker genes (Fabp4, PPARγ, Adipsin and FAS) and osteogenic differentiation marker genes (Runx2, Osx, alkaline phosphatase, and type I collagen). Kits were used to detect alkaline phosphatase activity and type I collagen levels. The relative expression levels of P-Erk and P-AMPKα were detected using signal pathway specific inhibitors (PD98059 and BML) and western blot assay. RESULTS AND CONCLUSION: (1) Uncarboxylated osteocalcin 3 μg/L promoted cell proliferation (P < 0.01). (2) Uncarboxylated osteocalcin promoted the formation of calcium nodules (P < 0.01) in bone marrow mesenchymal stem cells under high-glucose conditions but inhibited the formation of lipid droplets (P < 0.05), down-regulating the relative expression levels of adipogenic marker genes (PFabp4 < 0.01; PPPARγ < 0.05; PAdipsin < 0.01; PFAS < 0.01), but increasing the relative expression levels of osteogenic differentiation marker genes (PRunx2 < 0.05; POsx < 0.05; PALP < 0.01; PCOLI < 0.01). Uncarboxylated osteocalcin increased alkaline phosphatase activity (P < 0.01) and type I collagen level (P < 0.05). (3) Uncarboxylated osteocalcin up-regulated the expression levels of P-Erk (P < 0.01) and P-AMPKα (P < 0.01) under high-glucose conditions. (4) These results indicate that uncarboxylated osteocalcin promoted osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions through Erk/AMPKα signaling pathway and inhibited adipogenic differentiation.

Research progress on the balance regulation of osteogenic differentiation and adipogenic differentiation in bone marrow mesenchymal stem cell / 解剖学报

Osteoporosis (OP), a systemic bone metabolism disease, mainly manifested in the decrease of bone mass, the increase of bone fragility and the microstructure degeneration of the bone. Along with the in-depth research of the pathogenesis of osteoporosis, the imbalance differentiation of bone marrow mesenchymal stem cell (BMSCs) (Osteogenic differentiation decrease and adipogenic differentiation increase) is the main reason that causes osteoporosis. In this paper, we summarize the signal pathways of osteogenic differentiation and adipogenic differentiation of BMSCs. Better understand these signal pathways is conducive to elucidate and treat osteoporosis.

MicroRNA-138 Suppresses Adipogenic Differentiation in Human Adipose Tissue-Derived Mesenchymal Stem Cells by Targeting Lipoprotein Lipase

PURPOSE: Adipogenic differentiation of adipose tissue-derived mesenchymal stem cells (AMSCs) is critical to many disease-related disorders, such as obesity and diabetes. Studies have demonstrated that miRNA-138 (miR-138) is closely involved in adipogenesis. However, the mechanisms affected by miR-138 remain unclear. This work aimed to investigate interactions between miR-138 and lipoprotein lipase (LPL), a key lipogenic enzyme, in AMSCs. MATERIALS AND METHODS: Human AMSCs (hAMSCs) isolated from human abdomen tissue were subjected to adipogenic differentiation medium. Quantitative real-time polymerase chain reaction and Western blot assay were applied to measure the expressions of miR-138, LPL, and the two adipogenic transcription factors cytidine-cytidine-adenosine-adenosine-thymidine enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). The relationship between miR-138 and LPL was predicted utilizing the miRTarBase database and validated by dual luciferase reporter assay. RESULTS: Showing increases in C/EBPα and PPARγ expression levels, hAMSCs were induced into adipogenic differentiation. During adipogenesis of hAMSCs, miR-138 expression was significantly downregulated. Overexpression of miR-138 by transfection inhibited hAMSCs adipogenic differentiation in vitro. Mechanically, LPL was a target of miR-138. LPL expression was upregulated during adipogenesis of hAMSCs, and this upregulation was reversed by miR-138 overexpression. Functionally, silencing of LPL by transfection exerted similar inhibition of the expressions of C/EBPα and PPARγ. Meanwhile, LPL ectopic expression was able to partly abolish the suppressive effect of miR-138 overexpression on adipogenic differentiation of hAMSCs. CONCLUSION: Upregulation of miR-138 inhibits adipogenic differentiation of hAMSCs by directly downregulating LPL.

Relationship between Wnt10b and bone morphogenetic protein-9 induced osteogenic differentiation of mesenchymal stem cells / 中国药理学通报

Aim To study the relationship between WntlOb and bone morphogenetic protein-9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs), and the molecular mechanisms underlying this process. Methods PCR, Western blot and histochemical staining were used to detect the effect of BMP9 on WntlOb and the effect of WntlOb on BMP9-induced osteogenic differentiation in MSCs. Meanwhile, real-time PCR, Western blot, oil red O staining, and flow cytometry assay were used to analyze the potential mechanism of Wnt10b affecting the function of BMP9. Results Wnt10b could be detected in C3H10T1/2, C2C12, MEFs and MC3T3-E1 cells. BMP9 up-regulated the expression of WntlOb in C3H10T1/2 cells. WntlOb enhanced the capability of BMP9 to increase the level of OCN and mineralization in C3H10T1/2 cells, and silencing Wnt10b attenuated these effects of BMP9. Wnt10b exhibited no substantial effect on cell cycle affected by BMP9, but it enhanced the effect of BMP9 on inducing phosphorylation of Smadl/5/8. While silencing Wnt10b attenuated this effect of BMP9. In addition, Wnt10b inhibited BMP9-induced adipogenic differentiation in C3H10T1/2 cells, and silencing Wnt10b promoted this effect of BMP9. Conclusions Wnt10b can promote BMP9-induced osteogenic differentiation of MSCs, which may be mediated through enhancing BMP/Smad signaling and reducing adipogenic differentiation.

Flavonoids kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone inhibit mitotic clonal expansion and induce apoptosis during the early phase of adipogenesis in 3T3-L1 cells / 中西医结合学报

OBJECTIVE@#Kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone (DTMC) are two major flavonoids found in Chromolaena odorata Linn. leaf extract. The aim of this study was to elucidate the mechanism by which these two flavonoids exerted their effect on adipogenesis. The inhibitory effect of kaempferide and DTMC on adipocyte differentiation and their mechanisms involving mitotic clonal expansion (MCE) and apoptosis during the early stage of adipogenesis were investigated.@*METHODS@#Confluent 3T3-L1 preadipocytes were induced to differentiate and exposed to the flavonoids during various phases of differentiation. Intracellular lipid accumulation, cell density and expression of the transcription factors peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins α were assessed using AdipoRed, Oil red O and Western blot assays. Effects of both flavonoids on cell proliferation and apoptosis were also determined by carboxyfluorescein diacetate succinimidyl ester and annexin V-fluorescein isothiocyanate/propidium iodide-staining assays, respectively.@*RESULTS@#Kaempferide and DTMC showed significant, concentration-dependent anti-adipogenic activity and effect on cell density in the early phase of adipogenesis. The expression of the transcription factors seemed to be reduced when the treatment was prolonged or in the early phase of adipogenesis. These flavonoids interrupted MCE via inhibition of preadipocyte proliferation and induction of apoptosis. DTMC was nearly three times more potent than kaempferide in inducing apoptosis.@*CONCLUSION@#Kaempferide and DTMC exerted their anti-adipogenic activity through inhibition of MCE, either by suppressing cell proliferation or by inducing apoptosis during the early phase of differentiation.

Molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rat bone marrow stromal stem cells (rBMSCs) / 中草药·英文版

Objective: The study was designed to investigate the molecular mechanism of quercitrin on osteogenic differentiation and adipogenic differentiation of rBMSCs. Methods: rBMSCs were harvested from SD rats, and determination of alkaline phosphatase (ALP) activity, quantification of mineralization by Alizarin Red S staining, and the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) by RT-PCR after rBMSCs stimulated by osteogenic induction with (0.1–10) µg/mL of quercitrin, quantification of Lipid droplet by Oil Red O staining and the mRNA expression of adipogenic differentiation marker (PPARγ C/EBPα and aP2) by RT-PCR after rBMSCs stimulated by adipogenic induction with (0.1-10) µg/mL of quercitrin. Results: Quercitrin can up-regulate the mRNA expression of osteogenic differentiation markers (Runx2, BMP-2, and OSX) and increase ALP activity and mineralization after osteogenic induction, on the other hand quercitrin can suppress the mRNA expression of adipogenic differentiation markers (PPARγ C/EBPα and aP2) and decrease lipid droplet after adipogenic induction. Conclusion: This study suggested that quercitrin not only stimulated osteogenic differentiation but also inhibited adipogenic differentiation of rBMSCs, which was associated with the up-regulation of Runx2, BMP-2, and OSX mRNA expression and the down-regulation of PPARγ C/EBPα and aP2 mRNA expression.

Effect of IL-1β on human synovial fluid-derived mesenchymal stem cells in the temporomandibular joint / 口腔疾病防治

Objective@#This study investigated the effects of interleukin-1β (IL-1β) on human synovial fluid-derived mesenchymal stem cells (hSFMSCs) in the temporomandibular joint.@*Methods @#hSFMSCs from synovial fluid samples of temporomandibular disorder (TMD) patients were cultured in vitro. hSFMSCs were divided into three groups with different concentrations of rhIL-1β in complete medium (α-MEM cell culture medium + 10% FBS + 1× GlutaMAX): 0 ng/mL IL-1β group, 1 ng/mL IL-1β group and 10 ng/mL IL-1β group. Changes in the rate of colony formation, growth curve, cell cycle and apoptosis of hSFMSCs under IL-1β stimulation were evaluated. The osteogenic, adipogenic and chondrogenic potential of the cells were also determined using histochemical and real-time fluorescence quantitative PCR methods. @*Results @#No significant differences in growth or proliferation capacity were observed in any IL-1β-stimulated group in comparisons of the colony-formation rate (F = 0.665, P=0.548), growth curve (F=0.001, P=0.999), cell cycle (FG1=0.773, PG1=0.503; FS =0.636, PS =0.562) or apoptosis (F=0.196, P=0.827) of the cells. However, the multidifferentiation capacity of hSFMSCs was affected in the inflammatory environment. Mineralized nodule and lipid clusters decreased significantly, and the gene expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), peroxisomal proliferative receptor G2 (PPARG2) and lipoprotein lipase (LPL) were suppressed significantly in IL-1β-mediated induction medium (P < 0.05). In general, cartilage pellets formed in all the IL-1β-mediated chondrogenic differentiation groups. The gene levels of sex-determining region Y-related high-mobility group box-9 (SOX9) and collagen II were decreased (P < 0.05), while that of matrix metalloproteinase 13 (MMP13) was increased significantly in the presence of IL-1β (P < 0.05). @*Conclusion@# IL-1β directly affects the multidifferentiation potential of hSFMSCs but not their cell growth or proliferation ability.

Effect of sinapine on H2O2-induced adipogenic differentiation of bone marrow mesenchymal stem cells / 中国病理生理杂志

AIM:To investigate the effects of sinapine,an effective monomer of Chinese medicine,on hydro-gen peroxide(H2O2)-induced adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry.The adipogenic differentiation of BMSCs was induced by H2O2,and the toxicity of sinapine on BMSCs was tested by CCK-8 assay.After the modeling method and the concentration range of sinapine were determined,the lipid droplets in the cells were detected by Oil Red O semi-quanti-tative assay,and the optimal drug concentration was selected.Finally,Oil Red O assay was observed 24 h after drug inter-vention,and the expression of adipogenic differentiation-related proteins,adipocyte protein 2(aP2),peroxisome prolifera-tor-activated receptor γ(PPARγ)and glucose transporter 4(Glut4),at mRNA and protein levels in the BMSCs was deter-mined by qPCR and Western blot.RESULTS:Treatment with H2O2at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes.Below the concentration of 40 μmol/L,sinapine had no toxicity to BMSCs.The best inhibitory concentra-tion of sinapine on adipogenic differentiation was at 15 μmol/L.The number of lipid droplets in sinapine(15 μmol/L) group was significantly lower than that in model group.In sinapine group,the expression of aP2,PPARγand Glut4 at mR-NA and protein levels was lower than that in model group(P<0.01).CONCLUSION: Sinapine inhibits H2O2-induced adipogenic differentiation of rat BMSCs.The mechanism may be related to the PPARγ/AMPK signaling pathway.

Experimental research of adipogenic differentiative ability of adipose tissue-derived stromal cells in patients with type 2 diabetes mellitus / 重庆医学

Objective To investiagte the adipogenic differentiative ability of adipose tissue-derived stromal cells(ADSCs) between the patients with type 2 diabetes mellitus(T2DM) and healthy persons.Methods The adipose tissues were taken from the adipose tissue in T2DM patients and healthy persons for separating and culturing ADSCs.The cells of third generation were taken for inoculation.The difference in cellular phenotype and growth speed were compared between the two groups.Adding adipogenesis inducing fluid,the adipogenic differentiative situation was observed in the two groups.The oil red O was added on 14 d for conducting the cell staining and observation.The oil red O was extracted by isopropanol,and the cellular absorbances were compared between two groups.Meanwhile,the expression of PPAR-γ,C/EBP-α and C/EBP-β on 14 d of adipogenic differentiation were compared between two groups by using qPCR method.Results The cellular phenotype and growth speed of ADSCs had no statisticat difference between T2DM patients and healthy persons.On 14 d of adipogenic differentiation,the oil red O absorbance value of AD-SCs in T2DM patients was significantly higher than that in the healthy persons,and the expression of PPAR-γ,C/EBP-α and C/EBP-β were significantly higher than those in the healthy persons.Conclusion The adipogenic differentiative ability of ADSCs in T2DM patients is obviously higher than that in healthy persons,which may be one of causes easy to be obese in T2DM patients.

Effects of Astragalus polysaccharides on adipogenic differentiation of bone marrow mesenchymal stem cells in low oxygen environment / 生物工程学报

To study the effect of Astragalus polysaccharide (APS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) cultured in hypoxic environment. The optimal APS concentration, which could promote the proliferation of BMSCs, was screened by methyl thiazolyl tetrazolium method. The concentration was used to intervene in BMSCs-induced by adipogenic differentiation fluid growing in different oxygen concentrations (3%, 6%, 10% and 20%). The formation of lipid droplets in the BMSCs-intervened was observed by oil red O staining under the optical microscope. The mRNA and protein levels of the lipid relating genes peroxisome proliferator activated receptor gamma 2 (PPAR-γ₂) and lipoprotein lipase (LPL) were detected by Real-time PCR and Western blotting, respectively. The results showed that, comparing with the control group, 40 μg/mL APS could significantly promote the proliferation of BMSCs under low oxygen concentration. A large amount of lipid droplets existed in BMSCs growing in the adipogenic inducing fluid containing 40 μg/mL APS and the hypoxic environment, and the protein and mRNA levels of PPAR-γ₂ and LPL also raised. It was worth noting that the phenomenon was more significant in 10% oxygen concentration, and the difference was statistically significant (P<0.05). 40 μg/mL APS had effect on promoting the proliferation and adipogenic differentiation of BMSCs cultured in hypoxic environment, and the effect was related to the concentration of oxygen of BMSCs-cultured.

Mesenchymal Stem Cell Differentiation into Adipocytes Is Equally Induced by Insulin and Proinsulin In Vitro

BACKGROUND AND OBJECTIVES: In advanced β-cell dysfunction, proinsulin is increasingly replacing insulin as major component of the secretion product. It has been speculated that proinsulin has at least the same adipogenic potency than insulin, leading to an increased tendency of lipid tissue formation in patients with late stage β-cell dysfunction. METHODS AND RESULTS: Mesenchymal stem cells obtained from liposuction material were grown in differentiation media containing insulin (0.01 μmol), proinsulin (0.01 μmol) or insulin+proinsulin (each 0.005 μmol). Cell culture supernatants were taken from these experiments and an untreated control at weeks 1, 2, and 3, and were stored at −80°C until analysis. Cell differentiation was microscopically supervised and adiponectin concentrations were measured as marker for differentiation into mature lipid cells. This experiment was repeated three times. No growth of lipid cells and no change in adiponectin values was observed in the negative control group (after 7/14/12 days: 3.2±0.5/3.3±0.1/4.4±0.5 ng/ml/12 h). A continuous differentiation into mature adipocytes (also confirmed by Red-Oil-staining) and a corresponding increase in adiponectin values was observed in the experiments with insulin (3.6±1.9/5.1±1.4/13.3±1.5 ng/ml/12 h; p < 0.05 week 1 vs. week 3) and proinsulin (3.3±1.2/3.5±0.3/12.2±1.2 ng/ml/12 h; p < 0.05). Comparable effects were seen with the insulin/proinsulin combination. CONCLUSIONS: Proinsulin has the same adipogenic potential than insulin in vitro. Proinsulin has only 10~20% of the glucose-lowering effect of insulin. It can be speculated that the adipogenic potential of proinsulin may be a large contributor to the increased body weight problems in patients with type 2 diabetes and advanced β-cell dysfunction.

Capsanthin Inhibits both Adipogenesis in 3T3-L1 Preadipocytes and Weight Gain in High-Fat Diet-Induced Obese Mice

Adipogenesis in murine preadipocyte 3T3L-1 has been used as a model system to study anti-obese bioactive molecules. During adipogenesis in 3T3-L1 preadipocytes, we found that capsanthin inhibited adipogenesis (IC₅₀; 2.5 μM) and also showed lipolytic activity in differentiated adipocytes from the preadipocytes (ED₅₀ ; 872 nM). We identified that the pharmacological activity of capsanthin on adipogenesis in 3T3-L1 was mainly due to its adrenoceptor-β2-agonistic activity. In high-fat diet animal model study, capsanthin significantly enhanced spontaneous locomotive activities together with progressive weight-loss. The capsanthin-induced activation of kinetic behavior in mice was associated with the excessive production of ATP initiated by both the enhanced lipolytic activity together with accelerated oxidation of fatty acids due to the adrenoceptor β2-agonistic activity of capsanthin. Capsanthin also dose-dependently increased adiponectin and p-AMPK activity in high fat diet animals, suggesting that capsanthin has both anti-obesity and insulin sensitizing activities.

Isolating culture of adipose mesenchymal stem cells in psoriasis vulgaris patients and differentiation into immune regulation function / 基础医学与临床

Objeetive To study the differences of the biological characteristics and immune regulation function of adipose mesenchymal stem cells (AMSCs)from psoriasis patients and healthy people.Methods AMSCs were isolated and cultured from human psoriatic and healthy adipose tissue,the phenotypes and cell cycle of AMSCs taken from three generation were detected by flow eytometry.Alkaline phosphate enzyme staining and oil red o staining were used respectively to identify their adipogenic and osteogenic capacity.Next,the levels of inflammation antimicrobial proinflammatory factor were detected by PCR and ELISA.Then gene expression profile of AMSCs were screen by gene expression profile chip,as so to bolting the the gene array related with immunology gene.Results There was no significant change in cell morphology,and cell surface markers were expressed high for CD29,CD44,CD73,while lower for CD31,CD45 and HLA-DR.AMSCs of psoriasis patients and healthy people both had the ability of adipogenic and osteogenic differentiation.But the cell cycle showed the third generation AMSCs proliferation rates were slower than that of normal control,as compared with healthy controls,adipogenic differentiation ability was stronger.What'more,the level of inflammatory cytokines in psoriasis group was lower than that in controls such asIL-10,IDO,TGF-β,on the contrary the levels of proinflammatory factor in psoriasis group were higher than that in controls,such as TNF-α,IFN-γ.In addition,gene chip results suggested that psoriasis group AMSCs had obvious expression differences on JAK-STAT pathway with healthy controls.Conclusions Compared with the control,there are significant differences in patients AMSCs proliferation and adipogenic differentiation ability,immune inflammation suppression control ability is weaken,this phenomenon may be associated with JAK-STAT immune pathways related to downgrade.

Effect of pinostrobin chalcone on adipogenic differentiation of mesenchymal stem cell C3H10T1/2 / 中国中药杂志

Chalcones is a flavonoid wildly presented in many herbs. It has the effect to inhibit cells adipogenic differentiation. In order to study the effect of pinostrobin chalcone extracted and isolated from leaves of hickoryes on the adipogenic differentiation of murine embryonic mesenchymal stem cell (C3H10T1/2), MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium] method was used to detect the cell proliferation; adipogenic differentiation was characterized by oil red O staining and isopropanol extraction; the triglyceride content was detected by GAP-PAP enzyme method; and the C3H10T1/2 cell differentiation into adipocytes was also examined by the mRNA and protein expression of PPARγ, C/EBPα and FABP4 by RT-PCR and Western blot respectively. Results indicated that pinostrobin chalcone almost had no effect on cell proliferation activity when the concentration was less than or equal to 50 μmol•L⁻¹; the oil red O staining, isopropanol extraction and GAP-PAP enzyme method showed that pinostrobin chalcone significantly decreased the C3H10T1/2 adipogenic differentiation and triglyceride content in the cytoplasm of adipocytes; the RT-PCR and Western blot analysis showed that pinostrobin chalcone can down-regulate the mRNA and protein levels of FABP4, PPARγ and C/EBPα in C3H10T1/2 cells(P<0.05 or P<0.01). The experiment results suggest that pinostrobin chalcone can inhibit C3H10T1/2 adipogenic differentiation.