Young and old adipocytes have differential influence on the development of osteoblasts

ObjectiveThe aim of the current study was to investigate the effect of adipocytes on the differentiation of osteoblasts at different stages of adipocyte development.MethodsBMSCs were isolated from 4-week-old male wistar rat femurs and tibias, and flow cytometry was performed. Adipocytes were derived from BMSCs, cell morphology was continually observed from day 21 to day 50. Adipocyte medium was collected once every 2 days (d) and ELISA kits were used for detection of triglycerides (TG), tumor necrosis factor-α (TNF-α), and interleukin-6(IL-6) expression level. 21 d and 40 d old adipocyte and osteoblast cells were co-cultured, and alizarin red staining was performed after 21 d. After co-culture, the adherent cells were collected, and the expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) was detected by real time PCR.ResultResults of cell characterisation showed that the cells had positive expression of CD29 (97.92%) and CD44 (89.32%). With the increase of the induction time of mature adipocytes, the number of adipocyte on 21th d was significantly higher than 40th d, while the volume of adipocyte was significantly lower than 40th d (P < 0.05). The levels of TG(2.6 ± 0.83 mmol/l VS 3.8 ± 0.66 mmol/l), TNF-α(30.5 ± 2.53 pg/ml VS 57.6 ± 5.1 pg/ml), and IL-6(32.5 ± 1.42 pg/ml VS 55.1 ± 5.97 pg/ml) secreted by adipocytes increased with induction time: 40th d was significantly higher than 21th d (P < 0.01). When 21th d adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly increased over that of the positive control group, When 40th d adipocytes and osteoblasts were co-cultured, the number of calcium nodules significantly decreased over that of the positive control group (P < 0.05). The OPG(68.9 ± 5.39 VS 1.00 ± 0.36) expression was significantly increased, and the expression of RANKL (2.0 ± 0.84 VS 34.4 ± 2.01) was significantly decreased from the 21th d adipocytes co-cultured group compared with the 40th d adipocytes co-cultured group (P < 0.001).ConclusionThe differential size of adipocytes in the bone marrow can affect bone metabolism by regulating the expression of OPG/RANKL.     

促酰化蛋白受体C5L2在前脂肪细胞分化过程和脂肪酸负荷下的表达研究

目的 观察3T3-L1前脂细胞分化和游离脂肪酸（FFA）对3T3-L1（前）脂肪细胞C5L2基因和蛋白表达的影响。方法采用逆转录（RT）-PCR和流式细胞仪检测不同分化时段和FFA处理后（前）脂肪细胞C5L2mRNA和蛋白表达。结果 3T3-L1脂肪细胞C512mRNA表达呈分化依赖性增加,而C5L2蛋白表达水平在分化早期显著增强,诱导分化6hC5L2蛋白表达增加了21％（61％±18％VS51％±15％,P〈0．05）;分化12h达高峰,增加了38％（70％±12％VS51％±15％,P〈0．01）;分化3d基本恢复至0d水平。在3T3-L1成熟脂肪细胞,0．5mmol／L和1．0mmol／L油酸分别抑制46％（0．58±0．21 vs 1．08±0．46,P〈0．05）和84％（0．18±0．04VS1．08±0．46,P〈0．05）C5L2 mRNA表达,而0．125mmol／L油酸即能显著下调36％（35％±8％vs54％±7％,P〈0．01）C5L2蛋白表达。低浓度棕榈酸均能明显抑制C5L2mRNA和蛋白的表达,1．0mmol／L时c512mRNA和蛋白表达分别减少了41％（0．57±0．28vs0．97±0．41,P〈0．05）和55％（24％±13％VS54％±7％,P〈0．01）。油酸和棕榈酸对前脂肪细胞C512表达差异无统计学意义。结论促酰化蛋白／C5L2途径参与了脂肪细胞分化的调控过程。C512mRNA和蛋白表达的下调可能参与了油酸和棕榈酸诱导的成熟脂肪细胞胰岛素抵抗的发生。     

脂肪干细胞复合脱细胞真皮基质用于声带注射的实验研究

目的 探索脂肪干细胞(adipose-derived stem cell,ASC)与脱细胞真皮基质微粒(micronized acellular dermal matrix,MADM)复合培养后用于声带内注射的可行性.方法 体外分离培养兔ASC,取第3代细胞,用细胞膜红色荧光探针(DiI)标记,并与已制备好的MADM复合培养,形成细胞和材料复合体,应用荧光显微镜、扫描电镜了解ASC黏附材料情况,四唑类化合物3-(4,5-dimethylthizazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium,inner salt(MTS)比色法检测细胞增殖情况.ASC-MADM复合体培养3 d后与适量胶原混合后注入异体兔单侧声带内,术后2、4、8周内镜观察,并分别处死动物,检测ASC在声带中存活、分布情况.结果 MADM所含胶原纤维松散、多孔隙,具有良好三维结构;ASC可黏附其生长并能不断增殖,与没有复合MADM的对照组比较细胞增殖水平差异有统计学意义(P值均＜0.05或＜0.01).ASC-MADM复合体注入兔声带肌后均存活良好,连续内镜观察、冰冻切片、HE染色显示声带局部无不良反应,ASC-MADM复合体在兔声带内基本无炎症细胞浸润,ASC可在声带内存活8周.结论 MADM是较理想的细胞支架材料,ASC能黏附MADM生长并增殖.ASC-MADM复合体异体动物声带内注射可存活且无不良反应.

Abstract：

Objective To explore the feasibility of adipose-derived stem cells (ASC) combined with micronized acellular dermal matrix (MADM) for vocal cord injection. Methods The adipose-deprived stem cells were harvested from rabbit adipose tissue in vitro. The 3rd generation of ASC was labeled with DiI ( 1, 1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate) and cultured with MADM to form a complex. The adhesion of ASC to MADM was observed by fluorescence microscope and electron microscope.The proliferation of ASC on MADM was evaluated by 3-(4, 5-dimethylthizazol-2-yl)-5-( 3-carboxy methoxyphenyl)-2-(4-sulfonyl )-2H- tetrazolium, inner salt (MTS). Three days after the culture, the complex was mixed with appropriate amount of collagen, and then injected into the unilateral vocal cord of the rabbit. The animals were sacrificed 2, 4, 8 weeks after injection, the survival time and distribution of ASC in vocal fold were tested, and the responses of vocal cord to ASC-MADM and the degradation of MADM were observed. Results The ASC adhered to MADM and grew well (P ＜0. 05 or ＜0. 01 ), showing good compatibility with MADM in vocal cord tissue. The complex of ASC-MADM could be injected into the rabbit vocal cords, while no adverse reactions was observed in the vocal cord by endoscope, frozen section and HE staining. ASC could survive for 8 weeks in vocal cords, and no inflammatory cell infiltration was observed.Conclusions MADM is an ideal scaffold material and shows perfect compatibility with ASC which can adhere and proliferate well on it. The complex of ASC-MADM can be injected into the vocal cord and can survive. There is no adverse reaction in vocal cords.     

炎症转录因子C/EBPβ在间质细胞分化中作用与机制

免疫炎症性转录因子C/EBPβ在多种细胞的分化过程中起着重要作用.如在间充质细胞中,决定着间充质干细胞(MSCs)的分化方向,诱导脂肪细胞的分化,调节成骨细胞和软骨细胞分化.了解C/EBPβ及其不同亚型的在不同的间充质细胞中的作用,尤其是在成纤维细胞、脂肪细胞、软骨细胞和成骨细胞分化过程中的调控机制很有必要,随着间充质干细胞被再生医学重视,转录因子C/EBPβ有望成为未来研究的热点.     

Rosiglitazone balances insulin-induced exo- and endocytosis in single 3T3-L1 adipocytes

Rosiglitazone (Rosi) improves insulin sensitivity and increases the translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This involves the fusion of membrane-bound compartments with the plasma membrane, thus increasing the plasma membrane area. However, recent work has shown that in Rosi-pretreated 3T3-L1 adipocytes membrane area did not increase following insulin application, suggesting that the rates of exo- and endocytosis are balanced. Here we examined whether Rosi differentially affects the rates of exo- and endocytosis in 3T3-L1 adipocytes. The immunolabelling of GLUT4 revealed the 3.1-fold increase in PM-resident GLUT4 in Rosi-pretreated, insulin-stimulated cells. By monitoring cumulative exocytosis and endocytosis we found that in Rosi-pretreated cells insulin substantially stimulated the rate of exocytosis and to a similar extent also the rate of endocytosis. We conclude that Rosi-pretreatment balances insulin-stimulated exocytosis and endocytosis, which may prevent insulin-mediated adipocyte cell size increase.     

脂肪细胞线粒体功能与肥胖

线粒体主要存在于能量需求高、代谢强的组织,被认为是细胞的"动力工厂",为细胞的代谢活动提供能量.肥胖的发生主要是脂质积聚过多,而脂肪细胞线粒体是脂肪酸β氧化的场所之一,脂肪细胞线粒体的脂肪酸氧化功能对于维持脂肪细胞内甘油三酯代谢有重要作用,其功能失调与肥胖的关系成为新的研究热点.

Abstract：

Mitochondria are usually described as the "powerhouse unit" of the cell, which exist in tissues with a high energy demand and a high metabolic activity, and provide energy for the metabolic activity of cell. More lipid accumulation can lead to obesity. Mitochondria are the main sites of fatty acid β oxidization which are very important to adipocyte triglyceride metabolism. The relationship between mitochondrial dysfunction and obestiy has become the new research derection.     

骨关节炎患者髌下脂肪垫前脂肪细胞成脂诱导模型的建立

目的 观察骨关节炎患者髌下脂肪垫前脂肪细胞的原代培养及成脂诱导过程.方法 采用酶消化法分离得到前脂肪细胞,进行细胞形态学观察、CCK-8法测定生长曲线;成脂诱导过程中,油红O染色方法鉴定脂肪细胞、酶联免疫吸附法测定细胞上清脂联素.结果 原代培养的前脂肪细胞呈梭形,与成纤维细胞类似.成脂诱导第3天胞质内出现反光脂质小滴,第21天80%以上的前脂肪细胞可分化为脂肪细胞.细胞上清脂联素随脂肪细胞数量的增多而升高,3周时达到稳定,证实成脂诱导的细胞为功能活跃的脂肪细胞.结论 成功建立了骨关节炎患者髌下脂肪垫前脂肪细胞的原代培养及成脂诱导方法,为研究髌下脂肪垫的内分泌代谢功能及其在骨关节炎发病机制中的作用提供较为理想的细胞模型.

Abstract：

Objective To investigate the primary culture and adipogenic process of pre-adipocytes from infrapatellar fat pad of osteoarthritic patients. Methods The pre-adipocytes were isolated by enzymatic digestion. The morphological changes of cultured cells were observed and the growth curve was drawn by CCK-8 method. During the adipogenic process, the intracytoplasmic lipid of differentiated cells was determined by oil red O staining. And the adiponectin levels in the culture supernatants were measured by ELISA (enzyme-linked immunosorbent assay). Results The primary cultured fibroblast-like cells were spindle-shaped. In the process of adipogenesis, the intracytoplasmic lipid droplets were observed at Day 3and over 80% of the cells differentiated into adipocytes at Day 21. With the increasing number of adipocytes, the adiponectin levels in the culture supemant elevated and peaked at Week 3. The differentiated cells were proven to be adipocytes functioning actively. Conclusion The primary culture and adipogenic process of pre-adipocytes in infrapatellar fat pad of osteoarthritic patients has been successfully established. Thus it may provide an ideal model for the study of endocrine function of infrapatellar fat pad and understanding its role in the pathogenesis of osteoarthritis.     

Constitutively active Stat5A and Stat5B promote adipogenesis

Objective  

The metabolic syndrome is an important social problem affecting many people in developed countries. Obesity is a leading cause of this syndrome, hence understanding molecular mechanisms underlying obesity is of prime importance for preventive medicine to develop novel methods to alleviate the corresponding social cost as well as for pharmaceutical companies to develop antimetabolic drugs.     

De Novo and Depot-Specific Androgen Production in Human Adipose Tissue: A Source of Hyperandrogenism in Women with Obesity

IntroductionObesity in women is often associated with hyperandrogenism, but the role of adipose tissue (AT) in androgen synthesis remains unclear. Therefore, we studied whether AT could be a source of androgens promoting hyperandrogenism.MethodsSubcutaneous and visceral (visc) AT was collected from lean and obese women. Androgen levels were evaluated in serum, AT, and cell-culture supernatant. Gene and protein expression of steroidogenic enzymes were determined.ResultsObese subjects had elevated serum androgen levels, which reduced after weight loss. Androgens were measurable in AT and in cell-culture supernatants of adipocytes. Steroids were higher in AT from obese women, with the highest difference for testosterone in visc AT (+7.9-fold, p = 0.032). Steroidogenic enzymes were expressed in human AT with depot-specific differences. Obese women showed a significantly higher expression of genes of the backdoor pathway and of CYP19 in visc AT.ConclusionThe whole steroidogenic machinery of the classical and backdoor pathways of steroidogenesis, and the capacity for androgen biosynthesis, were found in both AT depots and cultured adipocytes. Therefore, we hypothesize that AT is a de novo site of androgen production and the backdoor pathway of steroidogenesis might be a new pathomechanism for hyperandrogenism in women with obesity.