复方补肾活血颗粒含药血清通过Trb3调控hBMSCs成骨成脂分化

目的 研究复方补肾活血颗粒含药血清通过Trb3调节人骨髓间充质干细胞(human bone mesenchymal stem cells, hBMSCs)成骨/成脂分化。方法 不同浓度复方补肾活血颗粒含药血清干预hBMSCs,通过CCK8法检测细胞活力,ALP活性、茜素红染色观察hBMSCs成骨分化,油红O染色鉴定脂肪分化。Western blot检测成骨/成脂标志物蛋白表达。Western blot和qPCR检测Trb3蛋白和基因表达。Trb3 siRNA转染hBMSCs后通过Western blot观察成骨成脂相关因子蛋白表达情况。结果复方补肾活血颗粒含药血清以浓度依赖性促进hBMSCs增殖。复方补肾活血颗粒含药血清增强hBMSCs中ALP活性及矿化结节,上调Runx2、Osterix蛋白表达并抑制PPARγ、FABP4蛋白表达和脂质积累。机制研究发现,复方补肾活血颗粒含药血清促进Trb3表达,当内源性Trb3敲低时,逆转了复方补肾活血颗粒含药血清促进成骨分化抑制脂肪分化的作用。结论 复方补肾活血颗粒含药血清通过Trb3以牺牲脂肪分化为代价,促进hBMSCs成骨分化。     

骨髓脂肪细胞在激素性骨坏死发生过程中的时序变化特点

目的血管外脂肪堆积如何参与激素性骨坏死的发病机制目前仍然不清楚。本课题应用建立的激素性骨坏死家兔模型研究骨髓脂肪细胞在骨坏死发生过程中组织病理形态学上的时序变化特点。方法32只28-32周龄成年新西兰大白兔分为正常对照组（n=16）和激素处理组（n=16）。激素处理组动物接受1次耳静脉内毒素注射和连续3次肌内糖皮质激素注射诱导骨坏死,正常对照组注射相应剂量的生理盐水。各组动物在最后一次注射糖皮质激素2周（早期）和4周（晚期）以后分别处死8只动物,获取双侧近端股骨,应用组织形态计量学技术分别测定股骨髓内脂肪细胞密度、脂肪细胞直径和脂肪细胞面积,同时进行骨坏死的组织病理学评价。结果激素处理早期组和晚期组骨髓脂肪细胞密度分别明显高于正常对照组67.1%和54.4%,但是早期组与晚期组之间差异不显著。激素处理晚期组骨髓脂肪细胞直径明显高于正常对照组,但是早期组骨髓脂肪细胞直径与正常对照组比较有减小趋势;激素处理早期组和晚期组骨髓脂肪细胞面积都分别明显高于正常对照组44%和83.6%,且晚期组亦显著高于早期组;脂肪细胞直径的频数分布显示,正常对照组峰值介于40-50μm,激素处理早期组较正常对照组增多的脂肪细胞直径介于30-40μm,晚期组较正常对照组增多的脂肪细胞直径介于50-60μm。组织病理学评价显示早期组骨坏死发生率为25%（2/8）,晚期组骨坏死发生率为87.5%（7/8）。结论在激素性骨坏死发生过程中,早期血管外脂肪堆积表现为大量生成的小脂肪细胞,后期血管外脂肪堆积表现为脂肪细胞出现肥大。     

血管瘤演变过程中过氧化物酶体增殖物激活受体-γ基因表达规律及与脂肪形成的关系

目的 探讨血管瘤演变过程中,过氧化物酶体增殖物激活受体-γ基因(Peroxisome proliferator activated receptors-γ,PPAR-γ)表达规律及其和脂肪形成的关系.方法 以Perilipin A为标志蛋白进行常规免疫组化染色,观察各阶段血管瘤中的脂肪形成.应用荧光免疫组化染色,观察控制脂肪形成的关键转录因子PPAR-γ在血管瘤组织中的表达定位,以α-SMA和CD31作共染色.应用RT-PCR检测PPAR-γ基因在各阶段血管瘤组织中的表达.结果 在血管瘤演变过程中,瘤体内脂肪组织逐渐增多,最终被纤维脂肪组织代替.PPAR-γ主要表达于微血管周围的细胞核.增生期、消退期和消退完成期血管瘤组织的PPAR-γ基因相对表达强度逐渐增高,但低于正常皮下脂肪组织.结论 PPAR-γ主要表达于微血管周围细胞,提示可能与血管瘤消退期脂肪形成有关.     

CCNG2 and CDK4 is associated with insulin resistance in adipose tissue

BackgroundThe involvement of cyclin G2 (CCNG2) and cyclin-dependent kinase-4 (CDK4), cell cycle regulatory proteins, in adipose tissue metabolism and insulin resistance is still unknown. The objective of this study was to analyze CCNG2 and CDK4 levels in visceral (VAT) and subcutaneous adipose tissue (SAT) from nonobese and morbidly obese patients and their relationship with insulin resistance.MethodsWe studied the mRNA and protein levels of CCNG2 and CDK4 in VAT and SAT from 12 nonobese and 23 morbidly obese patients (11 with low [MO-L-IR] and 12 with high insulin resistance [MO-H-IR]).ResultsThe nonobese patients had a significantly greater CCNG2 expression in VAT (P = .004) and SAT (P<.001) than the MO-L-IR and MO-H-IR patients. The MO-H-IR patients had a significantly lower CDK4 expression in VAT than the MO-L-IR (P = .026), but similar to the nonobese patients. CDK4 and CCNG2 expression correlated significantly in VAT (r = 0.511, P<.001) and SAT (r = .535, P = .001). In different multiple regression analysis models, CCNG2 and CDK4 expression in VAT was mainly predicted by glucose (P = .047 and P = .008, respectively), and CCNG2 expression in SAT was mainly predicted by body mass index (P = .041). No significant associations were found with CDK4 expression in SAT. Moreover, VAT CCNG2 expression was the main determinant of the improvement in the homeostasis model assessment of insulin resistance index at 3 months after bariatric surgery (B = -271.7, P = .026).ConclusionOur data show for the first time that the human CCNG2 and CDK4 expression of VAT are inversely associated with glucose and insulin resistance.     

Effects of sulfonylurea drugs on adiponectin production from 3T3-L1 adipocytes: implication of different mechanism from pioglitazone

Adiponectin is a fat-derived cytokine with anti-diabetic and anti-atherogenic properties. In this study, effects of sulfonylureas (SUs) on adiponectin production and the action mechanism were evaluated using 3T3-L1 adipocytes. The cells were incubated with glimepiride, glibenclamide, gliclazide, pioglitazone, metformin and the medium only as the control. In the control, the adiponectin level evaluated as the production rate per 24 h was not changed, while pioglitazone significantly increased the adiponectin level. SUs also increased the adiponectin level, but metformin failed to show any increase in adiponectin production. SUs induced adiponectin gene expression as well as pioglitazone. Pioglitazone significantly increased adipogenesis, but glimepiride did not. The aP2 gene expression was increased by pioglitazone, but not by glimepiride. Forskolin, a protein kinase A stimulator, reduced the adiponectin production stimulated by glimepiride but not by pioglitazone. These observations strongly suggest that SUs stimulate the adiponectin production through a different mechanism from pioglitazone, namely an interaction with protein kinase A activity. The significance of the extrapancreatic action of SUs observed in this study should be further evaluated in the clinical field.     

Effect of Ichnocarpus frutescens (L.) R. Br. hexane extract on preadipocytes viability and lipid accumulation in 3T3-L1 cells

ObjectiveTo investigate the crude extracts of Ichnocarpus frutescens (I. frutescens) for antiobesity effect.MethodsLeaves of I. frutescens were sequentially extracted with hexane, ethyl acetate, and methanol and their effect on viability of 3T3-L1 preadipocytes were evaluated. Based on this the apoptosis on preadipocytes was confirmed by DNA fragmentation and LDH (Lactate dehydrogenase) leakage assays. Anti-adipogenesis was performed by oil red O (ORO) staining and free glycerol release in the medium of differentiated adipocytes.ResultsThe hexane extract of I. frutescens (IFHE) inhibited cell viability in a time- and dose-related manner. An increased release of LDH, as a marker of membrane integrity, was observed at a dose of 200 μ g/mL. The discontinuous DNA fragments on agarose gel electrophoresis showed the apoptotic effect of the IFHE. Morphological observations of cells stained with ORO showed a decrease in cellular lipid content at the concentrations tested compared to the induced control cells. In the experiment of lipolytic activity, treatment with IFHE enhanced glycerol secretion with the rates of approximately 28%, 55%, and 46% at the concentrations of 100, 200 and 300 μ g/mL, respectively.ConclusionsThe observed properties clearly revealed the medicinal property of I. frutescens in the treatment of obesity.     

Adipose tissue and adipocyte dysregulation

Obesity-associated insulin resistance is a complex disorder involving a number of candidate molecules, pathways and transduction systems possessing potential causal actions. Inflammation in adipose tissue (AT) is one mechanism proposed to explain the development of insulin resistance, while identification of factors that lead to or cause AT dysfunction when it reaches its limit of expansion represents an important challenge. Pathological expansion of AT is characterized by changes in its blood flow, and the presence of enlarged and dysfunctional adipocytes that begin an inflammatory campaign of altered adipokine and cytokine secretions. Adipocyte senescence, necrosis and death are associated with increased immune cell and macrophage infiltration of AT in obesity. This can boost inflammation and reinforce fat cell dysfunction and death. In addition, pathological fat mass expansion is also related to limited recruitment of fat cell progenitors able to proliferate and differentiate into healthy small fat cells to compensate for cell death and preserve adipocyte numbers. Limiting vascular development and enhancing fibrotic processes worsen inflammation towards chronic irreversibility. The AT expandability hypothesis states that failure of AT expansion is one of the key factors linking positive energy balance and cardiometabolic risks, not obesity per se. Besides the usual treatment of obesity based on behavioral approaches (specific dietary/nutritional approaches together with increased physical activity), a number of questions remain concerning the possible recovery of metabolic health after inflammation-preventing interventions.     

Role of microRNAs in obesity and the metabolic syndrome

Obesity and the metabolic syndrome are major public health concerns, and present a formidable therapeutic challenge. Many patients remain recalcitrant to conventional lifestyle changes and medical therapies. Bariatric surgery has made laudable progress in the treatment of obesity and its related metabolic disorders, yet carries inherent risks. Unravelling the molecular mechanisms of metabolic disorders is essential in order to develop novel, valid therapeutic strategies. Mi(cro)RNAs play important regulatory roles in a variety of biological processes including adipocyte differentiation, metabolic integration, insulin resistance and appetite regulation. Investigation of these molecules and their genetic targets may potentially identify new pathways involved in complex metabolic disease processes, improving our understanding of metabolic disorders and influence future approaches to the treatment of obesity. This review discusses the role of miRNAs in obesity and related components of the metabolic syndrome, and highlights the potential of using miRNAs as novel biomarkers and therapeutic targets for these diseases.     

大鼠前脂肪细胞的原代培养分化

 目的 建立一种简便的大鼠前脂肪细胞的原代培养方法。 方法 无菌取成年Wistar大鼠的腹股沟纯脂肪颗粒，采用酶消化法进行原代培养。在倒置显微镜下观察细胞形态及生长情况，以油红O脂肪染色法进行原代培养细胞的鉴定。 结果 培养细胞的形态学观察显示，脂肪细胞贴壁后初为类圆形，3 d 后渐成梭形；7~8 d 细胞增殖明显，形状由多角梭形变为椭圆形，并开始积聚脂肪颗粒；10 d 后细胞呈椭圆形、圆形，胞内积聚大量脂肪颗粒。培养 10 d 的细胞经油红 O 染色后于普通光镜下观察，见细胞内出现红染颗粒，可以鉴定细胞分化为前脂肪细胞。 结论 成功建立了大鼠前脂肪细胞的原代培养方法，为脂肪内分泌学研究提供了制备适用细胞模型的手段。