抗波形纤维蛋白单抗对大鼠前体脂肪细胞增殖分化及波形纤维形态的影响

将抗波形纤维蛋白单抗通过显微注射导入未分化的大鼠前体脂肪细胞的细胞质,观察其对细胞增殖、分化,及其中等纤维——内源波形纤维形态的影响。以注射人IgG和未注射的前体脂肪细胞作为对照,发现注射单抗的前体脂肪细胞呈现以下特征：(1)细胞呈典型的凋亡形态,经Hoechst33342着染可见胞核呈致密浓染的颗粒状或块状荧光,胞内脂滴几乎没有;(2)增殖的细胞数和甘油三酯值均极显著低于对照组;(3)波形纤维蛋白间接免疫荧光染色微弱,纤维束解聚成短小团块状,分布无序、弥散。上述结果表明,抗波形纤维蛋白单抗对前体脂肪细胞的增殖和分化有明显阻滞作用。同时为揭示波形纤维蛋白可能参与了脂肪细胞形成这一重要生理过程提供了证据[动物学报49(6)：807～812,2003]。     

过表达miR-103促进猪前体脂肪细胞分化

为阐明miR-103在猪前体脂肪细胞分化过程中的调控作用,采用Real-time PCR检测猪前体脂肪细胞成脂分化过程中的miR-103表达谱,明确了其在分化过程中的表达趋势;使用miR-103的腺病毒超表达载体感染猪原代脂肪细胞,随后采用Real-time PCR和Western blotting分别检测成脂标记基因PPARγ、aP2的mRNA和蛋白表达量变化;油红O染色观察腺病毒miR-103侵染的前体脂肪细胞诱导分化第8天的成脂情况。结果显示,miR-103的表达量随着脂肪细胞分化而增加,在miR-103超表达的猪原代脂肪细胞的诱导分化过程中,成脂标记基因PPARγ、aP2的表达量与对照相比显著升高,分化第8天观察到明显的脂滴。说明miR-103能够促进猪前体脂肪细胞分化。     

用成熟脂肪建立一种新的猪前体脂肪细胞培养模型

用去分化的成熟脂肪细胞建立一种新的具有再增殖和再分化能力的猪前体脂肪细胞模型. 用“天花板” 培养法分离、培养1～3日龄仔猪皮下成熟脂肪细胞, 显微镜下观察细胞形态变化并计数, 流式细胞术检测细胞周期;油红O染色法检测脂肪细胞分化率, RT-PCR分析前体脂肪细胞标志基因Pref-1及成熟脂肪细胞关键转录因子PPARγ和C/EBPα等mRNA表达情况. 发现刚贴壁的细胞为单室脂滴成熟脂肪细胞, 油红O染色完全阳性; 14d后这种成熟脂肪细胞完全去分化为无脂滴的纤维状细胞, 并表达前体脂肪细胞标志基因Pref-1, 油红O染色阴性. 这种去分化的前体脂肪细胞在成脂诱导剂作用下,可重新分化为成熟的脂肪细胞. 结果证实,成熟脂肪细胞去分化后的前体脂肪细胞可重新增殖、分化为成熟脂肪细胞, 是一种新的有效的前体脂肪细胞模型.     

黄芩素对猪前体脂肪细胞增殖分化的影响

研究黄芩素(BAI)对猪前体脂肪细胞增殖分化的影响,并探讨其可能的作用机制。原代培养猪前体脂肪细胞,采用油红O染色观察细胞分化的形态学变化;MTT检测细胞增殖状况;油红O染色提取定量分析细胞内脂肪生成及细胞分化程度;分光光度法测定脂肪酸合酶(FAS)的活性;逆转录-聚合酶链反应(RT-PCR)检测分化特异基因过氧化物酶体增殖物激活受体γ2(PPARγ2)mRNA表达变化。结果显示,前体脂肪细胞在分化成脂肪细胞的过程中,其形态由梭形变成椭圆形、圆形,细胞内充满大小不一的脂滴;BAI浓度在160～640μmol/L时显著抑制其增殖(P<0.05)、BAI浓度为40～320μmol/L时显著抑制PPARγ2mRNA表达和FAS的活性,并抑制细胞分化(P<0.05)。以上结果说明,BAI对前体脂肪细胞增殖分化均有一定抑制作用,BAI可能通过抑制PPARγ2mRNA表达和降低FAS活性,从而抑制猪前体脂肪细胞分化。     

CRISPR/Cas9敲除PLIN1基因增强3T3-L1脂肪细胞的脂解作用

应用CRISPR/Cas9技术敲除3T3-L1前脂肪细胞plin1,观察PLIN1缺失对脂肪细胞中脂肪水解的影响并探究可能机制。常规培养3T3-L1前脂肪细胞,电穿孔法转染plin1敲除载体,嘌呤霉素培养基挑选plin1敲除细胞,观察转染及筛选后的细胞存活率。"鸡尾酒"法诱导3T3-L1前脂肪细胞分化,酶法测定甘油和TG含量,油红O染色观察脂滴形态及数目的变化。Western blotting检测PLIN1、PPARγ、Fsp27和脂肪酶的蛋白表达;RT-PCR检测PLIN1和脂肪酶的mRNA表达。对照组细胞诱导分化后,微小脂滴数目较少,单房脂滴数目较多并围绕细胞核呈环型排列。相较于对照组,敲除组细胞诱导分化后微小脂滴数目增加,单房脂滴体积缩小,数目减少;细胞中PLIN1mRNA及蛋白表达被显著抑制(P0.05);甘油水平显著上升(0.0984±0.0076),TG含量显著下降(0.031 0±0.005 3);HSL和ATGL两种脂肪酶的mRNA及蛋白表达均升高(P0.05);PPARγ和Fsp27的表达未有明显变化。上述结果表明plin1敲除后通过暴露脂滴中脂质以及上调脂肪酶等效应增强了3T3-L1脂肪细胞的脂解作用。     

长链非编码RNA AK043773在脂肪细胞分化中的作用

探索长链非编码RNA(lnc RNA)AK043773在脂肪细胞分化过程中的表达变化和调控作用。将小鼠前脂肪细胞成脂诱导,利用实时定量PCR技术检测成脂分化调控基因KLF7及AK043773的表达,构建AK043773过表达载体检测其对成脂分化的影响。经UCSC数据库检索发现AK043773位于KLF7基因4号外显子的3'端下游,骨髓基质细胞系ST2和前脂肪细胞系3T3-L1成脂诱导分化后,实时定量PCR检测结果显示AK043773及KLF7表达协同下调;构建p CDNA3.1-AK043773过表达载体,转染ST2细胞后能显著提高AK043773的表达,进行脂肪细胞分化条件诱导,通过油红O染色及OD520nm吸光值检测可见AK043773过表达显著抑制ST2细胞中脂滴产生。AK043773及KLF7在脂肪细胞分化过程中协同下调,上调AK043773的表达可显著抑制脂肪细胞分化。     

CTSB超表达促进体外培养的猪前体脂肪细胞分化

为研究溶酶体组织蛋白酶B(cathepsin B,CTSB)对脂肪细胞分化的影响,本实验构建了Ctsb重组腺病毒超表达载体,包装并侵染体外培养的猪前体脂肪细胞,采用油红O染色,油红O提取比色法检测猪前体脂肪细胞分化的情况,并通过real-time PCR法检测成脂关键基因mRNA水平的变化.结果显示,重组腺病毒Ctsb载体构建成功,转染猪前体脂肪细胞后,使Ctsb的mRNA和蛋白质表达量分别提高了约16倍和12倍. CTSB超表达能促进脂肪细胞的分化和脂质积累,成脂关键基因过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma, PPARγ)、脂肪酸结合蛋白2(adipocyte protein 2, aP2)的表达量均有显著升高. 研究表明,提高Ctsb的表达能促进猪前体脂肪细胞分化,揭示了Ctsb在猪前体脂肪细胞分化过程中可能发挥关键作用. 研究结果为进一步研究其作用机制奠定了基础.     

Leptin过表达对猪前脂肪细胞脂滴形成的研究

研究Leptin过表达对猪前脂肪细胞脂滴形成的影响,旨为进一步研究Leptin与脂质代谢相关的分子机制奠定理论基础。选取Leptin过表达与野生型猪皮下脂肪组织,在无菌条件下分离前脂肪细胞进行传代培养并诱导分化形成脂滴,通过油红O和Bodipy染色后观察脂滴面积并分析脂质的含量,利用Q-PCR检测脂滴形成相关基因mRNA的表达水平。诱导4 d后可分化形成脂滴,油红O和Bodipy染色的结果显示,Leptin过表达猪前脂肪细胞脂滴数量和甘油三酯含量显著低于野生型(P0.05);且脂质合成相关基因PPARγ、SCAP、SREPB1和PLIN2的表达水平显著低于野生型(P0.01)。结果表明,过表达Leptin可促使猪前脂肪细胞中PPARγ、SREPB1、SCAP、PLIN2基因的表达下调,进而抑制脂滴形成。     

罗格列酮和血清脂对绵羊前体脂肪细胞分化的影响

目的探讨罗格列酮(rosiglitazone,Ros)和血清脂(serum lipid,Lip)对绵羊前体脂肪细胞分化的影响及不同组织来源的前体脂肪细胞分化影响的差异。方法用不同浓度的Ros和(或)Lip培养绵羊皮下前体脂肪细胞和肾周前体脂肪细胞,通过测量3-磷酸甘油脱氢酶(GPDH)活性和油红O染色萃取液A值分析前体脂肪细胞的分化程度和脂肪细胞充脂量的变化,应用实时荧光定量PCR检测PPARγ和LPL mRNA的表达水平。结果 Ros和Lip提高细胞GPDH活性和脂滴的沉积量(P<0.05),上调LPL mRNA表达(P<0.05),最佳浓度分别为100nmol/L和20μL/mL;最佳浓度条件下Ros的诱导作用强于Lip(P<0.05),Ros显著提高了PPARγmRNA表达量(P<0.05),而Lip对PPARγmRNA的表达没有明显影响(P>0.05);Ros和Lip共同诱导与Ros单独作用之间没有明显差异(P>0.05);在相同诱导分化条件下,皮下前体脂肪细胞的分化程度高于肾周前体脂肪细胞(P<0.05)。结论研究结果表明Ros和Lip可促进绵羊前体脂肪细胞的分化,在相同条件下,皮下前体脂肪细胞的分化能力强于肾周前体脂肪细胞。     

MiR-130a 在猪皮下脂肪细胞分化中的调节作用

为研究miR-130a对猪皮下脂肪细胞分化的影响及可能机制,本试验分离猪皮下脂肪前体细胞,诱导分化为成熟脂肪细胞,检测脂肪细胞分化过程中脂滴变化及miR-130a及其可能靶基因TNF α和PPARγ的表达模式.同时合成miR-130a mimics及inhibitor 对细胞进行转染,并以乱序序列作为阴性对照（NC）.细胞转染24 h后进行诱导分化,连续诱导8 d,检测各处理细胞的聚脂情况及甘油三酯含量变化,荧光定量PCR检测脂肪细胞分化相关基因的表达变化.结果显示,猪皮下脂肪前体细胞分化过程中脂滴逐渐变大增多,miR-130a、TNF α和PPARγ的表达模式具有一定的相似性.转染结果显示,相对于对照组,miR 130a mimics转染组细胞脂滴减少变小,甘油三酯含量降低（P<0.05）,脂肪细胞分化相关基因LPL、PPARγ、adiponectin、FASN和葡萄糖转运相关基因GLUT1,GLUT4以及JNK通路上的PDE3B的表达均比对照组显著下调（P<0.01）;而miR-130a inhibitor转染组细胞则脂滴增多,甘油三酯含量提高（P<0.05）,但大部分分化相关基因的表达与对照组无显著差异,提示miR-130a可能不只通过单一的靶基因影响脂肪细胞分化.其结果为后续深入研究miR-130a调节猪脂肪细胞分化的通路及机制奠定基础.     

Rearrangement of the vimentin cytoskeleton during adipose conversion: formation of an intermediate filament cage around lipid globules

During adipose conversion of murine 3T3-L1 cells, the arrangement of vimentin intermediate filaments (IFs) changes from an extended fibrillar state to a complex cage formation tightly associated with the forming lipid globules. The fully developed cage complex surrounding the lipid globule consists of a monolayer of groups of regularly spaced vimentin IFs that in turn is closely ensheathed by a special endoplasmic reticulum cisterna. The same IF cage is also seen in other adipocytes in culture and in tissues. The specificity of the association of lipid globules with vimentin IFs during adipose conversion is discussed as a special form of compartmentalization supporting adipogenesis and is taken as an example of a possible IF function in relation to a cell differentiation process.     

Ultrastructural analysis of the in vitro differentiation of female rat preadipocytes

In an attempt to characterize the preadipocytes of the adipose tissue of female rat, we studied by electron microscopy the differentiation of the cells into mature adipocytes in in vitro cultures. The preadipocytes arose from the stroma-vascular fraction of perirenal and perigenital adipose tissue. Culture of the preadipocytes in an enriched medium consisting of Dulbecco's medium supplemented with 10% fetal calf serum, antibiotics, rat triglycerides (0.5%), insulin (290 nM) and Tween 80 (0.1 mg/ml) induced their adipose conversion. The morphology of preadipocytes changed progressively. They accumulated fat granules, droplets and finally globules, which fused together. The cell organelles featured qualitative and quantitative modifications. The nucleus migrated with most mitochondria and a part of the Golgi system towards the cell periphery; the rough endoplasmic reticulum, dilated at the initial stage of differentiation became less and less conspicuous; the perinuclear Golgi system was dispersed between lipid droplets during fat accumulation; thick bundles of microfilaments, localized beneath the plasma membrane disappeared; large lipid droplets were surrounded by a network of microfilaments; many microvesicles and some "rosettes" typical of mature adipocytes could be observed. Nevertheless, the ultrastructural criteria did not allow to clearly discriminate the undifferentiated cells: early preadipocytes (without lipid droplets), adipoblasts and fibroblasts, all of these being probably present in the culture system.     

Effects of angiotensin II on adipose conversion and expression of genes of the renin-angiotensin system in human preadipocytes.

A complete functional renin-angiotensin system exists in human adipose tissue, but its regulation and the effects of angiotensin II on cells from this tissue are only beginning to be understood. In this study, we examined the effects of angiotensin II on changes in lipid accumulation, specific glycerol-3-phosphate dehydrogenase activity, and the expression of five genes of the renin-angiotensin system during the adipose conversion of human primary cultured preadipocytes. Angiotensin II leads to a distinct reduction in insulin-induced differentiation, but only has a marginal effect on the adipose conversion of cells stimulated with insulin, cortisol, and isobutyl methyl xanthine. During differentiation, angiotensinogen mRNA levels rise, renin mRNA levels decline, whereas renin-binding protein and angiotensin-converting enzyme levels are unaffected. Angiotensin II downregulates angiotensinogen and renin gene expression, but it does not affect renin-binding protein and angiotensin-converting enzyme levels. Angiotensin II thus prevents the development of adipocytes in contact with high insulin levels, while not inhibiting differentiation, which is further stimulated. Therefore, angiotensin II could be a protective factor against uncontrolled expansion of adipose tissue. Further studies are needed to find out whether the effects of angiotensin II on the renin-angiotensin system are direct feedback loops or secondary to changes in the differentiation program.     

Expression of angiotensin II receptors type 1 and type 2 in human preadipose cells during differentiation.

Human adipose tissue expresses all the components necessary for the production of angiotensin peptides. Although local effects of angiotensin II on cells from adipose tissue are beginning to be recognised, the expression of angiotensin receptors on human preadipocytes and adipocytes is still controversial. This study addresses the issue by monitoring the mRNA levels as well as the protein production of angiotensin II receptors of type 1 and 2 (AT 1 and AT 2 ) during differentiation of primary human preadipocytes in culture and in mature adipocytes. mRNA levels of the two receptor types are inversely correlated during adipose conversion. AT 1 receptor mRNA is greatly diminished within 12 days after induction of differentiation, while AT 2 receptor mRNA is elevated. mRNA levels of mature adipocytes confirm this trend. The regulation is not seen as strongly on the protein level. The amount of AT 2 receptor protein is increased, correlating well with the rise in specific glycerol-3-phosphate dehydrogenase activity of the cells, but the AT 1 receptor protein does not vary during the whole differentiation period. As the functional role of AT 2 receptors in adipose tissue is not known to date, further studies have to show if the AT 1 -mediated inhibitory actions on adipose conversion are downregulated in differentiating cells through decreased AT 1 /AT 2 receptor ratio.     

Differential regulation of the stearoyl-CoA desaturase genes by thiazolidinediones in 3T3-L1 adipocytes

Two stearoyl-CoA desaturase (SCD) isoforms can be expressed during the differentiation of 3T3-L1 preadipocytes into adipocytes. Here we report on the effects of the peroxisome proliferator-activated receptor gamma ligand troglitazone (TRO) on scd1 and scd2 mRNA levels as determined by Northern blotting, on SCD protein expression as determined by Western blotting, and on total lipid composition as determined by GC during differentiation. In preadipocytes, scd1 mRNA and SCD protein were not detected, whereas scd2 mRNA was detected. These cells have high levels of palmitate (16:0), stearate (18:0), and monounsaturated oleate (Delta(9)-18:1) and low levels of monounsaturated palmitoleate (Delta(9)-16:1). In MDI (methylisobutylxanthine, dexamethasone, and insulin)-treated cells, scd1 mRNA and SCD protein were increased approximately 100-fold relative to preadipocyte levels, the scd2 mRNA level was increased 2-fold, Delta(9)-16:1 was increased approximately 20-fold, and 18:0 was decreased approximately 3-fold. In TRO-treated cells, the scd1 mRNA level was lower than that observed in preadipocytes, while the scd2 mRNA level was similar. TRO also decreased scd1 mRNA in primary adipocytes. The TRO-treated cells contained a Delta(9)-18:1 level typical of MDI-treated cells whereas, conversely, these cells also contained a low Delta(9)-16:1 level typical of preadipocytes. The implications of these correlations for the regulatory and enzymatic mechanism(s) used to establish and maintain lipid composition are discussed.     

Regulation of fibronectin, integrin and cytoskeleton expression in differentiating adipocytes: inhibition by extracellular matrix and polylysine

The differentiation of 3T3 preadipocytes into adipocytes is characterized by major changes in cell morphology from a fibroblastic to a rounded shape and by the induction of gene expression related to lipid metabolism. We have studied the synthesis and mRNA levels of proteins involved in the formation of cell-matrix contacts and in defining cell shape to determine the role and molecular basis of these morphological changes during adipose conversion. When confluent preadipocyte cultures were stimulated with adipogenic medium there was a gradual decrease in the expression of fibronectin, beta-integrin, actin and in the microfilament-associated proteins vinculin, alpha-actinin and tropomyosin. The changes in extracellular matrix and cytoskeletal mRNA levels were apparent before the accumulation of glycerophosphate dehydrogenase (GPD) mRNA and continued during the massive increase in GPD mRNA level. The culturing of preadipocytes on an extracellular matrix deposited on the dish by corneal endothelial cells, or on substrata coated with polylysine, prevented the morphological changes, the decrease in the level of assembled actin, the accumulation of lipid and the shifts in the expression of integrin, cytoskeletal proteins and GPD. In cells cultured on malleable hydrated collagen gels, adipocyte differentiation proceeded at normal rates. The results suggest that the regulated expression of proteins involved in the formation of the transmembrane linkage between the extracellular matrix and the microfilaments are programmed regulatory events that affect cell adhesion and thereby cell shape during adipocyte differentiation.     

Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.     

Postnatal catch-up growth after fetal protein restriction programs proliferation of rat preadipocytes

We studied the in vitro proliferation and differentiation of rat preadipocytes to investigate whether catch-up growth after prenatal protein restriction may program adipose precursor cells leading to development of increased adipose tissue mass. Pregnant rat dams were fed either an isocaloric low-protein diet (LP-8%) or control diet (C-20%). During lactation, in order to induce catch-up growth, dams from LP group were fed with the C diet and litter size was reduced to four pups instead of eight. Preadipocytes were isolated from weanling male pups (28 days of age). Differentiation and proliferation were assessed across time. At late stages of preadipocyte differentiation, no difference was observed in lipid accumulation of C or LP cultures but the mRNA expression of leptin was enhanced in LP cells. At early stages of culture, a higher DNA and protein content accompanied by a higher rate of proliferation was measured in adipocytes from LP cultures. Moreover, the mRNA expression of cyclin D1 was increased in these cells whereas the expression of peroxisome proliferators-activated receptor gamma (PPARgamma) and steroyl regulatory element binding protein (SREBP-1c) was significantly reduced during early stages. The results suggest that prenatal exposure to a LP followed by rapid catch-up growth is associated with a higher rate for proliferation in preadipocytes.     

FGF-10 is a growth factor for preadipocytes in white adipose tissue

FGF-10 is a mesenchymal factor affecting epithelial cells during pattern formation. However, the expression and physiological role of FGF-10 in adults remains to be elucidated. We examined the expression of FGF-10 mRNA in a variety of adult rat tissues, and found to be most abundant in white adipose tissue. In white adipose tissue, FGF-10 mRNA was expressed in preadipocytes but not in mature adipocytes. The expression in white adipose tissue during postnatal development was also examined. The expression level was low at postnatal day 10 (P10). However, FGF-10 mRNA was abundantly detected later on (P28 and P48) when white adipose tissue growth was stimulated. We also examined the activity of recombinant FGF-10 for primary rat preadipocytes. FGF-10 showed significant mitogenic activity for primary preadipocytes, but did not affect the differentiation of preadipocytes. The expression profile of FGF-10 mRNA and the activity of FGF-10 reported here indicate that FGF-10, a unique secreted factor produced in white adipose tissue, acts as a growth factor for preadipocytes in white adipose tissues.