The antiadipogenic effect of macrophage-conditioned medium depends on ERK1/2 activation

The proatherogenic state of obesity is associated with hypertrophied adipocytes that may arise because of deficient adipogenesis. Macrophages infiltrate adipose tissue as a function of obesity and may release factors that attenuate adipogenesis. Macrophage-conditioned medium inhibits human and 3T3-L1 adipocyte differentiation in culture, but underlying molecular mechanisms have yet to be defined. Exposure of 3T3-L1 cells throughout the 8-day period of differentiation to medium conditioned by THP-1 macrophages (THP-1-MacCM) blocked adipogenesis. Triacylglycerol (TG) accumulation and induction of peroxisome proliferator-activated receptor gamma and fatty acid synthase protein levels were inhibited by 59% (n = 4, P < .001), 29% (n = 4, P < .01), and 47% (n = 4, P < .01), respectively. THP-1-MacCM had no effect when added after the first 2 days of differentiation, indicating that early exposure of its targets must be needed to inhibit 3T3-L1 adipogenesis. Cell enumeration revealed a 44% decrease in clonal expansion compared with standard differentiation (n = 3, P < .01). Addition of THP-1-MacCM to 3T3-L1 preadipocytes increased ERK1/2 phosphorylation by 6.5-fold (n = 3, P < .01). PD98059 (an inhibitor of the ERK1/2 pathway) impaired the negative effect of THP-1-MacCM on TG accumulation, indicated by an inhibition of 25% vs 69% (n = 3, P < .001), without altering fatty acid synthase or peroxisome proliferator-activated receptor gamma levels. Our data implicate ERK1/2 as an important signaling mediator for the inhibitory effect of THP-1-MacCM on TG accumulation during 3T3-L1 adipogenesis.     

The activation state of macrophages alters their ability to suppress preadipocyte apoptosis

Adipose tissue contains macrophages whose state of activation is regulated as obesity develops. Macrophage-secreted factors influence critical processes involved in adipose tissue homeostasis, including preadipocyte proliferation and differentiation into adipocytes. Macrophage-conditioned medium (MacCM) from J774A.1 macrophages protects 3T3-L1 preadipocytes from apoptosis through platelet-derived growth factor (PDGF) signaling. Here, we investigated the effect of macrophage activation on MacCM-dependent preadipocyte survival. MacCM was prepared following activation of either J774A.1 macrophages with lipopolysaccharide (LPS) or human primary monocyte-derived macrophages (MD-macrophages) with LPS or interleukin 4 (IL4). 3T3-L1 and human primary preadipocytes were induced to undergo apoptosis in MacCM, and apoptosis was quantified by cell enumeration or Hoechst nuclear staining. Preadipocyte PDGF signaling was assessed by immunoblot analysis of phosphorylated PDGF receptor, Akt, and ERK1/2. Pro-inflammatory activation of J774A.1 macrophages with LPS inhibited the pro-survival activity of MacCM on 3T3-L1 preadipocytes, despite intact PDGF signaling. Upregulation of macrophage tumor necrosis factor a (TNFα) expression occurred in response to LPS, and TNFα was demonstrated to be responsible for the inability of LPS-J774A.1-MacCM to inhibit preadipocyte apoptosis. Furthermore, MacCM from human MD-macrophages (MD-MacCM) inhibited apoptosis of primary human preadipocytes. MD-MacCM from LPS-treated macrophages, but not IL4-treated anti-inflammatory macrophages, was unable to protect human preadipocytes from cell death. In both murine cell lines and human primary cells, pro-inflammatory activation of macrophages inhibits their pro-survival activity, favoring preadipocyte death. These findings may be relevant to preadipocyte fate and adipose tissue remodeling in obesity.     

Effect of collagen I and aortic carboxypeptidase-like protein on 3T3-L1 adipocyte differentiation

Aortic carboxypeptidase-like protein (ACLP) is a secreted protein expressed in preadipocytes and down-regulated during adipogenesis. Results from previous studies on the influence of ACLP overexpression on adipogenesis vary from no effect to complete inhibition. We hypothesized that ACLP may modulate adipogenesis in the presence of collagen I, a protein to which it binds. We compared control (pLXSN) 3T3-L1 preadipocytes with 3T3-L1 preadipocytes stably overexpressing ACLP (pLXSN-ACLP) that were grown in standard vs collagen I-coated dishes. Aortic carboxypeptidase-like protein overexpression, via retroviral transduction, resulted in a 3.2-fold increase in ACLP cellular levels and a 2.1-fold increase in ACLP levels released into medium. Aortic carboxypeptidase-like protein overexpression did not inhibit differentiation in standard dishes. In collagen I-coated dishes compared with standard dishes, control preadipocytes, when induced to differentiate, exhibited the same increase in triacylglycerol accumulation, but showed a significantly higher induction of fatty acid synthase (1.6-fold more), peroxisome proliferator-activated receptor γ (1.4-fold more), and CCAAT/enhancer-binding protein α (1.4-fold more). Aortic carboxypeptidase-like protein overexpression significantly reduced this enhanced induction of fatty acid synthase, peroxisome proliferator-activated receptor γ, and CCAAT/enhancer-binding protein α by 65%, 59%, and 66%, respectively, but had no effect on the accumulation of triacylglycerol during differentiation. Finally, studies on proadipogenic insulin signaling in ACLP-overexpressing preadipocytes demonstrated that insulin-stimulated Akt phosphorylation was significantly decreased by 27% in cells cultured in collagen I-coated dishes vs standard dishes. Our data suggest that ACLP inhibits certain aspects of 3T3-L1 adipogenesis in a collagen I-rich environment.     

Apoptosis of human abdominal preadipocytes before and after differentiation into adipocytes in culture

Differentiation of murine 3T3-L1 preadipocytes into adipocytes is associated with the acquisition of apoptotic resistance accompanied by the upregulation of cell survival genes. We have now examined the effect of adipogenesis on apoptotic susceptibility of human abdominal preadipocytes in primary culture. To induce apoptosis, human preadipocytes, or their differentiated counterparts, were serum-deprived for 24 or 48 hours. When indicated, ceramide was also used as an apoptotic trigger. Cell death was assessed by enumeration of adherent viable cells, and its apoptotic nature was verified by Hoechst staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After 48 hours of serum withdrawal, cell death was 26% +/- 4% in preadipocytes and was increased to 41% +/- 4% in differentiated adipocytes (mean +/- SE; n = 7 patients; P <.002). Under serum-free conditions for 24 hours, ceramide-induced cell death was 40% +/- 6% in preadipocytes and increased to 68% +/- 8% in adipocytes (mean +/- SE; P <.01; n = 8 patients). Neuronal apoptosis inhibitor protein (NAIP), an antiapoptotic protein cell survival that increases upon 3T3-L1 adipogenesis, was reduced in human preadipocytes undergoing differentiation (n = 6 patients). Preadipocytes derived from omental versus subcutaneous abdominal fat were more susceptible to apoptosis induced by serum deprivation, 16% +/- 4% versus 31% +/- 3% cell death, respectively (mean +/- SE; P <.02; n = 7 patients). Although the murine 3T3-L1 preadipocyte cell line is a useful model that approximates primary preadipocyte cell biology, our data derived from human preadipocyte studies suggest important differences with respect to the regulation of apoptosis.     

贝那普利、替米沙坦对人前脂肪细胞脂联素表达的影响

目的探讨贝那普利、替米沙坦对体外原代培养的人网膜和皮下来源的前脂肪细胞脂联素(APN)表达的影响。方法自12例行电切开腹部手术的健康成年女性腹部皮下和网膜分离前脂肪细胞,分为3组,即无干预正常对照组(NC组)、血管紧张素转换酶抑制剂类药物贝那普利组(ACEI组)和血管紧张素Ⅱ受体拮抗剂类药物替米沙坦组(ARB组),诱导分化共14 d。放射免疫法测定各组APN mRNA表达水平。结果与NC组比较,ACEI组、ARB组体外原代培养的人网膜和皮下前脂肪细胞中APN mRNA表达明显增强。结论贝那普利、替米沙坦可促进网膜来源的前脂肪细胞的分化,在以腹型肥胖为特征的代谢综合征干预方面可能具有广泛的应用前景。     

Phosphoinositide 3-kinase is required for human adipocyte differentiation in culture

OBJECTIVE: Phosphoinositide 3-kinase (PI3K) is required for murine adipocyte differentiation. However, a recent report concluded that PI3K was not involved in the differentiation of human preadipocytes into adipocytes. We have re-examined the role of PI3K in human preadipocyte differentiation, enrolling more patients and using more adipogenic indices. METHODS: Human preadipocytes, isolated from nine patients, were induced to differentiate in the presence or absence of 100 nM wortmannin. After 12-15 days, triacylglycerol accumulation and the expression of adipogenic markers (fatty acid synthase and adiponectin) were measured. RESULTS: A significant inhibition in triacylglycerol accumulation and in the induction of fatty acid synthase protein expression was observed, but there was no effect on adiponectin protein expression. CONCLUSION: Inhibition of PI3K reduces the differentiation of human preadipocytes into adipocytes, suggesting a role for this enzyme in the human adipogenic process.     

肿瘤坏死因子α对人脂联素3T3-L1细胞PPARγ2 mRNA表达的影响

目的探讨肿瘤坏死因子α（TNF-α）对稳定表达人脂联素3T3-L1细胞过氧化物酶体增殖物激活受体（PPAR）γ2 mRNA表达的影响，为进一步研究脂联素功能提供了实验基础。方法重组脂联素真核表达质粒（pcDNA3．1^＋-hADPN）脂质体法稳定转染3T3-L1细胞。用TNF-α（100ng／m1）处理未转染、转染空载载体、转染pcDNA3．1^＋-hADPN的3T3-L1细胞，RT-PCR检测PPARγ2 mRNA的表达量。结果（1）稳定转染了pcDNA3．1^＋-hADPN的未分化和已分化3T3-L1细胞中，PPARγ2表达量较未转染组明显增加（P〈0.01）。（2）TNF-α可明显抑制PPARγ2 mRNA的表达（P〈0.05）。（3）稳定转染pcDNA3．1^＋-hADPN可改善TNF-α抑制作用（P〈0.05）。结论稳定转染pcDNA3．1^＋-hADPN可明显增加未分化和已分化的3T3-L1细胞PPARγ2 mRNA表达。TNF-α抑制PPAR-γ2 mRNA表达，而转染pcDNA3．1^＋-hADPN可改善TNF-α抑制作用。     

S100A16基因促进3T3-L1前脂肪细胞分化

目的 研究S100A16基因在3T3-L1前脂肪细胞分化过程中的作用及机制.方法 构建过表达S100A16的慢病毒载体(PLJMI-S100A16-GFP),转染3T3-L1细胞.以Western印迹法检测S100A16正常3T3-L1细胞分化过程中S100A16的表达;采用油红O观察脂滴堆积情况;采用Western印迹和实时定量PCR方法检测前体脂肪细胞分化过程中相关基因的表达变化.免疫共沉淀方法检测S100A16是否与p53相互作用.结果 成功构建S100A16过表达3T3-L1细胞株;随着3T3-L1前脂肪细胞的分化,S100A16蛋白表达水平逐渐升高;高表达S100A16能够促进3T3-L1前脂肪细胞分化,促进甘油三酯在脂肪细胞内聚集(P＜0.01),同时上调脂肪细胞分化标志基因PPARy、CCAAT增强子结合蛋白α(C/EBP-α)、脂蛋白脂酶、脂肪细胞脂肪酸结合蛋白(aP2)及脂肪酸合成酶的表达(P＜0.05或P＜0.01);免疫共沉淀结果提示,S100A16蛋白与p53相互作用.结论 S100A16通过抑制p53活性进而促进3T3-L1前脂肪细胞的分化.     

Committed subcutaneous preadipocytes are reduced in human obesity

Aims/hypothesis The aim of this study was to test whether the availability of committed preadipocytes in abdominal and femoral subcutaneous adipose tissue varies with obesity and body fat distribution. Methods Body composition, fat cell size, committed preadipocytes and macrophages were measured in subcutaneous abdominal and femoral adipose depots of 17 lean, 16 upper-body-obese (UBO) and 13 lower-body-obese (LBO) women. Preadipocytes and macrophages were identified by simultaneous staining with the respective markers aP2 and CD68. In a subset of samples we measured preadipocyte proliferation, differentiation and susceptibility to apoptosis. Results Abdominal adipocytes were smaller in lean than in obese women. Committed preadipocytes represented a greater fraction of stromovascular cells in lean than in obese women but were similar between UBO and LBO women (abdomen: ∼30 ± 3 vs ∼17 ± 2%; thigh: ∼30 ± 3 vs ∼17 ± 2%). Preliminary data suggested that preadipocyte kinetics were similar in LBO and lean women, whereas preadipocytes of UBO women differentiated less and were more susceptible to apoptotic stimuli. The fraction of stromovascular cells that were macrophages was greater in both depots in obese women (UBO and LBO) than in normal-weight women, but the difference was not statistically significant. Conclusions/interpretation The proportion of subcutaneous adipose tissue stromovascular cells that are committed preadipocytes is reduced with obesity. This could be due to greater recruitment of preadipocytes to adipogenesis or greater preadipocyte apoptosis, depending upon the obesity phenotype. These data are consistent with the concept that body fat distribution may be regulated partly through differences in adipogenesis.     

RAS阻断剂对人前脂肪细胞分化和胰岛素敏感性的影响

目的 探讨肾素-血管紧张素系统(RAS)阻断剂对体外原代培养的人内脏和外周来源的前脂肪细胞分化和胰岛素敏感性的影响. 方法 自16例行电切开腹部手术的健康成年女性腹部皮下和网膜分离前脂肪细胞.分为4组,即无干预的正常对照(NC)组、噻唑烷二酮类约物吡格列酮(Pioglitazone)组、血管紧张素转换酶抑制剂(ACEI)类药物贝那普利(Benazapril)组和血管紧张素Ⅱ受体拮抗剂(ARB)类药物替米沙坦(Telmisartan)组,诱导分化共14 d.观察细胞活力、细胞内脂质含量和前脂肪细胞分化标志物--甘油-3-磷酸脱氧酶的活性.细胞的胰岛素敏感性通过葡萄糖消耗试验测定. 结果 对照组皮下来源的前脂肪细胞活力高于网膜,脂质含量反而低于网膜来源细胞,二者胰岛素敏感性无差别.与对照组相比,贝那普利、替米沙坦和吡格列酮均能明显提高网膜和皮下来源前脂肪细胞的细胞活力和脂质含量,并提高细胞的胰岛素敏感性;其中,替米沙坦组网膜前脂肪细胞的上述各指标均高于吡格列酮组.在网膜,替米沙坦和吡格列酬组的葡萄糖消耗量分别为(5.567±1.612)mmol/L和(4.418±1.572)mmol/L,P=0.020;皮下则相反,两组葡萄糖消耗量分别为(5.335±1.461)mmol/L和(7.506±1.615)mmol/L,P<0.01. 结论 RAS阻断剂(替米沙坦和贝那普利)可促进人前脂肪细胞分化并改善细胞的胰岛素敏感性,且较之吡格列酮在内脏发挥优势性作用.     

Knockdown of macrophage migration inhibitory factor disrupts adipogenesis in 3T3-L1 cells

Obesity is a condition in which adipose tissue mass is expanded. Increases in both adipocyte size and number contribute to enlargement of adipose tissue. The increase in cell number is thought to be caused by proliferation and differentiation of preadipocytes. Macrophage migration inhibitory factor (MIF) is expressed in adipocytes, and intracellular MIF content is increased during adipogenesis. Therefore, we hypothesized that MIF is associated with adipocyte biology during adipogenesis and focused on the influence of MIF on adipogenesis. To examine the effects of MIF on adipocytes, MIF expression in 3T3-L1 preadipocytes was inhibited by RNA interference, and cell differentiation was induced by standard procedures. The triglyceride content of MIF small interfering RNA (siRNA)-transfected 3T3-L1 cells was smaller than that of nonspecific siRNA-transfected cells. In addition, MIF knockdown apparently abrogated increases in adiponectin mRNA levels during differentiation. Gene expression of peroxisome proliferator-activated receptor (PPAR)gamma, CCAAT/enhancer binding protein (C/EBP)alpha, and C/EBPdelta decreased with MIF siRNA transfection, but C/EBPbeta expression increased. Cell number and incorporation of 5-bromo-2-deoxyuridine into cells decreased from 1-3 d and from 14-20 h, respectively, after induction of differentiation in MIF siRNA-transfected cells, thus suggesting that MIF siRNA inhibits mitotic clonal expansion. Taken together, these results indicated that MIF regulates differentiation of 3T3-L1 preadipocytes, at least partially, through inhibition of mitotic clonal expansion and/or C/EBPdelta expression.     

荧光标记胆固醇用于测定人单核巨噬细胞脂质外流

目的利用荧光标记的胆固醇(NBD-胆固醇)建立测定人巨噬细胞胆固醇外流的新方法,并与传统同位素标记胆固醇的测定方法进行比较。方法提取受试者外周血单核细胞分化为人巨噬细胞。体外培养人单核细胞系THP-1及鼠单核细胞系J774。以不同浓度的NBD-胆固醇孵育THP-1细胞,观察胆固醇浓度和孵育时间对细胞摄取的影响。用荧光酶标仪测定荧光信号强度以反映胆固醇摄取和外流。分别在三种细胞中测量NBD-胆固醇外流率,并在原代人巨噬细胞中观察与同位素标记胆固醇外流率的相关性。结果 NBD-胆固醇孵育细胞后迅速分布于细胞器中,但不进入细胞核。细胞对NBD-胆固醇的摄取有浓度依赖性,但达峰时间不受浓度影响。NBD-胆固醇外流率在各种巨噬细胞中呈现相似趋势,由载脂蛋白A1和高密度脂蛋白诱导的外流率在诱导外流4 h达到平台期。3H-胆固醇外流率在人巨噬细胞中与NBD-胆固醇外流率显著相关(r=0.968,P<0.001)。结论 NBD-胆固醇是一种有效的、敏感且环保的探针,可替代同位素标记的胆固醇用于巨噬细胞外流率的测定。     

Mechanism of adult primitive mesenchymal ST-13 preadipocyte differentiation

Convincing evidence supports the idea that adipogenesis occurs throughout the life of organisms. However, little is known about the adipogenesis program for adult adipocytes. We examine this issue using mouse adult primitive mesenchymal ST-13 preadipocytes that express the peroxisome proliferator-activated receptor-gamma (PPARgamma) gene while in a predifferentiated state. The gene expression of PPARgamma was sustained throughout differentiation when ST-13 preadipocytes were induced to become adipocytes by a PPARgamma ligand. However, the differentiation of pluripotent C3H10T1/2 stem cells and 3T3-L1 embryonic fibroblastic cells was associated with enhanced expression of the PPARgamma gene. Immunoblotting analysis revealed that C3H10T1/2 and 3T3-L1 cells expressed low levels of PPARgamma1 from the early stage, and the amount increased during differentiation, whereas PPARgamma2 appeared at the late stage. In contrast, ST-13 preadipocytes expressed an appreciable amount of PPARgamma1 that significantly decreased on differentiation, and a small amount of PPARgamma2 appeared late in the differentiation process. Furthermore, the standard hormone cocktail containing dexamethasone, methylisobutylxanthine, and insulin induced an increase in PPARgamma1 protein only at the early stage, and a low level of PPARgamma2 protein appeared late in ST-13 cells. However, levels of both PPARgamma1 and PPARgamma2 proteins were significantly induced within 2 d in 3T3-L1 cells in this hormonal adipogenesis. Moreover, exposing ST-13 preadipocytes to dexamethasone and insulin induced differentiation, but failed to induce adipogenesis in 3T3-L1. Adipogenesis in adult rat primary preadipocytes was also induced in a similar manner to that of ST-13. Our results indicate that ST-13 cells and primary preadipocytes derived from adults possess an adipogenesis program distinct from that of 3T3-L1 and C3H10T1/2 cells, and that it may represent the adipogenesis program for adult-specific adipocytes.     

Lipopolysaccharides Reduce Adipogenesis in 3T3-L1 Adipocytes Through Activation of NF-κB Pathway and Downregulation of AMPK Expression

Lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria serve as endotoxin to exert potent immune responses. However, the effect of LPS on adipogenesis has not been elucidated. The present study was designed to examine the effect of LPS on adipogenesis in 3T3-L1 preadipocytes and possible mechanism(s) of action involved. Our results revealed that LPS challenge significantly suppressed adipogenesis in 3T3-L1 preadipocytes mainly through downregulated expression of the late adipogenic markers PPARγ and aP2 as well as AMP-activated protein kinase (AMPK) expression and activity. As an inflammatory factor, LPS was found to lead to an overt reduction in IκBα levels compared with the time-matched controls, consolidating its pro-inflammatory property in 3T3-L1 preadipocytes. Our data also revealed that LPS retarded adipogenesis, the effect of which was partially reversed by the selective inhibitor of IKKβ. IκBα was found to be involved in the anti-adipogenic effect of LPS. In conclusion, LPS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through activation of NF-κB and inhibition of AMPK. With the activation of NF-κB pathway and inhibition of AMPK, LPS suppresses C/EBP α DNA-binding activity and the expression of late adipogenic markers PPARγ and aP2.     

GW4064, a farnesoid X receptor agonist,upregulates adipokine expression in preadipocytes and HepG2 cells

AIM: To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells.METHODS: The mRNA expression of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor-gamma 2 (PPAR-γ2), adiponectin, leptin, resistin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and the long isoform of leptin receptor (OB-Rb) and protein levels of adiponectin, leptin, and resistin were determined using fluorescent real-time PCR and enzyme linked immunosorbent assay, respectively, on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes exposed to GW4064. Moreover, mRNA expression of AdipoR2 and OB-Rb was also examined using fluorescent real-time PCR at 0, 12, 24, and 48 h in HepG2 cells treated with GW4064.RESULTS: The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, resistin, AdipoR1, AdipoR2, and OB-Rb and protein levels of adiponectin, leptin, and resistin increased along with differentiation of 3T3-L1 preadipocytes (P < 0.05 for all). The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, and AdipoR2 in 3T3-L1 preadipocytes, and AdipoR2 and OB-Rb in HepG2 cells was significantly increased after treatment with GW4064, when compared with the control group (P < 0.05 for all). A similar trend was observed for protein levels of adipokines (including adiponectin, leptin and resistin). However, the expression of resistin, AdipoR1, and OB-Rb in 3T3-L1 cells did not change after treatment with GW4064.CONCLUSION: The FXR agonist through regulating, at least partially, the expression of adipokines and their receptors could offer an innovative way for counteracting the progress of metabolic diseases such as nonalcoholic fatty liver disease.     

分泌型卷曲相关蛋白/β-联蛋白信号通路对脂肪形成的影响

目的:探讨分泌型卷曲相关蛋白(sFRP5)和经典Wnt信号通路在脂肪细胞形成中的作用及机制。方法:诱导3T3-L1前体脂肪细胞分化,用过表达sFRP5的携带绿色荧光蛋白基因的重组腺病毒(Ad-sFRP5-GFP)感染3T3-L1细胞并诱导成熟,利用实时荧光定量PCR检测经典Wnt下游靶基因和脂肪分化相关基因mRNA水平的变化。3T3-L1细胞分化成熟后,进行油红染色,观察过表达sFRP5对脂滴形成的影响;提取核浆蛋白,用蛋白质印迹法检测sFRP5对β-联蛋白核转位的影响。结果:3T3-L1细胞分化成熟后期,sFRP5、CCAAT增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物活化受体γ2(PPARγ2)表达均显著增加,同时,细胞周期蛋白D1表达显著下调。而诱导分化成熟后,过表达sFRP5的3T3-L1细胞周期蛋白D1无显著改变。与空载对照组相比,过表达sFRP5的3T3-L1细胞脂滴形成无明显变化。给予3T3-L1细胞重组小鼠sFRP5直接刺激或感染Ad-sFRP5-GFP,均未明显改变β-联蛋白核转位。结论:sFRP5的表达随着脂肪细胞分化而显著增加,但不影响体外脂肪细胞分化,sFRP5在体外不直接通过拮抗Wnt/β-联信号通路的方式发挥作用。