抑制Hedgehog信号通路促进C3H10T1/2间充质干细胞成脂分化

激活Hedgehog信号通路可抑制间充质干细胞成脂分化,但抑制Hedgehog信号通路是否可促进脂肪细胞分化研究结果却并不一致.本研究采用环靶明诱导C3H10T1/2细胞成脂分化,并以国际公认的成脂诱导剂混合物（胰岛素、地塞米松、吲哚美辛和IBMX）诱导细胞分化作为参考. qRT-PCR结果显示,在10 μmol/L环靶明(cyclopamine)处理的C3H10T1/2细胞中,Hedgehog信号通路各基因相对表达量显著下降,而成脂分化调控基因PPARγ,C/EBPα和成脂分化标志基因FABP4相对表达量显著升高（P < 0.05). 与此一致,Western印迹结果表明,在环靶明处理的C3H10T1/2细胞中,Hedgehog信号通路中的Shh蛋白和Gli1蛋白表达水平显著下降,成脂分化相关的PPARγ、C/EBPα和FABP4蛋白表达水平显著升高（P < 0.05）. 此外,油红O染色方法证明,环靶明处理可诱导C3H10T1/2细胞成脂分化.以上研究结果提示,抑制Hedgehog信号通路对小鼠胚胎间充质干细胞的成脂分化具有促进作用,并可能为瘦肉型猪的培育和猪肉品质调控研究提供参考依据.     

Ⅲ型纤连蛋白组件包含蛋白5通过抑制ERK1/2磷酸化抑制C3H10T1/2细胞成脂分化

旨在探究Ⅲ型纤连蛋白组件包含蛋白5（typeⅢ domain-containing protein5,FNDC5）对C3H10T1/2细胞成脂分化的调控作用。利用qRT-PCR和Western 印迹检测FNDC5在C3H10T1/2细胞成脂分化过程中的时序性表达规律;构建慢病毒包被的过表达/干扰FNDC5载体,转染C3H10T1/2细胞,采用qRT-PCR检测成脂分化关键基因的表达情况,油红O染色检测脂滴含量,利用Western 印迹检测细胞外信号调节激酶（extracellularregulatedkinase1/2,ERK1/2）及磷酸化ERK1/2（P-ERK1/2）的表达水平。C3H10T1/2细胞成脂诱导分化8d,Fndc5的表达量明显升高（P＜0.05）;C3H10T1/2细胞中过表达FNDC5,成脂分化关键基因过氧化物酶体增殖物激活受体-γ（peroxisome proliferator--activated receptor-γ,PPARγ）、脂肪酸结合蛋白4（fatty acid binding protein 4,FABP4）和CCAAT增强子结合蛋白α（CCAAT enhancer binding protein alpha,C/EBPα）的表达量显著降低（P＜0.01）,CCAAT增强子结合蛋白β（CCAAT enhancer binding protein beta,C/EBPβ）表达量明显降低（P＜0.05）,脂滴含量明显减少,P-ERK1/2的含量明显降低（P＜0.05）。C3H10T1/2细胞中干扰FNDC5,成脂分化关键基因PPARγ、C/EBPβ、FABP4和C/EBPα的表达量显著升高（P＜0.01）,脂滴含量明显增加,P-ERK1/2的含量明显升高（P＜0.05）。本研究发现,FNDC5可以通过抑制ERK1/2的磷酸化水平,抑制C3H10T1/2细胞的成脂分化,为FNDC5调控脂肪沉积的机制研究提供参考数据。     

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.     

新疆褐牛和安格斯牛公犊脂代谢相关肉品质性状的比较

动物体脂沉积具有明显的时空变化特征,且与其肉品质性状密切相关。新疆褐牛是我国著名的肉乳兼用培育品种,但与国外优良肉牛品种相比,其脂代谢相关肉品质性状的生长发育变化以及其相关调控基因的表达变化缺乏系统研究。本研究以相同饲养条件下安格斯肉牛和新疆褐牛3月龄公犊为对象,对其屠宰性状、不同脂肪组织形态测量指标、背最长肌和脂肪组织中脂肪酸合成酶(FASN)、脂肪酸结合蛋白4(FABP4)、激素敏感脂酶(HSL)、脂蛋白脂酶(LPL)和瘦素(LEP)基因的m RNA及其蛋白表达以及血清中其激素水平进行了测定。以期为后续的品种比较研究提供早期生长发育变化基础。结果为:(1)安格斯公犊肉骨比极显著高于新疆褐牛(p0.01),而肋脂厚和眼肌面积显著小于新疆褐牛(p0.05)。(2)安格斯肉牛心周脂肪和网膜脂肪组织中的脂肪细胞面积/单位面积脂肪细胞数量之比显著高于新疆褐牛(p0.05)。(3)在背最长肌和肾周脂肪中安格斯牛的FASN基因mRNA表达量均呈现出显著(p0.05)和极显著(p0.01)低于新疆褐牛的一致性变化;而在心周脂肪中则呈现安格斯公犊极显著高于新疆褐牛的相反变化(p0.01)。此外,在心周脂肪中安格斯牛的LEP和LPL基因的m RNA表达量极显著(p0.01)和显著(p0.05)高于新疆褐牛,而在肾周脂肪中安格斯牛的LPL、HSL和FABP4基因的mRNA表达量均极显著低于新疆褐牛(p0.01)。在心周脂肪组织和肾周脂肪中安格斯牛的LPL蛋白表达量极显著(p0.01)和显著(p0.05)高于新疆褐牛;(4)安格斯公犊血清5种激素浓度均显著(p0.05)或极显著(p0.01)低于新疆褐牛。除LEP外,该结果与肾周脂肪中其余4种激素编码基因的mRNA相对表达量变化一致。本研究结果表明,三月龄新疆褐牛体脂沉积能力大于安格斯牛,而产肉率有低于安格斯牛的趋势。体脂代谢相关基因及其蛋白表达的品种间差异主要体现在背最长肌、肾周、心周脂肪等组织。     

Rho激酶抑制剂Y-27632对C3H10T1/2细胞增殖与成脂分化的作用

探究Rho激酶抑制剂Y-27632对间充质干细胞（mesenchymal stem cells,MSCs）C3H10T1/2增殖和成脂分化的影响.实验分为对照组、成脂诱导组和Y-27632处理组（Y-27632+成脂诱导）. 利用MTT检测细胞增殖情况,油红O染色,异丙醇萃取法检测细胞成脂分化情况,半定量RT-PCR检测过氧化物酶体增殖物激活受体γ(peroxisome proliferator activiated receptor γ, PPARγ)和CCAAT增强子结合蛋白α (CCAAT enhancer binding protein α, C/EBPα)基因表达. 结果表明,Y-27632能够显著抑制C3H10T1/2细胞的增殖(P<0.05),并呈一定的浓度依赖性;高浓度Y-27632对C3H10T1/2细胞成脂分化具有显著抑制作用（P<0.05）;半定量RT-PCR结果显示,成脂诱导处理组PPARγ和C/EBPα表达量在第3 d、5 d和7 d显著低于成脂诱导组（P<0.05）. 综上所述,Y-27632能够抑制C3H10T1/2细胞增殖与成脂分化.     

绿原酸在小鼠3T3-L1前脂肪细胞分化进程中的作用

目的：探讨绿原酸（CGA）对小鼠3T3-L1前脂肪细胞分化的影响。方法：培养小鼠3T3-L1前脂肪细胞,分别设置空白对照组（CG）、阳性对照组罗格列酮组（RG）、阴性对照组GW9662组 （GG）和绿原酸组（CGA）。油红O染色观察小鼠3T3-L1前脂肪细胞分化后的细胞形态变化以及脂滴形成情况;组织细胞甘油三酯（TAG）酶法测定各组分化后的细胞TAG积累量;实时荧光定量PCR技术（qPCR）检测小鼠3T3-L1前脂肪细胞在分化过程中关键基因PPARγ2 mRNA表达水平。结果：CGA组被油红O染色的区域大于CG组和GG组,但CGA组颜色没有RG组鲜艳,且脂滴形状也与RG组存在差异。CGA组TAG积累量低于CG组和RG组,与CG组比较无显著性差异（P>0.05）,但与RG组比较有显著性差异（P<0.05）。在分化过程中,CGA组和RG组PPARγ2 mRNA的表达量高于CG组和GG组,有显著性差异（P<0.01）,GG组PPARγ2 mRNA的表达量自细胞分化第4d起低于CG组,有显著性差异（P<0.01）。结论：CGA可以促进小鼠3T3-L1前脂肪细胞的分化,同时降低分化后成熟脂肪细胞中TAG的积累量,其机制与分化相关因子PPARγ2的表达有关。     

高内涵脂滴三维影像定量分析研究细胞内的脂质储存

摘要 目的：研究细胞内脂滴含量的变化对肥胖、糖尿病等代谢性疾病发生发展的影响。方法：建立高内涵脂滴三维成像和定量分析系统,获得脂滴三维动态表型参数,例如细胞内脂滴的总体积量、脂滴平均体积、单一细胞内脂滴平均数量等指标。选择HeLa、AML-12、COS-7和3T3-L1四种细胞系进行油酸、基因沉默、酶活性抑制剂的处理,量化处理后四种细胞内的脂滴数量与大小的表型差异。结果：在加入油酸情况下,细胞随油酸浓度增加而生成更多、更大的脂滴,但AML-12细胞只有展现增加脂滴数量的变化表型;在HeLa细胞中进行19种中性脂合成通路上关键基因的转录表达沉默,发现需要同时双敲降两种甘油三酯合成酶DGAT1和DGAT2才能显着降低细胞内脂滴总体积储存量,但在COS-7细胞中只需要单敲降DGAT1即可降低脂滴存量;进一步使用了DGAT1/2抑制剂处理四种细胞后,发现对抑制剂响应可区分为两类细胞分组（HeLa、AML-12与COS-7、3T3-L1）的脂滴存量表型差异,其原因是DGAT1和DGAT2的转录表达谱在这两类细胞分组中的不同。结论：建立了高内涵脂滴三维成像和定量分析系统,量化了四种细胞系的脂滴数量与大小的表型差异,揭示了细胞的脂滴脂储存方式与蛋白酶表达谱的关系。     

钙蛋白酶抑制剂Calpeptin在脓毒症膈肌功能障碍中的作用研究

摘要 目的：探究钙蛋白酶抑制剂Calpeptin在脓毒症膈肌功能障碍中的保护作用及其分子机制。方法：通过腹腔内注射8 mg/kg LPS的方法构建大鼠脓毒症模型,将雄性SD大鼠分为3组（n=6/组）：正常对照组（Con组）、脓毒症组（Sepsis组）和钙蛋白酶抑制剂预处理组（Calpeptin组）。处死大鼠,快速分离大鼠膈肌组织,采用苏木精-伊红(HE)染色法检测膈肌组织病理学改变,通过实时荧光定量PCR法（qRT-PCR）分别检测膈肌组织中钙蛋白酶μ-Calpain、凋亡相关基因天冬氨酸特异性半胱氨酸蛋白酶-3（Caspase-3）、自噬相关蛋白Beclin-1、肿瘤坏死因子-α（TNF-α）和白介素-6（IL-6）mRNA表达水平。结果：与正常对照组相比,LPS处理24 h的脓毒症组大鼠膈肌组织HE染色未见明显膈肌萎缩改变,但膈肌收缩力下降,这与我们以往的研究结果一致。qRT-PCR法检测到脓毒症组大鼠膈肌组织中上述基因mRNA表达量明显增加（P<0.05）,而Calpeptin预处理后,上述基因mRNA表达水平显著下降（P<0.05）。结论：脓毒症时膈肌发生功能障碍,钙蛋白酶抑制剂Calpeptin可显著减轻LPS诱导的炎症、凋亡、自噬的激活。     

7-酮基胆甾醇-9-羧基壬烷通过SREBP1c通路缓解油酸诱导HepG2细胞脂质生成

本研究目的是为了探究7-酮基胆甾醇-9-羧基壬烷（OXL-1）对油酸诱导的HepG2细胞形成的非酒精性脂肪性肝病（NAFLD）细胞模型中脂质生成的潜在抑制作用。油红染色显示OXL-1能显著降低油酸诱导的甘油三酯（TG）和总胆固醇（TC）的脂质生成。基因芯片分析发现,与对照组相比,HepG2细胞经OXL-1处理后固醇调节元件结合蛋白1c（SREBP1c）、脂肪酸合酶（FAS）及乙酰辅酶a羧化酶α（ACCα）转录表达显著降低。相比较于对照组,OXL-1组甘油三脂减少56.87% ± 9.08%（P<0.01）,总胆固醇减少24.96% ± 5.45%（P<0.01）。同时也使SREBP1c、FAS和ACCα蛋白质表达水平降低。OXL-1组相比OA组,其SREBP1c、FAS和ACCα的蛋白质表达分别下调52.62% ± 6.38%（P<0.01）、51.14% ± 8.75%（P<0.01）和19.46% ± 3.64%（P<0.05）。结果说明,OXL-1可能经由SREBP1c、FAS和ACCα的转录和蛋白质水平的调控作用来阻止OA诱导的脂质蓄积。综上结果揭示,OXL-1可能在非酒精性脂肪肝病细胞模型中作为一种阻止脂质积累的新型化合物。     

Txnip基因siRNA慢病毒表达质粒构建及促进猪前体脂肪细胞分化

硫氧还蛋白互作蛋白(thioredoxin interacting protein, Txnip)是一种氧化还原调节蛋白质,与硫氧还蛋白结合并抑制其活性,调节细胞氧化还原状态,影响细胞多种生理过程,然而其在猪脂肪细胞分化中的作用尚不明确。本文设计合成3对靶向猪Txnip基因的shRNA寡核苷酸,分别连接于重组慢病毒载体pGLV_3/H_1/GFP+Puro构建siRNA表达质粒。测序验证后,与包装质粒共转染293T细胞,获得滴度1×10~8 pfu/mL的慢病毒干扰质粒。以MOI值100转染原代培养猪前体脂肪细胞,转染率均达80%以上,其中Txnip-shRNA-2转染细胞Txnip基因沉默率达75%。转染Txnip-shRNA-2的猪前体脂肪细胞用成脂分化培养液诱导后,每隔1 d检测细胞成脂分化及相关基因表达。结果发现,其分化比阴性对照质粒转染或未转染细胞显著增强(P<0.05),PPARγ和FAS mRNA表达水平显著提高(P<0.05)。本文构建siRNA慢病毒表达质粒能有效干扰猪Txnip基因表达,Txnip表达沉默可通过上调PPARγ表达促进猪前体脂肪细胞分化。本研究提示,Txnip可能是猪脂肪细胞分化的抑制因子。     

Gene expression profiling of subcutaneous adipose tissue in morbid obesity using a focused microarray: Distinct expression of cell-cycle- and differentiation-related genes

Background

Prevalence of obesity is increasing to pandemic proportions. However, obese subjects differ in insulin resistance, adipokine production and co-morbidities. Based on fasting plasma analysis, obese subjects were grouped as Low Acylation Stimulating protein (ASP) and Triglyceride (TG) (LAT) vs High ASP and TG (HAT). Subcutaneous (SC) and omental (OM) adipose tissues (n = 21) were analysed by microarray, and biologic pathways in lipid metabolism and inflammation were specifically examined.

Methods

LAT and HAT groups were matched in age, obesity, insulin, and glucose, and had similar expression of insulin-related genes (InsR, IRS-1). ASP related genes tended to be increased in the HAT group and were correlated (factor B, adipsin, complement C3, p < 0.01 each). Differences between LAT and HAT group were almost exclusively in SC tissue, with little difference in OM tissue. Increased C5L2 (p < 0.01), an ASP receptor, in HAT suggests a compensatory ASP pathway, associated with increased TG storage.

Results

HAT adipose tissue demonstrated increased lipid related genes for storage (CD36, DGAT1, DGAT2, SCD1, FASN, and LPL), lipolysis (HSL, CES1, perilipin), fatty acid binding proteins (FABP1, FABP3) and adipocyte differentiation markers (CEBPα, CEBPβ, PPARγ). By contrast, oxidation related genes were decreased (AMPK, UCP1, CPT1, FABP7). HAT subjects had increased anti-inflammatory genes TGFB1, TIMP1, TIMP3, and TIMP4 while proinflammatory PIG7 and MMP2 were also significantly increased; all genes, p < 0.025.

Conclusion

Taken together, the profile of C5L2 receptor, ASP gene expression and metabolic factors in adipose tissue from morbidly obese HAT subjects suggests a compensatory response associated with the increased plasma ASP and TG.     

The tyrosine kinase receptor HER2 (erbB-2): from oncogenesis to adipogenesis

Recent experimental evidences begin to support the notion that the proto-oncogene HER2 (erbB-2) might unexpectedly function to modulate the adipogenic conversion of preadipocytes. Two opposing scenarios have been proposed, however, to explain the influence of HER2 on adipocyte differentiation. In one hand, down-modulation of HER2 expression and pharmacological reduction of HER2 activity have been related to enhanced adipocyte differentiation. On the contrary, an increased abundance in HER2 has been described in differentiated adipocytes compared with preadipocytes. Considering that expression and activity of the lipogenic enzyme Fatty Acid Synthase (FASN) become up-regulated during adipogenic conversion, we recently hypothesized that a "HER2 --> FASN axis" -a "lipogenic benefit" that has been shown to enhance cancer cell proliferation, survival, chemoresistance and metastasis in biologically aggressive subgroups of breast carcinomas-might also naturally work during the differentiation of preadipocytes. To definitely clarify if the discrepancy between the opposing theories for a role of HER2 during adipocyte differentiation related to the experimental approach utilized to compare the abundance of HER2 in undifferentiated and differentiated adipocytes (i.e., cell lysates containing equivalent protein content versus cell lysates generated from similar cell numbers), we here took advantage of a high content microscopy approach. Using an automated confocal imaging platform, we monitored the expression status of the adipogenic marker FASN and its timing relationship with HER2 not only in individual 3T3-L1 cells but further in whole cultures of 3T3-L1 preadipocytes undergoing adipogenic conversion. Our findings not only confirm a non-oncogenic role for HER2 in the process of adipose differentiation but further suggest that HER2 might represent a previously unrecognized target to manage obesity via the lipogenic enzyme FASN.     

Differential Expression of Lipid Metabolism-Related Proteins in Different Breast Cancer Subtypes

Purpose

This study aimed to determine the expression and clinical significance of proteins that are involved in lipid metabolism in human breast tumors.

Methods

Tumors from 476 breast cancer patients were used to construct tissue microarrays. Then, immunohistochemistry (IHC) for hormone-sensitive lipase (HSL), Perilipin 1 (PLIN1), fatty acid binding protein 4 (FABP4), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX-1), and fatty acid synthase (FASN) was performed on these microarrays.

Results

Breast tumors were classified into 4 subtypes: luminal A (n = 242; 50.8%), luminal B (n = 134; 28.2%), human epidermal growth factor receptor 2 (HER2) (n = 50; 10.5%), and triple negative breast cancer (TNBC) (n = 50; 10.5%). The expression of PLIN1 (p < 0.001), FABP4 (p = 0.029), CPT-1A (p = 0.001), ACOX-1 (p < 0.001), and FASN (p < 0.001) differed significantly among these tumor subtypes. Notably, PLIN1, CPT-1A, and FASN expression was highest in HER2 tumors and lowest in TNBC tumors. Similarly, the expression of FABP4 and ACOX-1 was highest in HER2 tumors and lowest in luminal A tumors. In addition, ACOX-1 positivity was associated with significantly shorter overall survival (p = 0.018). When tumor subtype was considered, FABP4 positivity was associated with significantly shorter disease-free survival (p = 0.005) and overall survival (p = 0.041) in TNBC.

Conclusion

Lipid metabolism-related proteins are differentially expressed in different IHC subtypes of breast cancer and some are associated with decreased survival rates.     

Inhibition of fatty acid synthase suppresses U-2 OS cell invasion and migration via downregulating the activity of HER2/PI3K/AKT signaling pathway in vitro

FASN plays an important role in the malignant phenotype of various tumors. Our previous studies show that inhibition FASN could induce apoptosis and inhibit proliferation in human osteosarcoma (OS) cell in vivo and vitro. The aim in this study was to investigate the effect of inhibition FASN on the activity of HER2/PI3K/AKT axis and invasion and migration of OS cell. The expression of FASN, HER2 and p-HER2(Y1248) proteins was detected by immunohistochemistry in OS tissues from 24 patients with pulmonary metastatic disease, and the relationship between FASN and p-HER2 as well as HER2 was investigated. The results showed that there was a positive correlation between FASN and HER2 as well as p-HER2 protein expression. The U-2 OS cells were transfected with either the FASN specific RNAi plasmid or the negative control RNAi plasmid. FASN mRNA was measured by RT-PCR. Western blot assays was performed to examine the protein expression of FASN, HER2, p-HER2(Y1248), PI3K, Akt and p-Akt (Ser473). Migration and invasion of cells were investigated by wound healing and transwell invasion assays. The results showed that the activity of HER2/PI3K/AKT signaling pathway was suppressed by inhibiting FASN. Meanwhile, the U-2OS cells migration and invasion were also impaired by inhibiting the activity of FASN/HER2/PI3K/AKT. Our results indicated that inhibition of FASN suppresses OS cell invasion and migration via down-regulation of the “HER2/PI3K/AKT” axis in vitro. FASN blocker may be a new therapeutic strategy in OS management.     

组蛋白去乙酰化酶（HDAC）在间充质干细胞C3H10T1/2成脂分化过程中的表达及HDAC11的作用

组蛋白去乙酰化酶（histone deacetylase, HDAC）通过参与调节组蛋白乙酰化修饰调控基因表达. 研究发现多种HDAC参与成脂分化,但其机制尚不清楚. 本研究旨在探讨间充质干细胞C3H10T1/2成脂分化过程中组蛋白去乙酰化酶（HDAC）的表达变化及其对成脂分化的影响. 本研究首先建立了C3H10T1/2体外成脂分化的模型并以油红O染色鉴定成功诱导成脂分化. PCR检测C3H10T1/2细胞成脂分化过程中11种HDAC的变化趋势,发现成脂分化过程中,HDAC1、2、5、9和10的mRNA表达量下降而HDAC3、6、8和11的mRNA表达量明显上升,其中HDAC11上升最为显著. 进一步通过RNA干扰沉默HDAC11表达, PCR检测成脂分化的关键转录因子PPARγ2和成脂标志物Perilipin、Adipoq 的mRNA表达量下降,但Fabp4表达变化不明显. 油红O染色结果表明,诱导C3H10T1/2成脂分化过程中,干扰HDAC11表达,胞浆内脂滴形成数量减少,成脂分化受到抑制. 总之,我们实验的结果提示C3H10T1/2细胞成脂分化伴随着多种HDAC表达的变化,其中HDAC11的增加最显著,干扰HDAC11的表达可以抑制C3H10T1/2细胞的成脂分化.