凋亡蛋白酶活化因子1基因在脂肪细胞诱导分化中表达及肿瘤坏死因子-α对其调节作用

目的 观察3T3-L1脂肪前体细胞诱导分化过程中（0~10 d）凋亡蛋白酶活化因子1（APAF1）基因表达水平的变化趋势，探讨肿瘤坏死因子-α（TNF-α）对成熟脂肪细胞中APAF1基因表达水平的调节作用。方法 体外培养3T3-L1脂肪前体细胞，通过油红O染色鉴定其分化成熟的基础上，应用人重组TNF-α 1.0 ng·mL-1干预分化成熟的脂肪细胞，抽提脂肪细胞总RNA和总蛋白后，采用RT-PCR及Western blot技术检测诱导分化不同时段及TNF-α干预后不同时间（2、6、12和 24 h）脂肪细胞中APAF1基因的表达水平。结果 ① 3T3-L1 前体脂肪细胞诱导分化过程中（0~10 d），APAF1基因表达水平呈现逐渐减低的趋势;②人重组TNF-α对成熟脂肪细胞中APAF1基因的表达具有显著的增强作用，且TNF-α对APAF1基因表达的上调作用呈现随刺激时间延长而明显增强的总体趋势。结论 ① 在3T3-L1前体脂肪细胞分化过程中，APAF1基因的表达逐渐下调可能有利于脂肪细胞的分化成熟和脂质积聚；② APAF1基因在人重组TNF-α刺激成熟脂肪细胞过程中呈明显上调趋势，这种上调可能具有协同TNF-α促进成熟脂肪细胞去分化的作用。     

Ghrelin对3T3-L1前脂肪细胞增殖和分化的影响及相关机制探讨

目的：探讨ghrelin对3T3-L1前脂肪细胞增殖和分化的影响及作用机制。方法：体外培养3T3-L1前脂肪细胞,MTT法检测不同浓度ghrelin对其增殖活力的影响,半定量RT-PCR检测ghrelin对c-myc和胸苷激酶mRNA表达水平的影响;同时在利用胰岛素或ghrelin诱导分化的不同时段,通过油红O染色测定细胞分化程度,半定量RT-PCR检测过氧化物体增殖剂活化受体γ（PPARγ）、CAAT/增强子结合蛋白α（C/EBPα）mRNA的表达。结果：10-7~10-15 mol/L ghrelin作用24 h明显促进前脂肪细胞增殖,ghrelin组c-myc和胸苷激酶mRNA表达水平明显升高,与对照组相比差异均有显著性意义（P<0.05）。Ghrelin干预后,可诱导前脂肪细胞向成熟脂肪细胞的形态转变,但诱导分化率仍少于胰岛素;同时其PPARγ和C/EBPα mRNA表达水平较对照组明显升高（P<0.05）,ghrelin组和胰岛素组PPARγ和C/EBPα mRNA表达量随着分化时间的延长而增加,分化8 d与2 d相比,差异均有显著性意义（P<0.01）。结论：Ghrelin明显促进3T3-L1前脂肪细胞增殖与分化。Ghrelin可能通过增加c-myc的含量,进而引起胸苷激酶的活化,从而导致细胞周期的激活,促进细胞增殖;同时ghrelin可能通过增加PPAR-γ、C/EBP-α mRNA的表达,促进细胞分化,从而提高脂肪细胞对胰岛素的敏感性。［中国当代儿科杂志,2009,11（1）：69-73］     

TSG-6基因在3T3-L1脂肪细胞诱导分化中表达水平的变化及TNF-α对其调节作用的研究

目的观察3T3-L1脂肪细胞诱导分化过程中TSG-6基因mRNA表达水平的变化,探讨TNF-α对成熟脂肪细胞中TSG-6基因表达水平的调节作用.方法体外培养3T3-L1脂肪细胞,在诱导3T3-L1脂肪细胞分化成熟的基础上,应用重组TNF-α干预分化成熟的脂肪细胞,采用RT-PCR技术检测诱导分化不同时段及TNF-α干预后不同时间脂肪细胞中TSG-6基因的表达水平.结果 (1)随脂肪细胞逐渐分化成熟,TSG-6 基因mRNA表达水平逐渐升高.TSG-6 基因表达水平除在细胞分化第0～2天、第3～5天、第4～6天和第7～10天各时段内差异无显著意义(P>0.05)外,其余各时段之间表达水平,差异均有显著意义(P＜0.05);(2)不同浓度重组TNF-α(0.1～10.0 ng/ml)均能显著抑制成熟脂肪细胞中TSG-6基因mRNA的表达,除外TNF-α浓度1.0 ng/ml时6～24 h时段,其抑制作用呈现随TNF-α浓度增加和刺激时间延长而明显增强的总体趋势.TNF-α浓度为0.1 ng/ml时,TSG-6基因表达水平6 h内下降33.73%,12 h内下降97.39%;TNF-α浓度为1.0 ng/ml时,TSG-6基因表达水平6 h内下降78.68%,并持续至24 h;TNF-α浓度达10.0 ng/ml时,TSG-6基因表达水平2 h内即下降96.27%,TSG-6基因的表达几乎被完全抑制.结论 (1)TSG-6基因可能参与脂肪细胞分化及脂质形成过程;(2)TNF-α对脂肪细胞中TSG-6基因表达具有抑制作用,其抑制效应总体趋势上呈现剂量反应性特征.     

The role of hyperglycemia in FAT/CD36 expression and function

FAT/CD36 is a long-chain fatty acid transporter and scavenger receptor for oxidized LDL. Defects in FAT/CD36 have been linked to the hypertriglyceridemia and insulin resistance. Expression of FAT/CD36 was reported increase in type 1 diabetes; however, it remains unclear whether serum glucose or insulin plays an important role in this regulation. To elucidate the individual contribution of plasma glucose and insulin in the regulation of FAT/CD36 mRNA expression, we induced type 1 diabetes in male Sprague-Dawley rats using streptozotocin (STZ) and compared traditional insulin treatment with administration of the orally absorbed chemical agent vanadate, which reduces blood glucose levels via mechanisms that bypass insulin receptor action. STZ-exposed animals showed significant decreases in body weight (285.5 ± 2.8 vs. 233.1 ± 3.5 g, P < 0.001) and serum insulin levels (9.7 ± 0.7 vs. 2.8 ± 0.6 μU/ml, P < 0.05), accompanied by significant increases in blood glucose (71 ± 3 vs. 433 ± 11 mg/dl, P < 0.001), water intake (38.9 ± 0.9 vs. 205.9 ± 3.3 ml/day, P < 0.001) and food intake (22.0 ± 0.4 vs. 36.9 ± 1.0 g/day, P < 0.001). Diabetic animals demonstrated significant increases in FAT/CD36 mRNA levels in duodenum (2.2-fold), jejunum (1.8-fold), ileum (1.5-fold), adipose tissue (1.7-fold), and heart (2.5-fold) (P < 0.05). Insulin treatment reversed body weight loss and corrected hyperglycemia at diabetic rats as expected. Insulin treatment also corrected increased FAT/CD36 mRNA expression at diabetic rats. Vanadate significantly reduced serum glucose levels without increasing serum insulin or affecting body weight but reversed increased FAT/CD36 mRNA expression in diabetic rats. These data suggest that plasma glucose levels play more important role in the regulation of FAT/CD36 expression than concurrent changes in plasma insulin.     

鞘氨醇激酶基因在3T3-L1脂肪细胞诱导分化中的表达水平

目的 探讨鞘氨醇激酶基因(SPHK）在3T3-L1脂肪细胞诱导分化中表达水平的变化。方法采用细胞培养和RT-PCR技术检测细胞分化不同阶段脂肪细胞中SPHK基因表达水平。结果1．SPHK基因在3T3-L1脂肪前体细胞诱导分化初期表达呈明显上调趋势；2．随着脂肪前体细胞分化成熟，该基因表达水平明显低于诱导分化初期的基因表达水平。结论 SPHK基因可能参与脂肪细胞分化的调控过程，与肥胖发生有一定联系。     

TNFα对人脂肪细胞STEAP4基因表达的调控

目的：人STEAP4基因是一个参与胰岛素敏感性调节的肥胖相关差异表达基因,本研究旨在探讨胰岛素抵抗相关脂源性细胞因子TNFα对人成熟脂肪细胞中STEAP4基因的调节作用。方法：体外培养人前体脂肪细胞,在诱导人前体脂肪细胞分化成熟的基础上,应用不同浓度（0、5、10、25、50 ng/mL）人重组TNFα干预成熟脂肪细胞24 h,采用实时荧光定量RT-PCR技术、Western blot技术检测干预后人成熟脂肪细胞STEAP4基因在核酸和蛋白表达水平的变化。结果：不同浓度TNFα（5、10、25、50 ng/mL）干预24 h能显著上调人成熟脂肪细胞中STEAP4基因的mRNA表达,与未干预对照组差异有显著性（P<0.05）;50 ng/mL TNFα干预时STEAP4基因在人成熟脂肪细胞中的mRNA表达水平最高。与基因表达结果相一致,不同浓度TNFα（5、10、25、50 ng/mL）干预24 h能显著上调人成熟脂肪细胞中STEAP4蛋白的表达,与未干预对照组差异有显著性（P<0.05）;干预浓度提高到25 ng/mL 时干预效果最为明显。结论：TNFα能显著上调人成熟脂肪细胞中STEAP4基因核酸和蛋白的表达。［中国当代儿科杂志,2009,11（12）：1008-1011］     

NYGGF4基因过表达对脂肪细胞胰岛素敏感性及分泌功能的影响

目的：观察肥胖相关新基因NYGGF4过表达对脂肪细胞的胰岛素敏感性及分泌功能的影响。方法：体外培养稳定转染NYGGF4基因（NYGGF4-pcDNA3.1）的3T3-L1前体脂肪细胞,以转染空载体（pcDNA 3.1）的3T3-L1细胞为对照,胰岛素加地塞米松加1-甲基-3-异丁基黄嘌呤(MDI)方案诱导细胞分化成熟,液闪仪测定3H标记的葡萄糖摄取率,Western blot检测葡萄糖转运体4（GLUT4）的表达及转位;采用ELISA双抗体夹心法检测培养上清中TNF-α、IL-6、脂联素、抵抗素4种细胞因子的水平。结果：NYGGF4过表达显著降低胰岛素刺激的葡萄糖摄取率（P<0.05）。NYGGF4过表达对总的GLUT4的表达量没有影响,但明显降低胰岛素刺激的GLUT4由细胞浆向细胞膜的转位（P<0.05）。过表达NYGGF4的脂肪细胞其培养上清中TNF-α、IL-6、脂联素及抵抗素的分泌水平与对照组相比差异无显著性（P>0.05）。结论：NYGGF4基因过表达通过下调胰岛素刺激的GLUT4的转位而减少脂肪细胞的葡萄糖摄取率,提示脂肪细胞对胰岛素的敏感性降低,而对脂肪细胞的分泌功能无明显影响。［中国当代儿科杂志,2009,11（10）：846-849］     

Effect of bowel resection and high-fat diet on heart CD36/fatty-acid translocase expression in a rat model of short-bowel syndrome

Long-chain fatty acids (LCFA) are the major energy substrates for the heart. In short-bowel syndrome (SBS), LCFA delivery to the myocardium decreases due to fat malabsorption. Fatty-acid translocase (FAT)/CD36 has recently been identified as a LCFA-binding protein in heart tissue. To determine the effects of bowel resection and a high-fat diet (HFD) on myocardial CD36 expression, male Sprague-Dawley rats were randomly assigned to one of three groups: sham rats fed normal chow (Sham-NC); SBS rats fed NC (SBS-NC), and SBS rats fed a HFD (SBS-HFD). Control rats underwent transection and anastomosis; SBS animals underwent 75% small-bowel resection. Rats were killed at 3 or 14 days. Total body weight, heart weight, heart-tissue total lipid, serum cholesterol, and triglycerides were determined at death. Total RNA from the myocardium was extracted using TRIZOL reagent. Northern-blot analysis was used to determine FAT/CD36 mRNA. Statistical significance was determined by Student's t-test with P values below 0.05 considered significant. SBS-NC and SBS-HFD rats had significantly lower body weights compared with Sham-NC animals. The heart weights and myocardial total lipid did not vary among experimental groups. Decreases in plasma triglycerides (38.2 +/- 3.8 vs 58.8 +/- 5.5 mg/dl, P < 0.05) and cholesterol (38.2 +/- 6.9 vs 55.3 +/- 8.2 mg/dl, P < 0.05) in SBS-NC compared to Sham-NC rats on day 3 was accompanied by a twofold increase ( P < 0.05) in myocardial CD36/FAT mRNA levels. Early exposure to HFD led to increased (vs SBS-NC) plasma cholesterol (82.9 +/- 5.7 vs 38.2 +/- 6.9 mg/dl, P < 0.05) and triglycerides (62.5 +/- 15.6 vs 38.2 +/- 3.8 mg/dl, P < 0.05), and a concomitant decrease in CD36/FAT mRNA levels (45.1 +/- 17.8 vs 86.6 +/- 15%, respectively, P < 0.05). Plasma lipid concentration and myocardial CD36/FAT mRNA levels on day 14 were not significantly different among the experimental groups. In this rat model of SBS, the heart thus reacts to decreased LCFA delivery by increased tissue CD36/FAT mRNA levels and, consequently, active LCFA uptake. A HFD increased plasma lipid concentrations and decreased CD36/FAT levels.     

解偶联蛋白4基因在3T3-L1脂肪细胞诱导分化中的表达水平

目的　观察 3T3 L1脂肪前体细胞诱导分化过程中解偶联蛋白 4 (UCP4 )基因表达水平变化 ,探讨UCP4基因与肥胖发生之间的关系。方法　体外培养 3T3 L1细胞 ,诱导细胞分化 ,采用RT PCR技术在细胞分化成熟的不同时段检测脂肪细胞中UCP4基因mRNA表达水平。结果　UCP4基因高表达于 3T3 L1脂肪前体细胞中 ,随细胞分化成熟该基因表达水平渐下调。UCP4基因表达水平在诱导分化前 (d - 1)至d0、d0～ 3、d4～6、d7～ 8、d9～ 10内无显著性差异 (P >0 .0 5 ) ,余各时段表达水平均有显著差异 (P均 <0 .0 1)。结论　UCP4基因与肥胖发生相关 ,其在 3T3 L1细胞分化过程中表达逐渐下调可能有利于脂肪细胞的脂质积聚     

Effect of a high fat diet on lipid absorption and fatty acid transport in a rat model of short bowel syndrome

Long chain fatty acids (LCFAs) appear to be powerful stimulants for small bowel adaptation in patients with short bowel syndrome (SBS). However, the dietary lipid content may alter intestinal lipid transport. The aim of this study was to investigate the effects of a high fat diet (HFD) on in vivo lipid absorption and molecular and cellular mechanisms of LCFAs uptake by the remaining bowel. Male Sprague-Dawley rats (240-280) were randomly assigned to one of three groups: sham rats fed normal chow (sham-NC), SBS rats fed NC (SBS-NC) and SBS rats fed HFD (SBS-HFD). SBS rats underwent a 75% small bowel resection. Rats were sacrificed on day 3 or 14. Body weight, fat intake and fat clearance (total fecal fat) were measured twice a week. Fat absorbability was calculated as intake minus clearance and was expressed as percent of intake. Total RNA from the mucosa of duodenum, jejunum and ileum was extracted using TRIZOL Reagent. Northern blot analysis was performed to determine FAT/CD36 mRNA levels. Enterocyte LCFA transport was measured on day 14. LCFA uptake was determined by measuring cellular [3H]-oleate uptake over time (4-120 s). Mean (+/-SE) FAT/CD36 mRNA levels and oleate uptake kinetic parameters were analyzed using ANOVA. Fat absorbability diminished after bowel resection, suggesting fat malabsorption. Remaining bowel in SBS-NC rats responded by an increase in FAT/CD36 mRNA levels in the duodenum and ileum on day 3, and the duodenum and jejunum on day 14 compared to sham-NC animals, and was accompanied by an increase in enterocyte LCFA transport in all segments. Exposure to a HFD for 14 days resulted in significantly increased fat absorbability after 3 days compared to SBS-NC rats. However, FAT/CD36 mRNA levels (vs. SBS-NC) decreased in all segments on day 3. On day 14, FAT/CD36 mRNA levels were decreased in the duodenum and ileum and were accompanied by reduced oleate uptake by isolated enterocytes in the ileum (vs. SBS-NC). In a rat model of SBS, early high fat diet increased lipid absorptive capacity of the intestinal remnant as seen by increased fat absorbability. The main mechanisms of this effect may be an acceleration of structural intestinal adaptation resulting in an increased number of enterocytes. However, at molecular and cellular levels HFD decreased mucosal FAT/CD36 mRNA levels and oleic acid uptake by isolated enterocytes.     

Insulin-like growth factor binding protein-3 induces insulin resistance in adipocytes in vitro and in rats in vivo

Insulin-like growth factor binding protein (IGFBP)-3 binds to IGF and modulates their actions and also possesses intrinsic activities. We investigated its effects on insulin action and found that when IGFBP-3 was added to fully differentiated 3T3-L1 adipocytes in culture, insulin-stimulated glucose transport was significantly inhibited to 60% of control in a time- and dose-dependent manner. Tumor necrosis factor (TNF)-alpha treatment also inhibited glucose transport to the same degree as IGFBP-3 and, in addition, increased IGFBP-3 levels 3-fold. Co-treatment with TNF-alpha and IGFBP-3 antisense partially prevented the inhibitory effect of TNF-alpha on glucose transport, indicating a role for IGFBP-3 in cytokine-induced insulin resistance. Insulin-stimulated phosphorylation of the insulin receptor was markedly decreased by IGFBP-3 treatment. IGFBP-3 treatment suppressed adiponectin expression in 3T3-L1 adipocytes. Infusion of IGFBP-3 to Sprague-Dawley rats for 3 h decreased peripheral glucose uptake by 15% compared with controls as well as inhibiting glycogen synthesis. Systemic administration of IGFBP-3 to rats for 7 d resulted in a dramatic 40% decrease in peripheral glucose utilization and glycogen synthesis. These in vitro and in vivo findings demonstrate that IGFBP-3 has potent insulin-antagonizing capability and suggest a role for IGFBP-3 in cytokine-induced insulin resistance and other mechanisms involved in the development of type-2 diabetes.     

NYGGF4基因过表达对成熟脂肪细胞线粒体形态及动力学的影响

目的 探讨NYGGF4基因过表达对成熟3T3-L1脂肪细胞线粒体形态及动力学的影响.方法 以3T3-L1前体脂肪细胞为载体,建立NYGGF4基因稳定过表达细胞株,以空载质粒转染的细胞为对照,将前体脂肪细胞诱导分化为成熟脂肪细胞.采用透射电镜观察成熟脂肪细胞的线粒体形态,采用荧光定量PCR技术检测成熟脂肪细胞中线粒体融合基因(Mfn)1 mRNA、Mfn2 mRNA、线粒体动态相关基因(Drp)1 mRNA的表达水平.采用Western blot方法检测Mfn1蛋白、Mfn2蛋白、Drp1蛋白的表达水平.结果 1.电镜观察发现NYGGF4基因过表达的成熟脂肪细胞线粒体体积变小、数量明显减少,线粒体嵴断裂、减少、消失,部分线粒体肿胀,甚至呈空泡状;对照组成熟脂肪细胞的线粒体形态基本正常,线粒体嵴清晰可见,线粒体无明显肿胀、皱缩.2.NYGGF4基因过表达成熟脂肪细胞Mfn1 mRNA及Mfn1蛋白表达水平均显著高于对照组.3.NYGGF4基因过表达成熟脂肪细胞的Mfn2 mRNA、Drp1 mRNA及相应蛋白表达水平与对照组比较差异均无统计学意义.结论 在3T3-L1成熟脂肪细胞中,NYGGF4基因过表达可导致线粒体形态发生变化、数量减少,同时上调Mfn1 mRNA和Mfn1蛋白表达水平,提示NYGGF4基因在成熟脂肪细胞中过表达能影响细胞线粒体形态及动力学.     

3T3-L1脂肪细胞分化中促酰化蛋白对脂肪因子肾上腺髓质素mRNA表达的影响

目的 研究3T3-L1脂肪细胞分化过程中脂肪因子肾上腺髓质索(AM)mRNA表达变化及促酰化蛋白(ASP)对细胞分化过程中AM mRNA表达的影响,探讨ASP在脂肪代谢中的作用.方法 1.应用反转录(RT) -PCR法检测诱导分化4个时点(0d、1d、2d、3d)的3T3-L1脂肪细胞中AM mRNA的表达水平;2.对诱导分化过程中不同时间点(0h、12 h、24h、48 h)的3T3-L1脂肪细胞给予适合浓度的ASP处理,并设立相应空白对照,应用RT-PCR法检测细胞中AM mRNA的表达.结果 (1)脂肪细胞诱导分化各时间点(0d、1d、2d、3 d)AM mRNA均明显表达,且表达水平呈时间依赖性递减;(2)0 h、12 h和24h3个时点ASP组细胞中AMmRNA表达水平较空白对照组均显著下降(P＜0.05,0.01),而48 h时间点ASP组细胞中AM mRNA表达水平与空白对照组比较差异无统计学意义(P＞0.05).结论 ASP促成脂作用可能与其调节脂肪细胞分化过程中脂肪因子AM mRNA表达密切相关.     

Effect of low fat diet on lipid absorption and fatty-acid transport following bowel resection

 Low-fat diets (LFD) are used extensively in many different clinical conditions. However, the effect of this diet on lipid absorption and cellular long-chain fatty-acid (LCFA) transport is unknown. Fatty-acid translocase (FAT), the rat homologue of human CD36, is one of several LCFA plasma-membrane transport proteins that may play an important role in intestinal lipid uptake. The purpose of this study was to investigate the effects of a LFD on intestinal expression of FAT/CD36, enterocyte fatty-acid transport, and in-vivo lipid absorption in rats following bowel resection. Adult male Sprague-Dawley rats were divided into five experimental groups: normal rats fed normal chow(NR-NC) (10 kcal% fat), normal rats fed a LFD (NR-LFD) (3 kcal% fat), sham rats fed normal chow (Sham-NC), short-bowel syndrome rats fed normal chow (SBS-NC), and SBS rats fed a LFD (SBS-LFD). SBS rats underwent 75% small-bowel resection, while sham animals underwent bowel transection and re-anastomosis. Food intake, fecal mass, and fecal fat were measured over the last 3 days before death on day 14. Final body weight, plasma lipids and protein, and tissue total lipids in liver, adipose tissue, and intestine were determined at death. Total RNA from the mucosa of the duodenum, jejunum, and ileum was extracted for Northern blot analysis to determine fatty-acid translocase (FAT)/CD36 mRNA levels. An established cellular LCFA transport assay was used to determine isolated enterocyte [³H]-oleate uptake. Students's t-test was used to determine statistical significance (P < 0.05). NR-LFD rats demonstrated a small increase in overall food absorption and no change in fat absorption compared to NR-NC animals. A significant decrease in FAT/CD36 mRNA levels was seen in the duodenum and jejunum in NF-LFD rats (vs NR-NC) and was accompanied by reduced LCFA transport by isolated enterocytes from the jejunum and ileum. SBS-LFD rats demonstrated decreased FAT/CD36 mRNA levels in all three segments and a concomitant decrease in LCFA uptake enterocytes compared to the SBS-NC group. In addition, SBS-LFD rats showed significantly lower final body weight and plasma lipids compared to SBS-NC animals.     

The lipid fraction of human milk initiates adipocyte differentiation in 3T3-L1 cells

Background

The prevalence of childhood obesity has increased worldwide over the past decade. Despite evidence that human milk lowers the risk of childhood obesity, the mechanism is not fully understood.

Aims

We investigated the direct effect of human milk on differentiation of 3T3-L1 preadipocytes.Study design and subjects: 3T3-L1 preadipocytes were treated with donated human milk only or the combination of the standard hormone mixture; insulin, dexamethasone (DEX), and 3-isobututyl-1-methylxanthine (IBMX). Furthermore, the induction of preadipocyte differentiation by extracted lipids from human milk was tested in comparison to the cells treated with lipid extracts from infant formula. Adipocyte differentiation, specific genes as well as formation of lipid droplets were examined.

Results

We clearly show that lipids present in human milk initiate 3T3-L1 preadipocyte differentiation. In contrast, this effect was not observed in response to lipids present in infant formula. The initiation of preadipocyte differentiation by human milk was enhanced by adding the adipogenic hormone, DEX or insulin. The expression of late adipocyte markers in Day 7 adipocytes that have been induced into differentiation with human milk lipid extracts was comparable to those in control cells initiated by a standard adipogenic hormone cocktail.

Conclusions

These results demonstrate that human milk contains bioactive lipids that can initiate preadipocyte differentiation in the absence of the standard adipogenic compounds via a unique pathway.     

表没食子儿茶素-3-没食子酸酯对宫内生长受限大鼠肝脏脂代谢的影响和机制

目的 探讨表没食子儿茶素-3-没食子酸酯（EGCG）对宫内生长受限（IUGR）大鼠肝脏脂代谢的影响和机制。方法 采用母鼠孕期全程限食法建立IUGR大鼠模型,随机分为IUGR组和EGCG组,EGCG组大鼠在离乳后用含EGCG的饮用水喂养至10周,同时设立正常对照组,每组8只。13周龄时,测量各组大鼠体重后,采集大鼠血液及肝脏组织标本,检测各组大鼠血清空腹总胆固醇（TC）、甘油三酯（TG）、游离脂肪酸（FFA）、血糖（FPG）、胰岛素（FINS）和肝脏脂质水平,计算稳态模型评估胰岛素抵抗（HOMA-IR）和脂肪组织胰岛素抵抗（adipo-IR）,观察肝脏组织病理切片,并采用实时荧光定量PCR法检测肝脏相关基因的相对表达水平。结果 13周龄时,各组大鼠体重比较差异无统计学意义（P=0.067）。各组间的FPG、FFA、FINS、HOMA-IR和adipo-IR水平比较差异均有统计学意义（P < 0.05）。各组间的血清TC和TG水平比较差异无统计学意义（P > 0.05）,但在肝脏中IUGR组TC和TG水平均明显高于EGCG组（P < 0.05）。油红染色结果提示,IUGR大鼠的肝脏脂肪储积明显增加,而EGCG能够改善该现象。PCR结果显示,与对照组相比,IUGR组的Ampk mRNA及Adipor1 mRNA表达水平降低,Srebf1 mRNA表达水平增加（P < 0.05）,EGCG能逆转IUGR大鼠Ampk mRNA及Srebf1 mRNA的表达水平,且与对照组比较差异无统计学意义（P > 0.05）。结论 早期EGCG干预可能通过Ampk/Srebf1通路下调脂肪酸的从头合成,并通过改善肝细胞的胰岛素抵抗,从而降低IUGR大鼠的肝脏脂肪积累。     

肿瘤坏死因子-α对人脂肪细胞STEAP4蛋白膜转位的影响及其机制

目的 探讨肿瘤坏死因子-α (TNF-α)对人脂肪细胞STEAP4蛋白膜转位的影响,并分析其可能的机制.方法 应用1-甲基3-异丁基黄嘌呤(MIX)、地塞米松、胰岛素、罗格列酮方案诱导人前体脂肪细胞分化,以人重组TNF-α或加用细胞外信号调节蛋白激酶( ERK)抑制剂PD-98059干预人成熟脂肪细胞24 h,采用Western blot技术检测细胞膜蛋白与总蛋白中STEAP4蛋白的表达.结果TNF-α干预能显著促进人成熟脂肪细胞中STEAP4蛋白向细胞膜转位;ERK抑制剂PD-98059联合TNF-α与单独应用TNF-α诱导人成熟脂肪细胞的细胞膜蛋白和总蛋白中STEAP4蛋白的表达水平比较均无显著差异.结论 TNF-α干预显著促进人成熟脂肪细胞中STEAP4蛋白的膜转位,初步排除丝裂原活化蛋白激酶信号途径参与这一调控机制.     

PI3K抑制剂LY294002对鼠前脂肪细胞分化和C/EBPα及PPARγ表达的影响

目的：研究磷脂酰肌醇激酶（PI3K）抑制剂LY294002对小鼠前脂肪细胞分化和CCAAT增强子结合蛋白α（C/EBPα）及过氧化物酶体增殖物激活受体γ（PPARγ）表达的影响,探讨胰岛素受体底物（IRSs）/PI3K信号通路在前脂肪细胞分化中的作用。方法：小鼠3T3-L1细胞分为实验组和对照组,实验组加入LY294002（25 μmol/L）,对照组加入同体积DMSO。应用0.5 mmol/L 3-异丁基-1-甲基黄嘌呤(IBMX)、10-6 mol/L地塞米松和5 μg/mL胰岛素诱导两组前脂肪细胞分化,在培养的0 d、2 d、4 d、8 d分别收集细胞,实时PCR法及Western blot法检测细胞中C/EBPα和PPARγ表达水平。培养8 d油红O染色,观察细胞分化情况。结果：小鼠3T3-L1细胞基础状态下两组C/EBPα及PPARγ表达差异无统计学意义（P＞0.05）;诱导分化时实验组C/EBPα及PPARγ表达低于对照组（分别P＜0.05,P＜0.01）。油红O染色示实验组细胞无明显脂滴。结论：PI3K抑制剂LY294002能抑制小鼠前脂肪细胞分化及C/EBPα和PPARγ的表达,提示IRSs/PI3K信号通路可通过调节PPARγ和C/EBPα的表达在3T3-L1前脂肪细胞的分化中发挥重要作用。     

STEAP4基因在人前体脂肪细胞诱导分化过程中的表达

目的观察人肥胖相关全长新基因STEAP4在人前体脂肪细胞诱导分化过程中不同时段表达水平的变化,探讨STEAP4基因与脂肪细胞分化、脂质积聚的关系。方法体外培养人前体脂肪细胞,待细胞生长融合后以1-甲基-3-异丁基黄嘌呤、地塞米松、胰岛素及罗格列酮联合诱导方案,在体外诱导其分化为成熟脂肪细胞。在前体脂肪细胞向成熟脂肪细胞分化过程中观察细胞形态及脂质积聚变化;采用实时荧光定量RT-PCR技术检测诱导分化不同时段(前体,第0、4、6、8、11、14、17天)脂肪细胞中STEAP4基因mRNA的表达水平。结果STEAP4基因高表达于人前体脂肪细胞;加入脂肪细胞分化诱导剂后(第4天)表达略有上调,其后随脂肪细胞分化成熟该基因表达量呈逐渐下降趋势;至脂肪细胞完全分化成熟(第14-17天)表达量最低。STEAP4基因表达水平除在诱导分化前至第4天、第14-17天差异无统计学意义外,余各时间点的表达水平差异均有统计学意义(Pa<0.05)。结论STEAP4基因随脂肪细胞分化成熟表达逐渐下调,可促进脂肪细胞的分化成熟和脂质积聚,与肥胖的发生有关。