骨髓脂肪细胞多向分化潜能的初步探讨

目的 从人骨髓脂肪层中分离培养骨髓脂肪细胞,探讨其多向分化潜能.方法 体外贴壁培养和扩增人骨髓脂肪细胞,采用流式细胞仪检测骨髓脂肪细胞表面抗原CD105、CD166、CD49d等的表达情况.通过RT-PCR检测转录因子过氧化物酶增殖激活受体-γ2(PPAR-γ2)、CCAATT增强结合蛋白(C/EBPs)和瘦素(leptin)的表达.经体外诱导分化检测骨髓脂肪细胞的体外成脂和成骨能力.结果 流式细胞仪检测结果显示骨髓脂肪细胞与骨髓基质细胞具有相似的表面标志,但骨髓脂肪细胞特征性表达CD49d、C/EBPs以及leptin.体外诱导分化实验显示骨髓脂肪细胞具有成骨和成脂的能力.结论 骨髓脂肪细胞具有脂肪前体细胞的特性,但同时它具有同骨髓基质细胞相似的表面标志和多向分化潜能,提示可以作为一种新的组织工程种子细胞来源.     

瘦素对成骨细胞增殖及Collagen I和Cbfa1 mRNA表达的影响

目的 研究瘦素在体外对原代培养乳鼠颅盖骨成骨细胞增殖及对CollagenI(I型胶原蛋白)和Cbfa1 mRNA表达的影响.方法 培养乳鼠成骨细胞,以乳鼠成骨细胞为体外实验模型,采用MTT法检测不同浓度瘦素(0、40、80和160 ng/ml)作用于成骨细胞后的增殖情况;通过RT-PCR方法检测不同浓度瘦素对CollagenI和Cbfa1 mRNA的表达的影响.结果 瘦素作用于成骨细胞后,可促进其增值,OD值显著增加(P<0.01);瘦素增加CollagenI和Cbfa1 mRNA的表达,呈剂量效应关系.结论 瘦素促进成骨细胞增殖,同时通过调节CollagenI和Cbfa1的表达,促进成骨细胞分化,进而促进骨形成.     

瘦素对脂肪细胞的直接调节作用

目的:观察瘦素对人脂肪细胞分解代谢及脂肪蓄积的直接影响,探讨脂肪细胞的自分泌调节作用。方法:取正常成人皮下脂肪组织,常规提取和培养前脂肪细胞,待细胞融合后诱导分化为成熟脂肪细胞并随机分为四组:正常对照组、低浓度组、中浓度组和高浓度组,分别培养于含瘦素浓度为0ng/ml、10ng/ml、100ng/ml、1000ng/ml的培养液a中。培养4h后收集培养液检测游离脂肪酸和甘油浓度,取脂肪细胞经油红O染色后图像分析计算脂肪颗粒的积分光密度。结果:与对照组相比,低浓度组培养液中游离脂肪酸和甘油浓度分别增加87%(P<0.01)和5%(P<0.01),脂肪颗粒积分光密度减少12%(P<0.01);中浓度组培养液中游离脂肪酸和甘油浓度分别增加181%(P<0.01)和8%(P<0.01),脂肪颗粒光密度减少21%(P<0.01);高浓度组培养液中游离脂肪酸和甘油的浓度分别增加312%(P<0.01)和17%(P<0.01),脂肪颗粒的积分光密度减少35%(P<0.01)。游离脂肪酸和甘油浓度与瘦素浓度呈正相关,脂肪颗粒积分光密度与瘦素浓度呈负相关。结论:瘦素瞬时作用脂肪细胞,可促进脂肪分解代谢、减少脂肪蓄积;且随着瘦素浓度增加,作用更强,呈剂量依赖性。瘦素对脂肪细胞存在自分泌调节作用,可能在肥胖发病机制中起重要作用。     

瘦素对成骨细胞增殖及CollagenⅠ和Cbfa1 mRNA表达的影响

目的研究瘦素在体外对原代培养乳鼠颅盖骨成骨细胞增殖及对CollagenⅠ(Ⅰ型胶原蛋白)和Cbfa1 mRNA表达的影响。方法培养乳鼠成骨细胞,以乳鼠成骨细胞为体外实验模型,采用MTT法检测不同浓度瘦素(0、40、80和160ng/ml)作用于成骨细胞后的增殖情况;通过RT-PCR方法检测不同浓度瘦素对CollagenⅠ和Cbfa1 mRNA的表达的影响。结果瘦素作用于成骨细胞后,可促进其增值,OD值显著增加(P0.01);瘦素增加CollagenⅠ和Cbfa1 mRNA的表达,呈剂量效应关系。结论瘦素促进成骨细胞增殖,同时通过调节CollagenⅠ和Cbfa1的表达,促进成骨细胞分化,进而促进骨形成。     

高浓度瘦素自分泌诱导脂肪细胞瘦素抵抗的研究

目的:观察高浓度瘦素对人脂肪细胞分解代谢及脂肪蓄积的直接影响,探讨瘦素自分泌调节在肥胖发生中的作用。方法:取正常成人皮下脂肪组织,常规提取和培养前脂肪细胞,待细胞融合后诱导分化为脂肪细胞后分为二组:低浓度组、高浓度组;分别培养于瘦素终浓度为100ng/ml、1000ng/ml的培养液a中。于培养48h、72h收集培养液检测游离脂肪酸和甘油的浓度,取脂肪细胞行油红O染色并图像分析计算脂肪细胞中脂肪颗粒的积分光密度。结果:与低浓度组相比,瘦素作用48h、72h后,高浓度组培养液中游离脂肪酸浓度均下降[(0.16711±0.011900)mmol/L VS(0.20589±0.008738)mmol/L,(0.17544±0.013920)mmol/L VS(0.23567±0.026220)mmol/L,甘油含量均减少(28.1733±0.91377)μmol/L VS(30.2456±0.30084)μmol/L,(28.9367±0.79530)μmol/L VS(31.8567±0.79024)μmol/L],而脂肪颗粒积分光密度则升高(461136.89±12049.947 VS418570.33±5668.129,441566.56±5921.602 VS 390133.67±7001.304)。结论:高浓度瘦素长时程作用脂肪细胞,可延缓脂肪分解代谢,增加脂肪蓄积。高浓度瘦素经自分泌诱导脂肪细胞产生瘦素抵抗,可能对肥胖发生有重要影响。     

葛根素对大鼠乳房脂肪组织增殖的影响及其机制研究

目的:通过研究葛根素(Puerarin,Pur)对3T3-L1前脂肪细胞增殖分化及大鼠乳房脂肪组织的影响及其相关蛋白表达的调控,旨在探讨Pur对乳房脂肪组织的影响及其机制。方法:采用体外培养3T3-L1前脂肪细胞测定不同浓度Pur对3T3-L1脂肪细胞分化过程中胞浆脂滴积累的影响,通过Western blot、免疫荧光实验检测Pur对解偶联蛋白(Uncoupling protein 1,UCP-1)、重组人CCAAT增强子结合蛋白(CCAA Tenhancer-binding protein α,C/EBP-α)表达水平;进一步考察大鼠乳房涂抹不同浓度的Pur对脂肪组织增殖的影响,通过HE染色检测乳腺脂肪细胞体积、免疫组化检测乳腺脂肪蛋白,分析其增殖机制。结果:Pur通过上调3T3-L1细胞中脂肪分化相关蛋白UCP-1、C/EBPα的表达,显著促进3T3-L1脂肪细胞分化过程中胞浆脂滴积累。此外,体内实验结果显示Pur能促进大鼠乳房脂肪细胞增多,脂肪细胞直径增大,且促进乳腺UCP-1、C/EBPα的表达。结论:Pur显著促进大鼠乳房脂肪组织增殖,其作用机制可能与上调脂肪分化相关的蛋白 UCP-1、C/EBPα的表达有关。     

血管紧张素1-7对高糖诱导人肾小管上皮细胞转分化的影响及其机制

目的 研究血管紧张素1-7（Ang 1-7）对高糖诱导人肾小管上皮细胞（HK-2）转分化的影响及其可能机制。 方法 培养HK-2细胞分组如下：对照组（N组）、高糖组（H组）、高糖+Ang 1-7组（A组）、高糖+Ang 1-7+A779组（D组）、高糖+吡格列酮组（P组）。Western印迹检测各组HK-2细胞过氧化物酶体增殖物激活受体γ（PPAR-γ）及α平滑肌肌动蛋白（α-SMA）的蛋白表达;实时定量PCR检测HK-2细胞PPAR-γ及α-SMA的mRNA表达;免疫荧光检测α-SMA表达。 结果 Ang 1-7可上调高糖刺激下HK-2细胞PPAR-γ蛋白及mRNA表达（P < 0.05）;抑制高糖刺激的α-SMA蛋白及mRNA表达（P < 0.05）。这种作用与PPAR-γ激动剂吡格列酮类似。给予Mas受体抑制剂A779后,Ang 1-7的上述作用可被部分抑制。 结论 Ang 1-7在体外可通过上调PPAR-γ表达,从而部分抑制高糖诱导的α-SMA表达,实现其抑制转分化的作用,而这种作用部分通过Mas受体所介导。     

肿瘤坏死因子α对人肝癌多药耐药逆转作用的实验研究

目的研究肿瘤坏死因子α（TNF-α）对体外培养的人肝癌耐阿霉素细胞系（HepG2／ADM）多药耐药现象的逆转作用。方法不同浓度（100、500及2500U／ml）TNF-α作用于HepG2／ADM细胞72h后进行以下试验：用实时荧光定量聚合酶链反应技术检测各组多药耐药相关基因（MDR1）及脂质过氧化物酶体增殖物激活受体α（PPAR-α）基因的mRNA表达情况;用罗丹明外排法检测各组P-糖蛋白活性;用Annexin V检测0．5mg／L阿霉素诱导的各组细胞凋亡情况;利用MTF法检测各组耐药性的改变。结果TNF-α能诱导HepG2／ADM细胞的MDR1基因表达下调,PPAR-α基因表达上调,且能增加阿霉素诱导的凋亡细胞的比例及细胞毒作用。结论TNF-α可能分别通过抑制MDR1表达及促进PPAR-α表达而逆转HepG2／ADM细胞的耐药性。     

儿茶酚胺对人前脂肪细胞增殖与分化的作用

目的 研究去甲肾上腺素、肾上腺素和异丙肾上腺素这三种儿茶酚胺对人前脂肪细胞增殖与分化的作用。方法 培养人皮下脂肪的前脂肪细胞，在其增殖与分化的不同阶段分别加入肾上腺素、去甲肾上腺素和异丙肾上腺素这三种儿茶酚胺，观察其对人前脂肪细胞的增殖，分化过程中的磷酸甘油脱氢酶(GPDH)和细胞内脂肪积聚的作用，并探讨α受体和β受体阻断剂对这个过程的影响。结果:肾上腺素和异丙。肾上腺素对人前脂肪细胞的增殖有促进作用，对分化过程中的GPDH的升高有抑制作用；而对于分化过程中的脂肪积聚三者均有促进分解的作用；均通过前脂肪细胞的β受体完成。结论 人前脂肪细胞对儿茶酚胺的反应与成熟脂肪细胞相比有其特点，可望应用于脂肪分解药物的研究。     

bFGF对前脂肪细胞增殖和分化的作用

目的:探讨bFGF对体外培养前脂肪细胞增殖和分化活性的影响。方法:无菌条件下抽吸的脂肪组织进行前脂肪细胞的原代培养,加入生长因子bFGF 10ng/ml,用MTT法观察人前脂肪细胞生长状态,测定前脂肪细胞合成甘油三酯的总量。结果:bFGF有明显的促增殖作用,与对照组比较第1、2天差异不明显,随时间的推移,第6～7天到达高峰;到分化6天和9天时,甘油三酯总量明显升高,与对照组相比差异均有显著性(P<0.05)。结论:重组人bFGF可促进前脂肪细胞的增殖和分化。     

Differential effects of leptin on cancer in vitro

INTRODUCTION: Leptin, a protein produced by adipocytes, is an important signaling molecule in energy regulation and food intake. Many obese patients have leptin resistance associated with increased circulating leptin. Leptin receptor activation downregulates many regulatory genes, including STAT-3 and PAP 1. Certain cancers are associated with obesity, including breast, prostate, and colon. Recent studies have shown that leptin stimulates proliferation of human colon cancer in vitro. We hypothesized that leptin would have stimulatory effects on other human cancers. MATERIALS AND METHODS: Human cancer cell lines from esophagus (KYSE410 and 150), breast (ZR75-1 and MCF-7), prostate (DU145 and PC-3), and pancreas (PANC-1, Mia-PaCa) were cultured using standard techniques. Leptin (0.4 ng/ml and 4.0 ng/ml) was added for 24 h and 48 h. Cell growth was determined by MTT assay. Statistical analysis was performed using analysis of variance. RESULTS: Cancer cell lines demonstrated dose- and time-related responses to treatment. Leptin caused growth potentiation in breast, esophagus, and prostate cancer (P < 0.05). However, in both Mia-PaCa and PANC-1 pancreatic cancer cells, leptin inhibited growth (P < 0.05). This inhibitory effect peaked in PANC-1 at 48 h (78%). CONCLUSIONS: We have shown for the first time that human cancer cells exhibit differential responses to treatment with leptin, depending upon organ of derivation. Both leptin and leptin antagonism have potential efficacy in cancer therapy, based on cellular origin. Further studies are warranted and ongoing.     

痩素对小鼠破骨细胞分化及功能的影响

目的 观察瘦素（leptin）对体外骨髓诱导培养的小鼠破骨细胞分化和功能的作用效应,探索leptin和骨吸收之间的关联.方法 建立由巨噬细胞集落刺激因子（M-CSF）和骨保护素配体（RANKL）为共同细胞因子的小鼠破骨细胞骨髓诱导培养体系,将不同浓度的leptin作用于破骨细胞.实验中根据培养液中是否加入M-CSF和RANKL并依据leptin浓度的不同分为：A组,M-CSF和RANKL;B组,M-CSF、RANKL和leptin（80 ng/ml）;C组,M-CSF、RANKL和leptin（160 ng/ml）;D组,M-CSF、RANKL和leptin（240 ng/ml）;E组,M-CSF、RANKL和leptin（320 ng/ml）;F组,M-CSF、RANKL和leptin（400 ng/ml）;同时设立空白对照组G组.于作用后第7天取细胞玻片进行抗酒石酸酸性磷酸酶（TRAP）染色,观察破骨细胞并计数;于第10天取出骨片进行甲苯胺蓝染液染色,在光镜和扫描电镜观察骨吸收陷窝形态.结果：诱导培养的小鼠破骨细胞形态特征明显;A组在破骨细胞数量与D、E、F组相比较有明显的统计学差异（P＜0.05）;A组骨吸收面积比与B、C、D、E、F组都有明显的统计学差异（P＜0.05）.结论：leptin抑制体外培养的破骨细胞的分化和骨吸收功能.     

Leptin stimulates esophageal adenocarcinoma growth by nonapoptotic mechanisms

BACKGROUND: Leptin is a hormone primarily produced by adipocytes and serum leptin is elevated in obese persons. One risk factor associated with adenocarcinoma of the esophagus is obesity. We hypothesized that leptin would have stimulatory effects on esophageal adenocarcinoma and alter apoptosis in vitro. METHODS: Barrett's esophageal adenocarcinoma cells (BIC-1 and SEG-1) were cultured with human recombinant leptin (80 ng/mL) for 24 hours. Cell growth was determined by MTT assay. Apoptosis and necrosis was measured after 16 hours of treatment with leptin using a Cell Death Kit. RESULTS: Exogenous leptin stimulated cell proliferation in both cell lines. No changes in apoptosis or necrosis resulted between control and leptin-treated groups. CONCLUSIONS: We have shown that leptin increases the proliferation of human esophageal adenocarcinoma, but does not alter cell apoptosis or necrosis. The data suggest that leptin stimulates esophageal adenocarcinoma growth by nonapoptotic mechanisms. Leptin antagonism may have potential efficacy in esophageal cancer therapy.     

共轭三烯酸对人胶质瘤U251细胞的增殖抑制作用

目的 探讨共轭三烯酸(TCLA)对人胶质瘤细胞的增殖抑制作用及作用机制。方法 用噻唑蓝(MTT)比色法、克隆形成试验、5-溴脱氧尿嘧啶核苷(BrdU)掺人试验研究TCLA对人脑胶质瘤细胞的增殖抑制作用;Hoechst 33342/PI双染法、流式细胞术检测细胞凋亡指数和周期分布;逆转录-聚合酶链反应(RT-PCR)检测凋亡相关基因腺苷二磷酸核糖转移酶1(ADPRTL1)、细胞色素P4501A1( CYP1A1),过氧化物酶增殖体激活受体-γ(PPAR-γ)的表达。结果 共轭三烯酸对人胶质细胞瘤(U251)有明显抑制作用,呈剂量-时间-效应关系(P＜0.05)。克隆形成下降,40 μmol/LTCLA可完全抑制克隆形成。BrdU标记从(91.6±3.6)％下降到(14.4±4.4)％(P＜0.05),Hoechst 33342/PI双染试验,凋亡细胞增加(P＜0.05)。流式细胞仪检测显示,细胞凋亡指数从(7.3±1.2)％升高到(34.2±2.4)％(P＜0.05),Go/G1期细胞比例增加,S期细胞比例减少(P＜0.05);RT-PCR结果显示,凋亡相关基因ADPRTL1、CYP1 A1、PPAR-γ mRNA表达增强(P＜0.05)。结论 TCIA对人脑胶质瘤细胞具有增殖抑制和凋亡诱导作用,其作用机制可能与抑制肿瘤细胞DNA合成、细胞周期阻滞、上调凋亡相关基因(ADPRTL1、CYP1A1、PPAR-γ)表达有关。     

常用维生素对人前脂肪细胞增殖和分化的作用

目的研究维生素(A、B、C、D、E、K各族)对前脂肪细胞增殖和分化的作用。方法培养人前脂肪细胞，在其生长的不同阶段分别加入各种维生素，观察其对细胞的增殖、分化过程中的磷酸甘油脱氢酶(GPDH)和细胞内脂肪积聚的作用。结果维生素A对人前脂肪细胞增殖和分化均有抑制作用，维生素B6对人前脂肪细胞的分化有促进作用，维生素C对人前脂肪细胞增殖和分化均有明显的促进作用，维生素D3对人前脂肪细胞的分化有抑制作用，维生素E对人前脂肪细胞的分化有较强的抑制作用，维生素K1对人前脂肪细胞分化有极强的刺激作用，维生素K3对人前脂肪细胞的增殖和分化均有抑制作用。未发现其他维生素对人前脂肪细胞的明显作用。结论该研究结果可为人体合理地摄入维生素提供理论指导。     

Effects of leptin and neuropeptide Y on function of human ovarian granulosa cells in vitro

Objective: To investigate the effects of leptin and neuropeptide Y on steroidogenesis of human ovarian granulosa cells in vitro. Methods: Human ovarian granulosa cells were isolated from follicular fluid obtained during oocyte retrieval of in vitro fertilization-embryo transfer program and cultured for 2 days with various concentration of leptin(1,10,100 ng/ml) or neuropeptide Y (1×10-6, 1×10-7, 1×10-8 mol/L) alone or both,or with the combination of human chorionic gonadotropin (hCG 0, 20 IU/L). The medium was collected for estradiol (E2) and progesterone (P) measurements. Results: (1)Whether hCG existed or not, the adding of leptin did not alter estradiol and progesterone production by human granulosa cells (P>0.05).(2) Only when the concentration of neuropeptide Y was at 1×10-7mol/L,estradiol level was lower than that in the control (P<0.05).(3) The levels of estradiol in neuropeptide Y (1×10-7mol/L) plus hCG group were significantly higher than those with neuropeptide Y alone(P<0.05). (4) In the absence of hCG, the levels of estradiol in neuropeptide Y (1×10-7mol/L)plus leptin (10 ng/ml) group were significantly higher than those with neuropeptide Y(1×10-7mol/L)alone(P<0.05).Conclusions: (1)Leptin alone produced no direct effect on secretion of E2 and P from granulosa cells in vitro.(2)Neuropeptide Y alone may inhibit the secretion of E2, but the inhibition would probably be blocked with the presentation of hCG.(3)Leptin probably blocked the inhibition of neuropeptide Y on E2 secretion, and this may indicate that there were some coordination between leptin and neuropeptid Y on the level of ovarian function.     

Leptin inhibits osteoclast generation.

Originally, leptin was described as a product of adipocytes that acts on the hypothalamus to regulate appetite. However, subsequently, it has been shown that leptin receptors are distributed widely and that leptin has diverse functions, including promotion of hemopoietic and osteoblastic differentiation. It has been recognized for some time that both serum leptin and bone mass are correlated positively to body fat mass and, recently, we have shown a direct positive relationship between serum leptin and bone mass in nonobese women. We now report that leptin inhibits osteoclast generation in cultures of human peripheral blood mononuclear cells (PBMCs) and murine spleen cells incubated on bone in the presence of human macrophage colony-stimulating factor (hM-CSF) and human soluble receptor activator of NF-kappaB ligand (sRANKL). The half-maximal concentration inhibitory of leptin was approximately 20 nM in the presence of sRANKL at 40 ng/ml but decreased to approximately 2 nM when sRANKL was used at 5 ng/ml. The majority of the inhibitory effect occurred in the first week of the 3-week cultures. Inhibition did not occur when the PBMC cultures were washed vigorously to remove nonadherent cells or when purified CD14+ monocytes were used to generate osteoclasts, indicating an indirect or permissive effect via CD14- PBMC. Leptin increased osteoprotegerin (OPG) messenger RNA (mRNA) and protein expression in PBMC but not in CD14+ cells, suggesting that the inhibitory effect may be mediated by the RANKL/RANK/OPG system. Leptin may act locally to increase bone mass and may contribute to linkage of bone formation and resorption.     

Leptin promotes aggregation of human platelets via the long form of its receptor.

Plasma leptin levels are elevated in most obese individuals, and obesity is accompanied by a high incidence of cardiovascular disease. Therefore, leptin could be involved in the pathogenesis of cardiovascular disease. In the present study, the role of leptin was explored in the regulation of platelet function. The expression of the long form of the leptin receptor was detected in human platelets. At 50 ng/ml, human leptin induced phosphorylation of several proteins of platelets at the tyrosine residue. Neither leptin at concentrations < or = 100 ng/ml nor ADP at concentrations > or = 1 micromol/l affected platelet aggregation. However, after pretreatment with 100 ng/ml leptin for 5 min, 1 micromol/l ADP caused aggregation. Thus, leptin and ADP acted synergistically. At a concentration of 2 micromol/l, ADP induced platelet aggregation, which was markedly enhanced by 30-100 ng/ml leptin in a concentration-dependent manner. This concentration range corresponds to that of plasma leptin levels in obese individuals. At the lower concentrations (< 10 ng/ml) that are observed in normal individuals, leptin had no effect on platelet aggregation. In conclusion, leptin at high concentrations has the novel function of promoting platelet aggregation, which may be a key coupling factor between obesity and the cardiovascular disease associated with syndrome X and diabetes.