Ghrelin在肥胖发生机制中的实验研究及临床意义探讨
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摘要
儿童单纯性肥胖症是一种由于长期能量的摄入超过人体的消耗，体内的脂肪过度积聚、体质量超过一定范围的营养障碍性疾病。近年来，随着社会经济的发展，人民物质生活水平不断提高，生活现代化膳食结构发生改变，体力活动日益减少，儿童肥胖的患病率呈逐年增加的趋势，肥胖已成为一种全球性的流行病。儿童肥胖不仅损害儿童的身心健康，而且可能发展为成人肥胖，增加成人期心血管疾病发病的危险。由于其高发因素和高危后果，肥胖已经成为公共卫生领域的研究热点，预防肥胖的流行是21世纪前50年世界各国面临的最大公共卫生挑战之一。从儿童期着手预防和治疗肥胖显得十分重要。
肥胖是体脂的过多积聚，是脂肪细胞体积的增大和数目的增加。过量的能量合成甘油三酯储存于脂肪细胞中，甘油三酯的不断积累，使脂肪细胞体积增大。当前脂肪细胞增殖和分化异常增多或成熟脂肪细胞凋亡减少时，就会引起脂肪组织的过多堆积和脂肪细胞内分泌功能的紊乱，继而导致肥胖和胰岛素抵抗，最终引发糖尿病。因此，脂肪细胞生长、发育、分化及凋亡的分子调控机制已成为目前国际上研究肥胖及其相关疾病的热点方向。
Ghrelin是在1999年由Kojima等发现的生长激素促分泌素受体的第一个内源性配体，是一种胃源性促生长激素释放多肽，也是第一个被确认的末梢具有活性的促食欲因子。Ghrelin不仅能有效促进生长激素的分泌，还参与摄食、能量代谢、心血管和免疫功能的调节，是目前调节肽研究领域中的一个热点。Ghrelin可能是生长激素／胰岛素样生长因子-1轴和调节能量平衡的神经内分泌之间一个新的联结纽带。Ghrelin通过启动进食，增加食欲，增加营养物质、尤其是脂类的摄取，促进脂肪形成等多方面作用调节着机体能量代谢的平衡，在控制摄食与能量平衡及对饥饿的神经内分泌调节方面发挥重要的作用。
基于前脂肪细胞增殖、分化和凋亡在肥胖发生机制中的重要作用，本研究以3T3-L1前脂肪细胞为实验对象，观察ghrelin对其增殖、分化和凋亡的影响，并探讨其可能的分子生物学机制，进而深化对ghrelin这一目前已知的唯一能够促进食欲的外周激素的认识，深化对脂肪细胞增殖、分化和凋亡分子机制的认识，并进一步为肥胖及其相关疾病的预防和治疗提供新的研究方向。
第一部分生长激素促分泌素受体-1a基因在脂肪细胞分化成熟中的作用研究
目的通过观察生长激素促分泌素受体-1a(growth hormone secretagogue receptor,GHSR-1a)基因在3T3-L1前脂肪细胞诱导分化过程中不同时段表达水平的变化，从而进一步探讨GHSR-1a基因在脂肪细胞分化过程中的作用。
方法体外培养3T3-L1前脂肪细胞，采用经典的激素鸡尾酒诱导分化法诱导3T3-L1前脂肪细胞分化。在诱导其向成熟脂肪细胞分化的不同时段(从第1天到第8天)，采用光学显微镜观察脂肪细胞的形态学变化及细胞内脂滴的形成，通过油红O染色测定脂肪细胞分化程度及细胞内脂滴的聚积情况，采用化学比色法测定脂肪细胞中甘油三酯(triglyceride, TG)的总量，采用半定量逆转录-聚合酶链反应(RT-PCR)技术检测脂肪细胞中GHSR-1a基因mRNA表达水平的变化。
结果诱导分化前，3T3-L1前脂肪细胞呈梭形的成纤维细胞形态，胞浆内无脂滴；诱导分化第4天时，脂肪细胞变大、变圆，胞浆中出现脂滴；分化第6天时，细胞进一步变大、变圆，脂滴明显增多，分布于核周围，形成“戒环”样结构，从形态上由前脂肪细胞向成熟脂肪细胞转变；分化第8天时，胞浆中更多的脂滴积聚，大多数细胞分化成熟。分化第4天时，脂肪细胞的TG总量明显升高，分化第8天时TG总量进一步升高，与前脂肪细胞和对照组相比差异均有极显著的统计学意义(P＜0．01)。同时，GHSR-1a基因mRNA低表达于3T3-L1前脂肪细胞和分化第1天的脂肪细胞，随着脂肪细胞逐渐分化成熟，分化第4天和第8天时GHSR-1a基因mRNA的表达逐渐增加，分别是前脂肪细胞的1．7倍和1．9倍，差异均有显著的统计学意义(P＜0．05)；分化第8天时GHSR-1a基因mRNA的表达分别是分化第1天和第4天的1．3倍和1．1倍，差异均有显著的统计学意义(P＜0．05)；GHSR-1a基因mRNA的表达水平除在诱导分化第0～1天和第1～4天内差异无统计学意义(P＞0．05)外，其余各时间段间表达水平差异均有显著的统计学意义(P＜0．05)。
结论经典的激素鸡尾酒诱导分化法能够诱导3T3-L1前脂肪细胞向成熟脂肪细胞分化，并且能够显著增加前脂肪细胞分化过程中甘油三酯的积累。3T3-L1前脂肪细胞分化过程中GHSR-1a基因表达逐渐上调，其表达变化与脂肪细胞分化、脂质积聚过程相一致，可能参与了脂肪细胞分化过程。
第二部分Ghrelin对前脂肪细胞增殖的作用及相关机制的研究
目的观察不同浓度ghrelin对3T3-L1前脂肪细胞增殖活力的影响，进而探讨其可能的作用机制。
方法体外培养3T3-L1前脂肪细胞，采用MTT法检测不同浓度ghrelin对其增殖活力的影响，采用半定量RT-PCR技术检测ghrelin对前脂肪细胞c-myc和胸苷激酶基因mRNA表达水平的影响。
结果10~(-7)～10~(-15)mol／L ghrelin作用24h均能明显促进3T3-L1前脂肪细胞增殖(P＜0．05)，其中10~(-11)mol／L，时促增殖效应最明显(P＜0．01)，而且同一浓度ghrelin促进前脂肪细胞增殖的效应随作用时间的延长而提高；ghrelin干预后前脂肪细胞c-myc和胸苷激酶基因mRNA表达水平明显升高，与对照组相比差异均有显著的统计学意义(P＜0．05)。
结论Ghrelin能够促进3T3-L1前脂肪细胞增殖。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。
第三部分Ghrelin诱导前脂肪细胞分化过程中形态学观察、甘油三酯总量变化及分化转录因子表达的研究
目的通过观察ghrelin诱导3T3-L1前脂肪细胞分化过程中的形态学变化，测定分化过程中甘油三酯在细胞中的累积情况，检测分化过程中分化转录因子的表达情况，进而探讨ghrelin诱导脂肪细胞分化的作用机制。
方法体外培养3T3-L1前脂肪细胞，采用经典的激素鸡尾酒诱导分化法以及ghrelin诱导其分化。在诱导其向成熟脂肪细胞分化的不同时段(从第1天到第8天)，采用光学显微镜观察脂肪细胞的形态学变化及细胞内脂滴的形成，通过油红O染色测定脂肪细胞分化程度及脂滴的聚积情况，采用化学比色法测定脂肪细胞TG总量，采用半定量RT-PCR技术检测脂肪细胞中过氧化物酶体增殖剂活化受体γ(PPARγ)、CAAT／增强子结合蛋白α(C／EBPα)基因mRNA的表达情况。
结果3T3-L1前脂肪细胞呈典型的梭形，胞浆中无脂滴，形态与成纤维细胞相似。Ghrelin诱导分化第4天时，细胞形态开始变圆，仅少量细胞中出现细小的脂滴；分化第6天时，细胞变圆，体积稍变大，胞浆中脂滴增多，聚集在细胞核周围；分化第8天时，细胞明显变大、变圆，胞浆内出现明显的脂滴，脂滴分布于核周围，形成“戒环”样结构。10~(-11)mol／L ghrelin诱导3T3-L1前脂肪细胞的分化率可达70％，而激素鸡尾酒诱导分化组为90％。诱导分化第4天时，ghrelin组脂肪细胞的TG总量明显升高，与前脂肪细胞和对照组相比差异均有极显著的统计学意义(P＜0．01)；分化第8天时，TG总量进一步升高，与前脂肪细胞、分化第4天时的脂肪细胞和对照组相比差异均有极显著的统计学意义(P＜0．01)。Ghrelin诱导分化第4天时，PPARγ基因mRNA表达水平较对照组升高，是对照组的1．77倍，差异有极显著的统计学意义(P＜0．01)；分化第8天时，PPARγ基因mRNA表达进一步升高，分别是对照组和分化第4天时的1．62倍和1．81倍，差异均有极显著的统计学意义(P＜0．01)。而ghrelin组与同分化时间点Insulin组比较，差异没有显著的统计学意义(P＞0．05)。Ghrelin诱导分化第4天时，C／EBPα基因mRNA表达水平较对照组升高，是对照组的1．44倍，差异有极显著的统计学意义(P＜0．01)；分化第8天时，C／EBPα基因mRNA表达进一步升高，分别是对照组和分化第4天时的1．44倍和2．08倍，差异均有极显著的统计学意义(P＜0．01)。而ghrelin组与同分化时间点Insulin组比较，分化第4天时差异没有显著的统计学意义(P＞0．05)，分化第8天时差异有极显著的统计学意义(P＜0．01)。
结论Ghrelin具有诱导3T3-L1前脂肪细胞分化的生物学作用，PPARγ、C／EBPα随着脂肪细胞分化的进行表达水平逐渐升高，并发挥协同作用，直至终末分化期。这提示在ghrelin诱导前脂肪细胞分化过程中，PPARγ、C／EBPα对细胞完成终末分化具有重要意义。
第四部分Ghrelin对前脂肪细胞凋亡的作用及相关机制的研究
目的通过观察不同浓度ghrelin对3T3-L1前脂肪细胞凋亡率和细胞周期的影响，以及p-ERK1／2和p-Akt蛋白表达水平的变化，进而探讨ghrelin对3T3-L1前脂肪细胞凋亡的作用及分子机制。
方法体外培养3T3-L1前脂肪细胞，采用AnnexinⅤ-FITC／PI双染流式细胞仪分析不同浓度ghrelin对3T3-L1前脂肪细胞凋亡率的影响：采用PI单染流式细胞仪检测细胞周期的构成；采用Western blot方法检测ghrelin干预后脂肪细胞p-ERK1／2和p-Akt蛋白表达水平的变化。
结果10~(-7)～10~(-15)mol／L ghrelin组细胞凋亡率分别为(53．3±6．1)％，(48．2±5．6)％，(42．3±4．6)％，(45．5±4．3)％，(57．8±6．3)％，对照组细胞凋亡率为(58．2±6．3)％，10~(-11)mol／L和10~(-13)mol／L ghrelin干预后细胞凋亡率较对照组显著降低(P＜0．05)，其中10~(-11)mol／L时抑凋亡效应最明显。流式细胞仪细胞周期分析显示10~(-11)mol／Lghrelin干预后，3T3-L1前脂肪细胞处于亚二倍体峰的细胞数明显减少(P＜0．05)，而G_0／G_1，S和G_2／M期细胞数基本不变(P＞0．05)。10~(-7)～10~(-15)mol／Lghrelin均可以引起前脂肪细胞和成熟脂肪细胞ERK1／2快速而强烈的活化(P＜0．01)，然而ghrelin诱导的ERK1／2的活化在脂肪细胞分化早期虽然与对照组相比明显升高(P＜0．05)，但是与分化前和成熟脂肪细胞相比却是明显减弱的(P＜0．05)；同时在前脂肪细胞和成熟脂肪细胞中，ghrelin诱导的ERK1／2的活化可早至ghrelin刺激后2min，在5～10min达到作用高峰，持续至少30min以上，然而在脂肪细胞分化早期，ERK1／2的活化持续仅不足10min。10~(-9)～10~(-15)mol／L ghrelin均可以引起前脂肪细胞和成熟脂肪细胞Akt的活化(P＜0．05),同时在前脂肪细胞和成熟脂肪细胞中，ghrelin刺激30min后即可检测到p-Akt蛋白的高水平表达，并且持续高水平表达可达24h (P＜0．05)。
结论Ghrelin可以抑制3T3-L1前脂肪细胞凋亡，而ERK1／2和P13K／Akt信号通路可能参与了ghrelin调节细胞凋亡的过程。
Simple obesity is the consequence of a chronic positive energy balance. Childhoodobesity is a global epidemic and rising trends in overweight and obesity is apparent in bothdeveloped and developing countries. Simple obesity in particular constitutes a growinghealth problem throughout the world. It is widely accepted that childhood obesity is animportant risk factor for the appearance of obesity in adulthood. And the great increase inobesity portends future increases in incidence of heart diseases; diabetes, stroke, andpossibly cancers. There has been a growing concern about obesity worldwide.
On cellular level, obesity is caused by increasing amount and volume of adipocytesinduced by unceasing differentiation of preadipocytes. Abnormal differentiation,proliferation and apoptosis of preadipocytes will affect glucolipid metabolism, induceobesity and insulin resistance, and then influence the development of type 2 diabetes. It issignificant to study the effects of drugs on proliferation, differentiation and apoptosis ofpreadipocytes for prevention and cure of those metabolic diseases which have a closerelationship with obesity and insulin resistance. Therefore, many scholars have recentlyfocused on the regulation of the adipocyte proliferation, differentiation and apoptosis, andits relationship with obesity.
Ghrelin is a stomach-derived hormone. It was originally identified as an endogenousligand of growth hormone secretagogue receptor (GHSR). In addition to its predictable effect on growthhormone (GH) secretion, ghrelin has an important role in the short-termregulation of appetite and the long-term regulation of energy balance and glucosehomeostasis. It is reported that ghrelin could induce adipogenesis not only through centralmechanisms but also though its direct effects on adipocytes. Therefore in recent years,much-attention has been paid to the research on ghrelin.
In this study, 3T3-L1 preadipocytes were cultured in vitro. The effects of ghrelin onthe proliferation, differentiation and apoptosis of 3T3-L1 preadipocytes and the possiblemechanisms were investigated in order to analyze the relationship between ghrelin and thepathogenesis of obesity from cellular level.
PartⅠGrowth Hormone Seeretagogue Receptor-la Gene Expression
during the Period of 3T3-L1 Preadipoeyte Differentiation
Objective To investigate growth hormone secretagogue receptor-la (GHSR-la) geneexpression during the period of 3T3-L1 adipocyte differentiation and toexplore the relationship between the GHSR-la gene expression andadipocytes differentiation.
Methods 3T3-L1 preadipocytes were cultured in vitro and induced to differentiate intomatured adipocytes by lmg/L Insulin+0.5mM IBMX+1.0μM DEX. Themorphological changes of 3T3-L1 adipocytes were observed by microscope,the differentiation rate was assayed by Oil-Red O staining, and triglyceride(TG) mass was detected by chemical colorimetry methods during the periodof 3T3-L1 adipocyte differentiation. Total RNA was extracted fromadipocytes at various time, and the levels of GHSR-1a gene mRNAexpression were measured by RT-PCR.
Results 3T3-L1 preadipocytes were fibroblastic and had no obvious fat droplet incytoplasm. However, there were some fat drops in 3T3-L1 cells and thesecells became bigger and rounder on the forth day of differentiation. With timegone, more and more fat drops accumulated in these cells. Compared withpreadipocytes and control group, the concentration of TG mass began toincrease on the forth day of differentiation and increase more significantly onthe eighth day (P＜0.01). The GHSR-1a gene mRNA began to express low onthe first day of differentiation. From the fourth day to the eighth day, theGHSR-1a gene mRNA expression was obviously up-regulated (P＜0.05).There were significant differences between any two detected time points inthe levels of GHSR-1a gene mRNA expression (P＜0.05), except those pointsbetween 0 day to 1 day, and 1 day to 4 day (P＞0.05).
Conclusion Insulin+IBMX+DEX can induce 3T3-L1 adipocytes differentiate, andstimulate the TG mass accumulate. GHSR-1a gene mRNA expression isup-regulated during 3T3-L1 adipocyte differentiation. Up-regulation in thelevels of GHSR-la gene mRNA expression during 3T3-L1 adipocytedifferentiation may be involved in the adipogenesis of adipocytes.
PartⅡ
Effects of Ghrelin on the Proliferation of 3T3-L1 Preadipoeytes andIts Possible Mechanisms
Objective To investigate the effects of ghrelin on the proliferation of 3T3-L1preadipocyte and analyze the possible mechanisms.
Methods 3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of3T3-L1 preadipocytes that were treated with different concentrations ofghrelin were evaluated by MTT method and the levels of c-myc andthymidine kinase gene mRNA expression were detected by RT-PCR.
Results Ghrelin at concentrations of 10~(-7)mol/L to 10~(-15)mol/L significantly stimulatedpreadipocyte proliferation (P＜0.05), with the most pronounced effectobserved at 10~(-11)mol/L (P＜0.01). Treatment of 3T3-LI preadipocytes with10~(-9)mol/L and 10~(-11)mol/L ghrelin significantly increased the levels of c-mycand thymidine kinase gene mRNA expression (P＜0.01).
Conclusions Ghrelin can promote the proliferation of 3T3-L1 preadipocytes and enhancethe expression of c-myc, activate thymidine kinase which may play animportant role in the proliferation of 3T3-L1 preadipocytes.
PartⅢ
Effects of Ghrelin on the Differentiation of 3T3-L1 Preadipocytesand Its Possible Mechanisms
Objective To observe the morphological changes, the differentiation rate, TG mass andthe sequential expression of transcription factors in 3T3-L1 adipocytesdifferentiation induced by ghrelin.
Methods 3T3-L1 preadipocytes were cultured in vitro and induced to differentiate intomatured adipocytes by 1mg/L Insulin+0.5mM IBMX+1.0μM DEX and10~(-11)mol/L ghrelin respectively. The morphological changes of 3T3-L1adipocytes were observed by microscope, the differentiation rate was assayedby Oil-Red O staining, TG mass was detected by chemical colorimetry methods, and the levels of peroxisome proliferation activated receptorγ(PPART) and CAAT/enhancer binding protein (C/EBPα) gene mRNAexpression were detected by RT-PCR during the period of 3T3-L1preadipocyte differentiation.
Results 3T3-L1 preadipocytes were fibroblastic and had no obvious fat droplet incytoplasm. However, there were some fat drops in 3T3-L1 cells on the forthday induced by ghrelin. And there were a lot of fat drops in 3T3-L1 cells andthese cells became bigger and rounder on the sixth day induced by ghrelin.With time gone, more and more fat drops accumulated in these cells. The rateof differentiation induced by ghrelin was 70%, compared with that byInsulin+IBMX+DEX was 90%. Compared with preadipocytes and controlgroup, the concentration of TG mass began to increase on the forth day ofdifferentiation and increase more significantly on the eighth day (P＜0.01).Compared with the control group, 10~(-11)mol/L ghrelin significantly increasedthe levels of PPARy and C/EBPαgene mRNA expression during thedifferentiation (P＜0.05). There were significant differences between 4 day to8 day in the levels of PPARγand C/EBPαgene mRNA expression during thedifferentiation induced by ghrelin or Insulin+IBMX+DEX (P＜0.01).
Conclusions Ghrelin can induce 3T3-L1 preadipocytes differentiate. During the period ofadipocyte differentiation induced by ghrelin, PPARγand C/EBPαwereinduced to express and keep high levels until the preadipocytes werecompletely differentiated into adipocytes. It suggests that the sequentialexpression of these transcription factors induced by ghrelin could explain apart of the mechanism for ghrelin to induce the preadipocytes differentiation.
PartⅣ
Effects of Ghrelin on the Apoptosis of 3T3-L1 Preadipocytes and ItsPossible Mechanism
Objective To explore the possible mechanisms of ghrelin inhibiting apoptosis of 3T3-L1preadipocytes, and analyze the relationship between ghrelin and thepathogenesis of obesity from cellular level.
Methods 3T3-L1 preadipocytes were cultured in vitro. The apoptotic rates ofpreadipocytes that were treated with different concentrations of ghrelin weredetected by flow cytometry (FCM) with AnnexinⅤ-FITC and propidiumiodide (PI) double labeling technique. Cell cycle was analyzed by using FCMwith PI labeling technique. The levels of p-ERK1/2 and p-Akt proteinexpression were determined by Western blot.
Results The apoptotic rates of 3T3-L1 preadipocytes treated by 10~(-11)mol/L,10~(-13)mol/L ghrelin were (42.3±4.6) % and (45.5±5.3) % respectively.Compared with the control group, the apoptotic rates in the above groupsdecreased significantly (P＜0.05), with the most pronounced effect observed at10~(-11)mol/L. Cell cycle analysis showed that, with the treatment of 10~(-11)mol/Lghrelin, the cell number in sub-G_0 peak (apoptotic peak) decreasedsignificantly (P＜0.05), while the cell number in G_0/G_1, S and G_2/M had nosignificant changes (P＞0.05). The levels of p-ERK1/2 and p-Akt proteinexpression were significantly enhanced by treatment with ghrelin in 3T3-L1preadipocytes and terminally differentiated adipocytes (P＜0.01).
Conclusions Ghrelin can efficiently inhibit the apoptosis of 3T3-L1 preadipocytes withenhancing the expression of p-ERK1/2 and p-Akt.
肥胖是体脂的过多积聚，是脂肪细胞体积的增大和数目的增加。过量的能量合成甘油三酯储存于脂肪细胞中，甘油三酯的不断积累，使脂肪细胞体积增大。当前脂肪细胞增殖和分化异常增多或成熟脂肪细胞凋亡减少时，就会引起脂肪组织的过多堆积和脂肪细胞内分泌功能的紊乱，继而导致肥胖和胰岛素抵抗，最终引发糖尿病。因此，脂肪细胞生长、发育、分化及凋亡的分子调控机制已成为目前国际上研究肥胖及其相关疾病的热点方向。
Ghrelin是在1999年由Kojima等发现的生长激素促分泌素受体的第一个内源性配体，是一种胃源性促生长激素释放多肽，也是第一个被确认的末梢具有活性的促食欲因子。Ghrelin不仅能有效促进生长激素的分泌，还参与摄食、能量代谢、心血管和免疫功能的调节，是目前调节肽研究领域中的一个热点。Ghrelin可能是生长激素／胰岛素样生长因子-1轴和调节能量平衡的神经内分泌之间一个新的联结纽带。Ghrelin通过启动进食，增加食欲，增加营养物质、尤其是脂类的摄取，促进脂肪形成等多方面作用调节着机体能量代谢的平衡，在控制摄食与能量平衡及对饥饿的神经内分泌调节方面发挥重要的作用。
基于前脂肪细胞增殖、分化和凋亡在肥胖发生机制中的重要作用，本研究以3T3-L1前脂肪细胞为实验对象，观察ghrelin对其增殖、分化和凋亡的影响，并探讨其可能的分子生物学机制，进而深化对ghrelin这一目前已知的唯一能够促进食欲的外周激素的认识，深化对脂肪细胞增殖、分化和凋亡分子机制的认识，并进一步为肥胖及其相关疾病的预防和治疗提供新的研究方向。
第一部分生长激素促分泌素受体-1a基因在脂肪细胞分化成熟中的作用研究
目的通过观察生长激素促分泌素受体-1a(growth hormone secretagogue receptor,GHSR-1a)基因在3T3-L1前脂肪细胞诱导分化过程中不同时段表达水平的变化，从而进一步探讨GHSR-1a基因在脂肪细胞分化过程中的作用。
方法体外培养3T3-L1前脂肪细胞，采用经典的激素鸡尾酒诱导分化法诱导3T3-L1前脂肪细胞分化。在诱导其向成熟脂肪细胞分化的不同时段(从第1天到第8天)，采用光学显微镜观察脂肪细胞的形态学变化及细胞内脂滴的形成，通过油红O染色测定脂肪细胞分化程度及细胞内脂滴的聚积情况，采用化学比色法测定脂肪细胞中甘油三酯(triglyceride, TG)的总量，采用半定量逆转录-聚合酶链反应(RT-PCR)技术检测脂肪细胞中GHSR-1a基因mRNA表达水平的变化。
结果诱导分化前，3T3-L1前脂肪细胞呈梭形的成纤维细胞形态，胞浆内无脂滴；诱导分化第4天时，脂肪细胞变大、变圆，胞浆中出现脂滴；分化第6天时，细胞进一步变大、变圆，脂滴明显增多，分布于核周围，形成“戒环”样结构，从形态上由前脂肪细胞向成熟脂肪细胞转变；分化第8天时，胞浆中更多的脂滴积聚，大多数细胞分化成熟。分化第4天时，脂肪细胞的TG总量明显升高，分化第8天时TG总量进一步升高，与前脂肪细胞和对照组相比差异均有极显著的统计学意义(P＜0．01)。同时，GHSR-1a基因mRNA低表达于3T3-L1前脂肪细胞和分化第1天的脂肪细胞，随着脂肪细胞逐渐分化成熟，分化第4天和第8天时GHSR-1a基因mRNA的表达逐渐增加，分别是前脂肪细胞的1．7倍和1．9倍，差异均有显著的统计学意义(P＜0．05)；分化第8天时GHSR-1a基因mRNA的表达分别是分化第1天和第4天的1．3倍和1．1倍，差异均有显著的统计学意义(P＜0．05)；GHSR-1a基因mRNA的表达水平除在诱导分化第0～1天和第1～4天内差异无统计学意义(P＞0．05)外，其余各时间段间表达水平差异均有显著的统计学意义(P＜0．05)。
结论经典的激素鸡尾酒诱导分化法能够诱导3T3-L1前脂肪细胞向成熟脂肪细胞分化，并且能够显著增加前脂肪细胞分化过程中甘油三酯的积累。3T3-L1前脂肪细胞分化过程中GHSR-1a基因表达逐渐上调，其表达变化与脂肪细胞分化、脂质积聚过程相一致，可能参与了脂肪细胞分化过程。
第二部分Ghrelin对前脂肪细胞增殖的作用及相关机制的研究
目的观察不同浓度ghrelin对3T3-L1前脂肪细胞增殖活力的影响，进而探讨其可能的作用机制。
方法体外培养3T3-L1前脂肪细胞，采用MTT法检测不同浓度ghrelin对其增殖活力的影响，采用半定量RT-PCR技术检测ghrelin对前脂肪细胞c-myc和胸苷激酶基因mRNA表达水平的影响。
结果10~(-7)～10~(-15)mol／L ghrelin作用24h均能明显促进3T3-L1前脂肪细胞增殖(P＜0．05)，其中10~(-11)mol／L，时促增殖效应最明显(P＜0．01)，而且同一浓度ghrelin促进前脂肪细胞增殖的效应随作用时间的延长而提高；ghrelin干预后前脂肪细胞c-myc和胸苷激酶基因mRNA表达水平明显升高，与对照组相比差异均有显著的统计学意义(P＜0．05)。
结论Ghrelin能够促进3T3-L1前脂肪细胞增殖。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。
第三部分Ghrelin诱导前脂肪细胞分化过程中形态学观察、甘油三酯总量变化及分化转录因子表达的研究
目的通过观察ghrelin诱导3T3-L1前脂肪细胞分化过程中的形态学变化，测定分化过程中甘油三酯在细胞中的累积情况，检测分化过程中分化转录因子的表达情况，进而探讨ghrelin诱导脂肪细胞分化的作用机制。
方法体外培养3T3-L1前脂肪细胞，采用经典的激素鸡尾酒诱导分化法以及ghrelin诱导其分化。在诱导其向成熟脂肪细胞分化的不同时段(从第1天到第8天)，采用光学显微镜观察脂肪细胞的形态学变化及细胞内脂滴的形成，通过油红O染色测定脂肪细胞分化程度及脂滴的聚积情况，采用化学比色法测定脂肪细胞TG总量，采用半定量RT-PCR技术检测脂肪细胞中过氧化物酶体增殖剂活化受体γ(PPARγ)、CAAT／增强子结合蛋白α(C／EBPα)基因mRNA的表达情况。
结果3T3-L1前脂肪细胞呈典型的梭形，胞浆中无脂滴，形态与成纤维细胞相似。Ghrelin诱导分化第4天时，细胞形态开始变圆，仅少量细胞中出现细小的脂滴；分化第6天时，细胞变圆，体积稍变大，胞浆中脂滴增多，聚集在细胞核周围；分化第8天时，细胞明显变大、变圆，胞浆内出现明显的脂滴，脂滴分布于核周围，形成“戒环”样结构。10~(-11)mol／L ghrelin诱导3T3-L1前脂肪细胞的分化率可达70％，而激素鸡尾酒诱导分化组为90％。诱导分化第4天时，ghrelin组脂肪细胞的TG总量明显升高，与前脂肪细胞和对照组相比差异均有极显著的统计学意义(P＜0．01)；分化第8天时，TG总量进一步升高，与前脂肪细胞、分化第4天时的脂肪细胞和对照组相比差异均有极显著的统计学意义(P＜0．01)。Ghrelin诱导分化第4天时，PPARγ基因mRNA表达水平较对照组升高，是对照组的1．77倍，差异有极显著的统计学意义(P＜0．01)；分化第8天时，PPARγ基因mRNA表达进一步升高，分别是对照组和分化第4天时的1．62倍和1．81倍，差异均有极显著的统计学意义(P＜0．01)。而ghrelin组与同分化时间点Insulin组比较，差异没有显著的统计学意义(P＞0．05)。Ghrelin诱导分化第4天时，C／EBPα基因mRNA表达水平较对照组升高，是对照组的1．44倍，差异有极显著的统计学意义(P＜0．01)；分化第8天时，C／EBPα基因mRNA表达进一步升高，分别是对照组和分化第4天时的1．44倍和2．08倍，差异均有极显著的统计学意义(P＜0．01)。而ghrelin组与同分化时间点Insulin组比较，分化第4天时差异没有显著的统计学意义(P＞0．05)，分化第8天时差异有极显著的统计学意义(P＜0．01)。
结论Ghrelin具有诱导3T3-L1前脂肪细胞分化的生物学作用，PPARγ、C／EBPα随着脂肪细胞分化的进行表达水平逐渐升高，并发挥协同作用，直至终末分化期。这提示在ghrelin诱导前脂肪细胞分化过程中，PPARγ、C／EBPα对细胞完成终末分化具有重要意义。
第四部分Ghrelin对前脂肪细胞凋亡的作用及相关机制的研究
目的通过观察不同浓度ghrelin对3T3-L1前脂肪细胞凋亡率和细胞周期的影响，以及p-ERK1／2和p-Akt蛋白表达水平的变化，进而探讨ghrelin对3T3-L1前脂肪细胞凋亡的作用及分子机制。
方法体外培养3T3-L1前脂肪细胞，采用AnnexinⅤ-FITC／PI双染流式细胞仪分析不同浓度ghrelin对3T3-L1前脂肪细胞凋亡率的影响：采用PI单染流式细胞仪检测细胞周期的构成；采用Western blot方法检测ghrelin干预后脂肪细胞p-ERK1／2和p-Akt蛋白表达水平的变化。
结果10~(-7)～10~(-15)mol／L ghrelin组细胞凋亡率分别为(53．3±6．1)％，(48．2±5．6)％，(42．3±4．6)％，(45．5±4．3)％，(57．8±6．3)％，对照组细胞凋亡率为(58．2±6．3)％，10~(-11)mol／L和10~(-13)mol／L ghrelin干预后细胞凋亡率较对照组显著降低(P＜0．05)，其中10~(-11)mol／L时抑凋亡效应最明显。流式细胞仪细胞周期分析显示10~(-11)mol／Lghrelin干预后，3T3-L1前脂肪细胞处于亚二倍体峰的细胞数明显减少(P＜0．05)，而G_0／G_1，S和G_2／M期细胞数基本不变(P＞0．05)。10~(-7)～10~(-15)mol／Lghrelin均可以引起前脂肪细胞和成熟脂肪细胞ERK1／2快速而强烈的活化(P＜0．01)，然而ghrelin诱导的ERK1／2的活化在脂肪细胞分化早期虽然与对照组相比明显升高(P＜0．05)，但是与分化前和成熟脂肪细胞相比却是明显减弱的(P＜0．05)；同时在前脂肪细胞和成熟脂肪细胞中，ghrelin诱导的ERK1／2的活化可早至ghrelin刺激后2min，在5～10min达到作用高峰，持续至少30min以上，然而在脂肪细胞分化早期，ERK1／2的活化持续仅不足10min。10~(-9)～10~(-15)mol／L ghrelin均可以引起前脂肪细胞和成熟脂肪细胞Akt的活化(P＜0．05),同时在前脂肪细胞和成熟脂肪细胞中，ghrelin刺激30min后即可检测到p-Akt蛋白的高水平表达，并且持续高水平表达可达24h (P＜0．05)。
结论Ghrelin可以抑制3T3-L1前脂肪细胞凋亡，而ERK1／2和P13K／Akt信号通路可能参与了ghrelin调节细胞凋亡的过程。
Simple obesity is the consequence of a chronic positive energy balance. Childhoodobesity is a global epidemic and rising trends in overweight and obesity is apparent in bothdeveloped and developing countries. Simple obesity in particular constitutes a growinghealth problem throughout the world. It is widely accepted that childhood obesity is animportant risk factor for the appearance of obesity in adulthood. And the great increase inobesity portends future increases in incidence of heart diseases; diabetes, stroke, andpossibly cancers. There has been a growing concern about obesity worldwide.
On cellular level, obesity is caused by increasing amount and volume of adipocytesinduced by unceasing differentiation of preadipocytes. Abnormal differentiation,proliferation and apoptosis of preadipocytes will affect glucolipid metabolism, induceobesity and insulin resistance, and then influence the development of type 2 diabetes. It issignificant to study the effects of drugs on proliferation, differentiation and apoptosis ofpreadipocytes for prevention and cure of those metabolic diseases which have a closerelationship with obesity and insulin resistance. Therefore, many scholars have recentlyfocused on the regulation of the adipocyte proliferation, differentiation and apoptosis, andits relationship with obesity.
Ghrelin is a stomach-derived hormone. It was originally identified as an endogenousligand of growth hormone secretagogue receptor (GHSR). In addition to its predictable effect on growthhormone (GH) secretion, ghrelin has an important role in the short-termregulation of appetite and the long-term regulation of energy balance and glucosehomeostasis. It is reported that ghrelin could induce adipogenesis not only through centralmechanisms but also though its direct effects on adipocytes. Therefore in recent years,much-attention has been paid to the research on ghrelin.
In this study, 3T3-L1 preadipocytes were cultured in vitro. The effects of ghrelin onthe proliferation, differentiation and apoptosis of 3T3-L1 preadipocytes and the possiblemechanisms were investigated in order to analyze the relationship between ghrelin and thepathogenesis of obesity from cellular level.
PartⅠGrowth Hormone Seeretagogue Receptor-la Gene Expression
during the Period of 3T3-L1 Preadipoeyte Differentiation
Objective To investigate growth hormone secretagogue receptor-la (GHSR-la) geneexpression during the period of 3T3-L1 adipocyte differentiation and toexplore the relationship between the GHSR-la gene expression andadipocytes differentiation.
Methods 3T3-L1 preadipocytes were cultured in vitro and induced to differentiate intomatured adipocytes by lmg/L Insulin+0.5mM IBMX+1.0μM DEX. Themorphological changes of 3T3-L1 adipocytes were observed by microscope,the differentiation rate was assayed by Oil-Red O staining, and triglyceride(TG) mass was detected by chemical colorimetry methods during the periodof 3T3-L1 adipocyte differentiation. Total RNA was extracted fromadipocytes at various time, and the levels of GHSR-1a gene mRNAexpression were measured by RT-PCR.
Results 3T3-L1 preadipocytes were fibroblastic and had no obvious fat droplet incytoplasm. However, there were some fat drops in 3T3-L1 cells and thesecells became bigger and rounder on the forth day of differentiation. With timegone, more and more fat drops accumulated in these cells. Compared withpreadipocytes and control group, the concentration of TG mass began toincrease on the forth day of differentiation and increase more significantly onthe eighth day (P＜0.01). The GHSR-1a gene mRNA began to express low onthe first day of differentiation. From the fourth day to the eighth day, theGHSR-1a gene mRNA expression was obviously up-regulated (P＜0.05).There were significant differences between any two detected time points inthe levels of GHSR-1a gene mRNA expression (P＜0.05), except those pointsbetween 0 day to 1 day, and 1 day to 4 day (P＞0.05).
Conclusion Insulin+IBMX+DEX can induce 3T3-L1 adipocytes differentiate, andstimulate the TG mass accumulate. GHSR-1a gene mRNA expression isup-regulated during 3T3-L1 adipocyte differentiation. Up-regulation in thelevels of GHSR-la gene mRNA expression during 3T3-L1 adipocytedifferentiation may be involved in the adipogenesis of adipocytes.
PartⅡ
Effects of Ghrelin on the Proliferation of 3T3-L1 Preadipoeytes andIts Possible Mechanisms
Objective To investigate the effects of ghrelin on the proliferation of 3T3-L1preadipocyte and analyze the possible mechanisms.
Methods 3T3-L1 preadipocytes were cultured in vitro. The proliferation potentials of3T3-L1 preadipocytes that were treated with different concentrations ofghrelin were evaluated by MTT method and the levels of c-myc andthymidine kinase gene mRNA expression were detected by RT-PCR.
Results Ghrelin at concentrations of 10~(-7)mol/L to 10~(-15)mol/L significantly stimulatedpreadipocyte proliferation (P＜0.05), with the most pronounced effectobserved at 10~(-11)mol/L (P＜0.01). Treatment of 3T3-LI preadipocytes with10~(-9)mol/L and 10~(-11)mol/L ghrelin significantly increased the levels of c-mycand thymidine kinase gene mRNA expression (P＜0.01).
Conclusions Ghrelin can promote the proliferation of 3T3-L1 preadipocytes and enhancethe expression of c-myc, activate thymidine kinase which may play animportant role in the proliferation of 3T3-L1 preadipocytes.
PartⅢ
Effects of Ghrelin on the Differentiation of 3T3-L1 Preadipocytesand Its Possible Mechanisms
Objective To observe the morphological changes, the differentiation rate, TG mass andthe sequential expression of transcription factors in 3T3-L1 adipocytesdifferentiation induced by ghrelin.
Methods 3T3-L1 preadipocytes were cultured in vitro and induced to differentiate intomatured adipocytes by 1mg/L Insulin+0.5mM IBMX+1.0μM DEX and10~(-11)mol/L ghrelin respectively. The morphological changes of 3T3-L1adipocytes were observed by microscope, the differentiation rate was assayedby Oil-Red O staining, TG mass was detected by chemical colorimetry methods, and the levels of peroxisome proliferation activated receptorγ(PPART) and CAAT/enhancer binding protein (C/EBPα) gene mRNAexpression were detected by RT-PCR during the period of 3T3-L1preadipocyte differentiation.
Results 3T3-L1 preadipocytes were fibroblastic and had no obvious fat droplet incytoplasm. However, there were some fat drops in 3T3-L1 cells on the forthday induced by ghrelin. And there were a lot of fat drops in 3T3-L1 cells andthese cells became bigger and rounder on the sixth day induced by ghrelin.With time gone, more and more fat drops accumulated in these cells. The rateof differentiation induced by ghrelin was 70%, compared with that byInsulin+IBMX+DEX was 90%. Compared with preadipocytes and controlgroup, the concentration of TG mass began to increase on the forth day ofdifferentiation and increase more significantly on the eighth day (P＜0.01).Compared with the control group, 10~(-11)mol/L ghrelin significantly increasedthe levels of PPARy and C/EBPαgene mRNA expression during thedifferentiation (P＜0.05). There were significant differences between 4 day to8 day in the levels of PPARγand C/EBPαgene mRNA expression during thedifferentiation induced by ghrelin or Insulin+IBMX+DEX (P＜0.01).
Conclusions Ghrelin can induce 3T3-L1 preadipocytes differentiate. During the period ofadipocyte differentiation induced by ghrelin, PPARγand C/EBPαwereinduced to express and keep high levels until the preadipocytes werecompletely differentiated into adipocytes. It suggests that the sequentialexpression of these transcription factors induced by ghrelin could explain apart of the mechanism for ghrelin to induce the preadipocytes differentiation.
PartⅣ
Effects of Ghrelin on the Apoptosis of 3T3-L1 Preadipocytes and ItsPossible Mechanism
Objective To explore the possible mechanisms of ghrelin inhibiting apoptosis of 3T3-L1preadipocytes, and analyze the relationship between ghrelin and thepathogenesis of obesity from cellular level.
Methods 3T3-L1 preadipocytes were cultured in vitro. The apoptotic rates ofpreadipocytes that were treated with different concentrations of ghrelin weredetected by flow cytometry (FCM) with AnnexinⅤ-FITC and propidiumiodide (PI) double labeling technique. Cell cycle was analyzed by using FCMwith PI labeling technique. The levels of p-ERK1/2 and p-Akt proteinexpression were determined by Western blot.
Results The apoptotic rates of 3T3-L1 preadipocytes treated by 10~(-11)mol/L,10~(-13)mol/L ghrelin were (42.3±4.6) % and (45.5±5.3) % respectively.Compared with the control group, the apoptotic rates in the above groupsdecreased significantly (P＜0.05), with the most pronounced effect observed at10~(-11)mol/L. Cell cycle analysis showed that, with the treatment of 10~(-11)mol/Lghrelin, the cell number in sub-G_0 peak (apoptotic peak) decreasedsignificantly (P＜0.05), while the cell number in G_0/G_1, S and G_2/M had nosignificant changes (P＞0.05). The levels of p-ERK1/2 and p-Akt proteinexpression were significantly enhanced by treatment with ghrelin in 3T3-L1preadipocytes and terminally differentiated adipocytes (P＜0.01).
Conclusions Ghrelin can efficiently inhibit the apoptosis of 3T3-L1 preadipocytes withenhancing the expression of p-ERK1/2 and p-Akt.
引文
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