CIDEC在人脂肪细胞分化中的作用及其机制研究

发布时间：2018-07-01 12:58

本文选题：CIDEC + 肥胖　；参考：《第四军医大学》2010年博士论文

【摘要】：随着社会经济的发展,人们的生活方式的转变,肥胖(obesity)已成为当前世界性的健康问题,是诱发糖尿病、心脑血管疾病等多种代谢相关疾病的重要危险因素,其与吸烟、艾滋病并称为人类健康的三大杀手。研究发现,脂肪细胞的分化与肥胖的发生有着密切的关系。因此,研究脂肪细胞的分化及其机制,对于预防肥胖的发生,治疗肥胖及相关疾病具有重要意义。 CIDE(cell death-inducing DFF45-like effectors)家族是20世纪末发现的一组能够诱导细胞凋亡的新基因。CIDE家族主要包括CIDEA、CIDEB和CIDEC(在小鼠称为FSP27)。最近研究发现CIDE家族成员与肥胖的发生密切相关。CIDEA、CIDEB和FSP27敲除小鼠,均能抵制高脂饮食诱导的肥胖。其中FSP27是小鼠脂肪特异性蛋白,仅在成熟的脂肪细胞中表达,与脂肪细胞分化密切相关,并且具有抑制脂肪分解促进脂肪储积的作用。CIDEC,作为FSP27的人类同源物,其在人体内功能及机制尚不明确。因此本课题将着重研究CIDEC在人类脂肪细胞分化中的作用,期望为今后治疗肥胖提供新的靶点。【目的】 1、研究CIDEC的表达与脂肪细胞分化的关系;2、研究CIDEC的亚细胞定位;3、研究下调CIDEC表达对脂肪细胞分化的影响及机制。【方法】 1、利用免疫组织化学、Real-time PCR和Western blot方法检测CIDEC在不同发育阶段的人胚胎脂肪组织以及不同分化程度脂肪肿瘤组织中的表达变化;2、构建CIDEC与荧光蛋白融合表达的质粒,利用脂质体转染COS-7细胞,以确定CIDEC蛋白的亚细胞定位。3、体外原代培养人前脂肪细胞,并进行诱导分化,检测不同分化阶段CIDEC的表达水平。4、利用慢病毒siRNA沉默CIDEC的表达,以研究下调CIDEC表达对脂肪细胞分化的影响,并分析其机制。【结果】 1、CIDEC的表达水平与脂肪细胞的分化相关我们采用免疫组织化学、Real-time PCR和Western blot方法比较了3例不同发育阶段的胎儿脂肪组织中CIDEC的表达情况,同时检测调控脂肪细胞分化关键分子PPARγ的变化。结果显示,无论mRNA还是蛋白水平,CIDEC均呈现随着分化成熟逐渐增高的趋势,并且与PPARγ的表达趋势完全一致。对30例人体正常脂肪组织和45例不同分化程度的脂肪肿瘤组织的免疫组织化学染色结果显示,CIDEC随着脂肪细胞分化程度降低而表达降低。采用Real-time PCR和Western blot方法的研究结果显示,同正常脂肪组织相比,脂肪瘤中CIDEC的表达变化不显著,而分化好的脂肪肉瘤、黏液型脂肪肉瘤及去分化脂肪肉瘤中CIDEC的表达均显著降低(n=5, *p0.05)。此外,通过体外实验原代培养人前脂肪细胞,并诱导成为成熟脂肪细胞,随着脂肪细胞的诱导分化成熟,CIDEC和PPARγ的表达水平同诱导前相比急剧升高(*p0.05),同脂肪分化标志分子FABP4变化趋势一致。以上体内、外实验结果提示,CIDEC在脂肪细胞分化过程中发挥重要作用。 2、CIDEC蛋白主要定位于脂滴表面,与线粒体没有共定位我们采用脂质体转染法将重组质粒pShuttle-CMV-EGFP-CIDEC和pShuttle-CMV-DsRed1-CIDEC转染COS-7细胞。以GFP-adipophilin作为脂滴的标志物;Bodipy 493/503作为中性脂肪染料;MitoTracker Red CMXRos作为线粒体染料;Hoechst 33258作为细胞核染料。结果显示:CIDEC分布于脂滴周围,与adipophilin共定位,说明CIDEC是一个定位于脂滴表面的蛋白;同时我们发现CIDEC与线粒体不存在共定位。 3、下调CIDEC的表达水平,脂肪细胞的分化受到抑制我们首先构建了携带针对CIDEC的高效siRNA的慢病毒,将原代培养的人前脂肪细胞在诱导分化以前感染此慢病毒,观察下调CIDEC的表达对脂肪细胞分化的影响。结果发现,下调CIDEC表达水平后,脂肪细胞内脂滴体积明显减小,脂肪含量也显著降低,失去了成熟脂肪细胞的表型。进一步研究发现,CIDEC低表达后,细胞内甘油三酯含量降低,而培养液中甘油的含量却显著增高。分子水平研究发现,成熟脂滴标志分子perilipin以及脂肪细胞分化标志分子FABP4表达水平显著降低。以上实验说明,下调CIDEC的表达使甘油三酯合成降低,分解增强,此外脂滴的成熟也受到影响,从而抑制了脂肪细胞的分化。【结论】本研究发现CIDEC是一个脂滴表面蛋白,CIDEC的表达与脂肪细胞分化相关,CIDEC可能通过抑制脂肪的分解,促进脂滴的成熟,从而参与了脂肪细胞的分化过程。如果抑制了CIDEC的表达,可阻止脂肪细胞的分化,防止肥胖的发生,因此CIDEC有望成为治疗肥胖的新靶点。
[Abstract]:With the development of social economy, the transformation of people's life style, obesity (obesity) has become the current world health problem, is an important risk factor to induce diabetes, cardiovascular and cerebrovascular diseases and other metabolic related diseases, which are the three major killers of smoking, AIDS and human health. Research found that the differentiation and fertilizer of fat cells There is a close relationship between the occurrence of fat and the study of the differentiation and mechanism of adipocyte, which is of great significance in preventing the occurrence of obesity and in the treatment of obesity and related diseases.
The CIDE (cell death-inducing DFF45-like effectors) family was found in the late twentieth Century a group of new genes that can induce apoptosis, including CIDEA, CIDEB, and CIDEC (in mice called FSP27). Recent studies have found that CIDE family members are closely related to the occurrence of obesity. Diet induced obesity, in which FSP27 is a fat specific protein in mice, is expressed only in mature adipocytes, closely related to the differentiation of fat cells, and has the effect of inhibiting fat decomposition and promoting the accumulation of fat,.CIDEC. As a human homologue of FSP27, its function and mechanism in human body is not clear. Therefore, this topic will focus on the research. The role of CIDEC in human adipocyte differentiation is expected to provide a new target for future treatment of obesity.
[Objective]
1, we studied the relationship between the expression of CIDEC and adipocyte differentiation; 2, we studied the subcellular localization of CIDEC; 3, we studied the effect of down-regulation of CIDEC expression on adipocyte differentiation and its mechanism.
[method]
1, immunohistochemistry, Real-time PCR and Western blot were used to detect the changes in the expression of CIDEC in human fetal adipose tissue and different degree of differentiation of fat tumor tissues at different developmental stages. 2, the plasmids fused with CIDEC and fluorescent protein were constructed, and COS-7 cells were transfected by liposomes to determine the subcellular localization of CIDEC protein. .3, the primary human preadipocytes were cultured in vitro, and the differentiation was induced, the expression level of CIDEC in different stages of differentiation.4 was detected. The expression of CIDEC was silenced by lentivirus siRNA, in order to study the effect of down regulation of CIDEC expression on the differentiation of adipocytes and to analyze the mechanism.
[results]
1, the expression level of CIDEC is related to adipocyte differentiation.
We used immunohistochemistry, Real-time PCR and Western blot to compare the expression of CIDEC in 3 fetal adipose tissues at different developmental stages, and also detected the changes in the key molecule PPAR gamma regulating the differentiation of adipocyte differentiation. The results showed that CIDEC showed a gradual increase in the level of differentiation and maturity regardless of mRNA or protein levels. The trend of the expression was consistent with the expression trend of PPAR gamma. Immunohistochemical staining of 30 normal fat tissues and 45 adipose tumor tissues with different degrees of differentiation showed that the expression of CIDEC decreased with the decrease in the degree of adipocyte differentiation. The results of the Real-time PCR and Western blot methods showed that the same normal fat was the same as normal fat. The expression of CIDEC in lipoma was not significant in lipoma, but the expression of CIDEC in well differentiated liposarcoma, mucous liposarcoma and dedifferentiated liposarcoma was significantly decreased (n=5, *p0.05). In addition, human preadipocytes were cultured in vitro and induced to become mature adipocytes, with the induction of adipocytes. The expression level of CIDEC and PPAR gamma increased sharply compared with that before induction (*p0.05), which was the same as that of the fat differentiation marker FABP4. In vivo, the experimental results suggest that CIDEC plays an important role in the process of adipocyte differentiation.
2, CIDEC protein is mainly located on the surface of lipid droplets and is not Co located with mitochondria.
We used the liposome transfection to transfect the recombinant plasmid pShuttle-CMV-EGFP-CIDEC and pShuttle-CMV-DsRed1-CIDEC into COS-7 cells. GFP-adipophilin was used as a marker for lipid droplets; Bodipy 493/503 was used as a neutral fatty dye; MitoTracker Red CMXRos was used as a mitochondrial dye; Hoechst 33258 was used as a nuclear dye. The results showed: CIDEC distribution The location of adipophilin around the lipid droplet indicates that CIDEC is a protein located on the surface of lipid droplets. At the same time, we found that there is no co localization between CIDEC and mitochondria.
3, down regulate the expression level of CIDEC and inhibit the differentiation of adipocytes.
We first constructed a slow virus carrying high efficiency siRNA for CIDEC. The primary cultured preadipocytes infected the lentivirus before inducing differentiation and observed the effect of down regulation of the expression of CIDEC on the differentiation of adipocytes. The results showed that the fat droplet volume decreased obviously and the fat content was significant after the downregulation of CIDEC expression level. Further studies found that the content of triglycerides in the cells decreased and the content of glycerol in the culture medium increased significantly after the low expression of CIDEC. Molecular level studies found that the expression level of the mature lipid droplet marker molecule perilipin and the biomarker of adipocyte differentiation was significantly reduced. The above results showed that the level of FABP4 expression was significantly reduced. The results showed that the expression of CIDEC decreased, the triglyceride synthesis decreased, the decomposition increased, and the maturation of lipid droplets was also affected, thus inhibiting the differentiation of adipocytes.
[Conclusion]
This study found that CIDEC is a lipid droplet surface protein, and the expression of CIDEC is related to the differentiation of fat cells. CIDEC may inhibit the decomposition of fat and promote the maturation of lipid droplets, thus participating in the differentiation process of fat cells. If the expression of CIDEC is suppressed, the differentiation of adipocytes can be prevented and obesity is prevented, so CIDEC is expected to become a promising one. It is a new target for the treatment of obesity.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R363

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