人CIDE-3基因在脂肪细胞分化及代谢中作用的研究
发布时间：2018-07-26 15:11
【摘要】： 脂肪组织是机体能量储存及利用的重要场所。脂肪细胞分化和代谢异常可导致肥胖发生,目前肥胖已日趋成为世界性的健康问题,同时肥胖也是糖尿病和心脑血管疾病等多种慢性疾病的危险因素。因此研究其发病机理对于预防和治疗肥胖及其相关疾病具有非常重要的意义。 CIDE(cell death-inducing DFF45-like effector)家族是20世纪末发现的一组可诱导细胞凋亡的基因。近来发现,其家族成员与肥胖的发生密切相关。其中Fsp27(fat specific protein),小鼠脂肪特异蛋白,仅在成熟的脂肪细胞中表达。Fsp27定位于脂滴可以抑制脂肪分解促进脂肪积聚。CIDE-3是Fsp27在人的同源物,其主要分布在小肠、心脏、结肠和胃,在脑、肾脏和肝中低表达,并不是脂肪特异基因。那么CIDE-3在人体内生物学功能如何,是否也像FSP27一样在脂肪细胞分化代谢过程中发挥重要作用,这些问题并不十分清楚。 在本研究中,我们构建了EGFP-CIDE-3及DsRed1-CIDE-3融合基因真核表达载体,通过转染COS-7细胞确定CIDE-3蛋白的亚细胞定位。并构建携带EGFP-CIDE -3及DsRed1-CIDE-3融合基因的腺病毒,同时进行了人前脂肪细胞的体外分离和原代培养,并通过腺病毒感染过表达CIDE-3以探讨脂肪细胞的增殖分化机制和CIDE-3在脂肪细胞分化代谢过程中的作用。 【目的】 1、确定CIDE-3蛋白的亚细胞定位;2、构建携带EGFP-CIDE -3及DsRed1-CIDE-3融合基因的腺病毒;3、研究CIDE-3在脂肪细胞分化代谢过程中的作用。 【方法】 1、构建EGFP-CIDE-3及DsRed1-CIDE-3融合基因真核表达载体,通过脂质体转染COS-7细胞以确定CIDE-3蛋白的亚细胞定位;2、利用Stratagene公司的AdEasyTM XL腺病毒载体系统构建携带EGFP-CIDE -3及DsRed1-CIDE-3融合基因的腺病毒;3、从抽脂获得的脂肪乳液进行人前脂肪细胞的体外分离和原代培养;4、利用MTT方法绘制人前脂肪细胞的生长曲线;5、利用流式细胞方法测定前脂肪细胞的表面标志分子以鉴定细胞的来源;6、通过腺病毒感染的方法过表达CIDE-3以探讨CIDE-3在脂肪细胞分化代谢过程中的作用。 【结果】 1、CIDE-3蛋白主要定位于脂滴表面,部分位于内质网上 首先以真核表达质粒pShuttle-CMV作为载体,构建了重组质粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed1-CIDE-3,然后对重组质粒进行了酶切电泳及DNA测序等分析,证实我们在实验中成功地构建了重组质粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed1-CIDE-3。为了研究CIDE-3蛋白的亚细胞定位,我们以脂质体法将重组质粒转染COS-7细胞。采用GFP-CB5(Cytochrome B5,已知内质网定位)作为ER标志物;GFP-ADRP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。实验结果显示:CIDE-3与内质网标志物CB5存在部分共定位,CIDE-3分布于GFP-CB5所形成的网状结构上;CIDE-3分布于脂滴周围,与ADRP在脂滴周围完全重叠,说明CIDE-3是一个脂滴定位的蛋白;同时我们发现CIDE-3与线粒体不存在共定位。 2、携带CIDE-3融合基因的腺病毒构建成功 我们利用Stratagene公司的AdEasyTM XL腺病毒载体系统,经过克隆目的基因、构建重组穿梭质粒、在大肠杆菌BJ5183中进行同源重组获得重组腺病毒质粒及转染包装细胞AD293等步骤制备了携带CIDE-3基因的重组腺病毒。通过PCR扩增CIDE-3,倒置荧光显微镜下观察荧光融合蛋白表达等方法证实重组腺病毒Ad-EGFP-CIDE-3和Ad-DsRed1-CIDE-3构建成功。 3、CIDE-3可以抑制脂肪酸的氧化分解,促进脂滴成熟 我们采用抽脂术后的脂肪细胞乳液培养了原代人前脂肪细胞。并经生长曲线测定初步证明其确为前脂肪细胞,具有文献中提出的3个典型特征:1)来自于脂肪组织,形态为梭形,胞浆内无或很少有脂肪颗粒;2)增殖迅速,与成纤维细胞在倍增时间上接近;3)在形成单层汇合后能变为脂肪细胞,胞质内会出现脂肪颗粒。同时,流式细胞仪检测发现我们所分离培养的前脂肪细胞的CD系列抗原标记表达特点与文献报道一致。 在随后的试验中,我们在前脂肪细胞诱导分化的过程中分别加入重组腺病毒Ad-EGFP-CIDE-3和对照病毒Ad-EGFP。并在在荧光显微镜下观察脂滴的形成及大小。被Ad-EGFP-CIDE-3感染的前脂肪细胞在诱导分化过程中较对照组细胞脂滴聚集的数量增多,脂滴的体积明显增大,同时脂肪细胞甘油三酯合成速度明显升高。可见CIDE-3与Fsp27相同均可以通过抑制细胞甘油三酯分泌促进其积聚最终促进脂滴的成熟。 【结论】 本研究发现CIDE-3蛋白定位于脂滴表面并部分位于内质网上,是一个脂滴表面蛋白。人CIDE-3蛋白通过抑制脂肪酸的氧化分解,促进脂滴的成熟参与了脂肪细胞的分化过程。
[Abstract]:Adipose tissue is an important place for the energy storage and utilization of the body. Adipocyte differentiation and metabolic abnormalities can lead to obesity. At present, obesity has become a worldwide health problem. At the same time, obesity is also a risk factor for many chronic diseases such as diabetes and cardio cerebrovascular diseases. Therefore, the study of its pathogenesis is the prevention and treatment of the disease. Obesity and its related diseases are of great importance.
The CIDE (cell death-inducing DFF45-like effector) family is a group of genes that can induce apoptosis at the end of the twentieth Century. Recently, it has been found that family members are closely related to the occurrence of obesity. Among them, Fsp27 (fat specific protein), mouse fat specific protein, only in mature adipocytes,.Fsp27 is located in lipid droplets. Fat decomposition promotes fat accumulation.CIDE-3 is Fsp27 in human homologous, mainly distributed in the small intestine, heart, colon and stomach, low expression in the brain, kidney and liver, and is not a fat specific gene. Then how does CIDE-3 play an important role in the differentiation and metabolism of adipocyte like FSP27 in human body The problem is not very clear.
In this study, we constructed the eukaryotic expression vector of EGFP-CIDE-3 and DsRed1-CIDE-3 fusion gene, determined the subcellular localization of CIDE-3 protein by transfection of COS-7 cells, and constructed adenovirus carrying EGFP-CIDE -3 and DsRed1-CIDE-3 fusion gene, and carried out in vitro isolation and primary culture of human preadipose fat cells in vitro and through adenovirus. Overexpression of CIDE-3 was used to investigate the mechanism of adipocyte proliferation and differentiation and the role of CIDE-3 in adipocyte differentiation and metabolism.
[Objective]
1, determine the subcellular localization of CIDE-3 protein; 2, construct adenovirus carrying EGFP-CIDE -3 and DsRed1-CIDE-3 fusion gene; 3, to study the role of CIDE-3 in the process of adipocyte differentiation and metabolism.
[method]
1, construct the eukaryotic expression vector of EGFP-CIDE-3 and DsRed1-CIDE-3 fusion gene, transfect COS-7 cells through liposomes to determine the subcellular localization of CIDE-3 protein; 2, use Stratagene company's AdEasyTM XL adenovirus vector system to construct adenovirus carrying EGFP-CIDE -3 and DsRed1-CIDE-3 fusion basis; 3, fat emulsion obtained from liposuction In vitro separation and primary culture of prepedestrian adipocytes; 4, using MTT method to plot the growth curve of preadipocytes; 5, using flow cytometry to determine the surface markers of preadipocytes to identify the source of cells; 6, through the adenovirus infection method over expression of CIDE-3 to explore the process of CIDE-3 in the differentiation and metabolism of adipocytes. The role of it.
[results]
1, CIDE-3 protein is mainly located on the surface of lipid droplets and partly on the endoplasmic reticulum.
First of all, recombinant plasmid pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed1-CIDE-3 were constructed with eukaryotic expression plasmid pShuttle-CMV, and then the recombinant plasmid was analyzed by enzyme cut electrophoresis and DNA sequencing. It was proved that we successfully constructed the recombinant plasmid pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed1-CIDE-3. in the experiment. In order to study the subcellular localization of CIDE-3 protein, we transfected the recombinant plasmid into COS-7 cells using liposome method. Using GFP-CB5 (Cytochrome B5, known endoplasmic reticulum location) as a marker of ER; GFP-ADRP as a marker of lipid droplets; Bodipy 493/503 as a neutral fatty dye; MitoTracker Red CMXRos as a mitochondrial dyestuff; 33258 The experimental results showed that CIDE-3 was partially Co located with the endoplasmic reticulum marker CB5, and CIDE-3 was distributed on the net structure formed by GFP-CB5; CIDE-3 was distributed around the lipid droplets and overlapped completely around the lipid droplets, indicating that CIDE-3 was a lipid droplet localization protein; at the same time, we found no co determination between CIDE-3 and mitochondria. Position.
2, the construction of adenovirus carrying CIDE-3 fusion gene is successful
We use the AdEasyTM XL adenovirus vector system of Stratagene company to construct recombinant shuttle plasmid by cloning the target gene. The recombinant adenovirus carrying the recombinant adenovirus plasmid and the transfected packing cell AD293 in the Escherichia coli BJ5183 are prepared by the homologous recombination. CIDE-3 and inverted fluorescence by PCR are amplified by PCR. The expression of fluorescent fusion protein was confirmed by microscope. The recombinant adenovirus Ad-EGFP-CIDE-3 and Ad-DsRed1-CIDE-3 were successfully constructed.
3, CIDE-3 can inhibit the oxidative decomposition of fatty acids and promote the maturation of lipid droplets.
We used fat cell emulsion after liposuction to cultivate the original preadipocytes. The growth curve showed that it was a preadipocyte, with 3 typical features proposed in the literature: 1) from the adipose tissue, spindle shape, no or few fat particles in the cytoplasm; 2) proliferated rapidly with fibroblasts. The increase of time was close to; 3) after the formation of monolayer confluence, it could become adipocytes and fat particles appeared in the cytoplasm. At the same time, the flow cytometry showed that the expression of CD series antigen of the preadipocytes isolated from our isolated culture was in accordance with the literature.
In the subsequent experiment, we added the recombinant adenovirus Ad-EGFP-CIDE-3 and the control virus Ad-EGFP. to the preadipocyte differentiation process, and observed the formation and size of the lipid droplets under the fluorescence microscope. The number of lipid droplets accumulated in the induced differentiated Cheng Zhongjiao control group was increased by the Ad-EGFP-CIDE-3 infected preadipocytes. The volume of lipid droplets increased significantly and the rate of triglyceride synthesis in adipocytes increased significantly. The same CIDE-3 and Fsp27 could promote the accumulation of triglycerides in cells to promote their accumulation and eventually promote the maturation of lipid droplets.
[Conclusion]
This study found that CIDE-3 protein is located on the surface of lipid droplets and partly on the endoplasmic reticulum. It is a lipid droplet surface protein. Human CIDE-3 protein promotes the maturation of fat cells by inhibiting the oxidation decomposition of fatty acids and promoting the maturation of lipid droplets.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R589.2;R363
本文编号：2146412
[Abstract]:Adipose tissue is an important place for the energy storage and utilization of the body. Adipocyte differentiation and metabolic abnormalities can lead to obesity. At present, obesity has become a worldwide health problem. At the same time, obesity is also a risk factor for many chronic diseases such as diabetes and cardio cerebrovascular diseases. Therefore, the study of its pathogenesis is the prevention and treatment of the disease. Obesity and its related diseases are of great importance.
The CIDE (cell death-inducing DFF45-like effector) family is a group of genes that can induce apoptosis at the end of the twentieth Century. Recently, it has been found that family members are closely related to the occurrence of obesity. Among them, Fsp27 (fat specific protein), mouse fat specific protein, only in mature adipocytes,.Fsp27 is located in lipid droplets. Fat decomposition promotes fat accumulation.CIDE-3 is Fsp27 in human homologous, mainly distributed in the small intestine, heart, colon and stomach, low expression in the brain, kidney and liver, and is not a fat specific gene. Then how does CIDE-3 play an important role in the differentiation and metabolism of adipocyte like FSP27 in human body The problem is not very clear.
In this study, we constructed the eukaryotic expression vector of EGFP-CIDE-3 and DsRed1-CIDE-3 fusion gene, determined the subcellular localization of CIDE-3 protein by transfection of COS-7 cells, and constructed adenovirus carrying EGFP-CIDE -3 and DsRed1-CIDE-3 fusion gene, and carried out in vitro isolation and primary culture of human preadipose fat cells in vitro and through adenovirus. Overexpression of CIDE-3 was used to investigate the mechanism of adipocyte proliferation and differentiation and the role of CIDE-3 in adipocyte differentiation and metabolism.
[Objective]
1, determine the subcellular localization of CIDE-3 protein; 2, construct adenovirus carrying EGFP-CIDE -3 and DsRed1-CIDE-3 fusion gene; 3, to study the role of CIDE-3 in the process of adipocyte differentiation and metabolism.
[method]
1, construct the eukaryotic expression vector of EGFP-CIDE-3 and DsRed1-CIDE-3 fusion gene, transfect COS-7 cells through liposomes to determine the subcellular localization of CIDE-3 protein; 2, use Stratagene company's AdEasyTM XL adenovirus vector system to construct adenovirus carrying EGFP-CIDE -3 and DsRed1-CIDE-3 fusion basis; 3, fat emulsion obtained from liposuction In vitro separation and primary culture of prepedestrian adipocytes; 4, using MTT method to plot the growth curve of preadipocytes; 5, using flow cytometry to determine the surface markers of preadipocytes to identify the source of cells; 6, through the adenovirus infection method over expression of CIDE-3 to explore the process of CIDE-3 in the differentiation and metabolism of adipocytes. The role of it.
[results]
1, CIDE-3 protein is mainly located on the surface of lipid droplets and partly on the endoplasmic reticulum.
First of all, recombinant plasmid pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed1-CIDE-3 were constructed with eukaryotic expression plasmid pShuttle-CMV, and then the recombinant plasmid was analyzed by enzyme cut electrophoresis and DNA sequencing. It was proved that we successfully constructed the recombinant plasmid pShuttle-CMV-EGFP-CIDE-3 and pShuttle-CMV-DsRed1-CIDE-3. in the experiment. In order to study the subcellular localization of CIDE-3 protein, we transfected the recombinant plasmid into COS-7 cells using liposome method. Using GFP-CB5 (Cytochrome B5, known endoplasmic reticulum location) as a marker of ER; GFP-ADRP as a marker of lipid droplets; Bodipy 493/503 as a neutral fatty dye; MitoTracker Red CMXRos as a mitochondrial dyestuff; 33258 The experimental results showed that CIDE-3 was partially Co located with the endoplasmic reticulum marker CB5, and CIDE-3 was distributed on the net structure formed by GFP-CB5; CIDE-3 was distributed around the lipid droplets and overlapped completely around the lipid droplets, indicating that CIDE-3 was a lipid droplet localization protein; at the same time, we found no co determination between CIDE-3 and mitochondria. Position.
2, the construction of adenovirus carrying CIDE-3 fusion gene is successful
We use the AdEasyTM XL adenovirus vector system of Stratagene company to construct recombinant shuttle plasmid by cloning the target gene. The recombinant adenovirus carrying the recombinant adenovirus plasmid and the transfected packing cell AD293 in the Escherichia coli BJ5183 are prepared by the homologous recombination. CIDE-3 and inverted fluorescence by PCR are amplified by PCR. The expression of fluorescent fusion protein was confirmed by microscope. The recombinant adenovirus Ad-EGFP-CIDE-3 and Ad-DsRed1-CIDE-3 were successfully constructed.
3, CIDE-3 can inhibit the oxidative decomposition of fatty acids and promote the maturation of lipid droplets.
We used fat cell emulsion after liposuction to cultivate the original preadipocytes. The growth curve showed that it was a preadipocyte, with 3 typical features proposed in the literature: 1) from the adipose tissue, spindle shape, no or few fat particles in the cytoplasm; 2) proliferated rapidly with fibroblasts. The increase of time was close to; 3) after the formation of monolayer confluence, it could become adipocytes and fat particles appeared in the cytoplasm. At the same time, the flow cytometry showed that the expression of CD series antigen of the preadipocytes isolated from our isolated culture was in accordance with the literature.
In the subsequent experiment, we added the recombinant adenovirus Ad-EGFP-CIDE-3 and the control virus Ad-EGFP. to the preadipocyte differentiation process, and observed the formation and size of the lipid droplets under the fluorescence microscope. The number of lipid droplets accumulated in the induced differentiated Cheng Zhongjiao control group was increased by the Ad-EGFP-CIDE-3 infected preadipocytes. The volume of lipid droplets increased significantly and the rate of triglyceride synthesis in adipocytes increased significantly. The same CIDE-3 and Fsp27 could promote the accumulation of triglycerides in cells to promote their accumulation and eventually promote the maturation of lipid droplets.
[Conclusion]
This study found that CIDE-3 protein is located on the surface of lipid droplets and partly on the endoplasmic reticulum. It is a lipid droplet surface protein. Human CIDE-3 protein promotes the maturation of fat cells by inhibiting the oxidation decomposition of fatty acids and promoting the maturation of lipid droplets.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R589.2;R363
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