人CIDE-3基因在脂肪細(xì)胞分化及代謝中作用的研究
發(fā)布時間：2018-07-26 15:11
【摘要】： 脂肪組織是機(jī)體能量儲存及利用的重要場所。脂肪細(xì)胞分化和代謝異�？蓪�(dǎo)致肥胖發(fā)生,目前肥胖已日趨成為世界性的健康問題,同時肥胖也是糖尿病和心腦血管疾病等多種慢性疾病的危險因素。因此研究其發(fā)病機(jī)理對于預(yù)防和治療肥胖及其相關(guān)疾病具有非常重要的意義。 CIDE(cell death-inducing DFF45-like effector)家族是20世紀(jì)末發(fā)現(xiàn)的一組可誘導(dǎo)細(xì)胞凋亡的基因。近來發(fā)現(xiàn),其家族成員與肥胖的發(fā)生密切相關(guān)。其中Fsp27(fat specific protein),小鼠脂肪特異蛋白,僅在成熟的脂肪細(xì)胞中表達(dá)。Fsp27定位于脂滴可以抑制脂肪分解促進(jìn)脂肪積聚。CIDE-3是Fsp27在人的同源物,其主要分布在小腸、心臟、結(jié)腸和胃,在腦、腎臟和肝中低表達(dá),并不是脂肪特異基因。那么CIDE-3在人體內(nèi)生物學(xué)功能如何,是否也像FSP27一樣在脂肪細(xì)胞分化代謝過程中發(fā)揮重要作用,這些問題并不十分清楚。 在本研究中,我們構(gòu)建了EGFP-CIDE-3及DsRed1-CIDE-3融合基因真核表達(dá)載體,通過轉(zhuǎn)染COS-7細(xì)胞確定CIDE-3蛋白的亞細(xì)胞定位。并構(gòu)建攜帶EGFP-CIDE -3及DsRed1-CIDE-3融合基因的腺病毒,同時進(jìn)行了人前脂肪細(xì)胞的體外分離和原代培養(yǎng),并通過腺病毒感染過表達(dá)CIDE-3以探討脂肪細(xì)胞的增殖分化機(jī)制和CIDE-3在脂肪細(xì)胞分化代謝過程中的作用。 【目的】 1、確定CIDE-3蛋白的亞細(xì)胞定位;2、構(gòu)建攜帶EGFP-CIDE -3及DsRed1-CIDE-3融合基因的腺病毒;3、研究CIDE-3在脂肪細(xì)胞分化代謝過程中的作用。 【方法】 1、構(gòu)建EGFP-CIDE-3及DsRed1-CIDE-3融合基因真核表達(dá)載體,通過脂質(zhì)體轉(zhuǎn)染COS-7細(xì)胞以確定CIDE-3蛋白的亞細(xì)胞定位;2、利用Stratagene公司的AdEasyTM XL腺病毒載體系統(tǒng)構(gòu)建攜帶EGFP-CIDE -3及DsRed1-CIDE-3融合基因的腺病毒;3、從抽脂獲得的脂肪乳液進(jìn)行人前脂肪細(xì)胞的體外分離和原代培養(yǎng);4、利用MTT方法繪制人前脂肪細(xì)胞的生長曲線;5、利用流式細(xì)胞方法測定前脂肪細(xì)胞的表面標(biāo)志分子以鑒定細(xì)胞的來源;6、通過腺病毒感染的方法過表達(dá)CIDE-3以探討CIDE-3在脂肪細(xì)胞分化代謝過程中的作用。 【結(jié)果】 1、CIDE-3蛋白主要定位于脂滴表面,部分位于內(nèi)質(zhì)網(wǎng)上 首先以真核表達(dá)質(zhì)粒pShuttle-CMV作為載體,構(gòu)建了重組質(zhì)粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed1-CIDE-3,然后對重組質(zhì)粒進(jìn)行了酶切電泳及DNA測序等分析,證實我們在實驗中成功地構(gòu)建了重組質(zhì)粒pShuttle-CMV-EGFP-CIDE-3和pShuttle-CMV-DsRed1-CIDE-3。為了研究CIDE-3蛋白的亞細(xì)胞定位,我們以脂質(zhì)體法將重組質(zhì)粒轉(zhuǎn)染COS-7細(xì)胞。采用GFP-CB5(Cytochrome B5,已知內(nèi)質(zhì)網(wǎng)定位)作為ER標(biāo)志物;GFP-ADRP作為脂滴的標(biāo)志物;Bodipy 493/503作為中性脂肪染料;MitoTracker Red CMXRos作為線粒體染料;Hoechst 33258作為細(xì)胞核染料。實驗結(jié)果顯示:CIDE-3與內(nèi)質(zhì)網(wǎng)標(biāo)志物CB5存在部分共定位,CIDE-3分布于GFP-CB5所形成的網(wǎng)狀結(jié)構(gòu)上;CIDE-3分布于脂滴周圍,與ADRP在脂滴周圍完全重疊,說明CIDE-3是一個脂滴定位的蛋白;同時我們發(fā)現(xiàn)CIDE-3與線粒體不存在共定位。 2、攜帶CIDE-3融合基因的腺病毒構(gòu)建成功 我們利用Stratagene公司的AdEasyTM XL腺病毒載體系統(tǒng),經(jīng)過克隆目的基因、構(gòu)建重組穿梭質(zhì)粒、在大腸桿菌BJ5183中進(jìn)行同源重組獲得重組腺病毒質(zhì)粒及轉(zhuǎn)染包裝細(xì)胞AD293等步驟制備了攜帶CIDE-3基因的重組腺病毒。通過PCR擴(kuò)增CIDE-3,倒置熒光顯微鏡下觀察熒光融合蛋白表達(dá)等方法證實重組腺病毒Ad-EGFP-CIDE-3和Ad-DsRed1-CIDE-3構(gòu)建成功。 3、CIDE-3可以抑制脂肪酸的氧化分解,促進(jìn)脂滴成熟 我們采用抽脂術(shù)后的脂肪細(xì)胞乳液培養(yǎng)了原代人前脂肪細(xì)胞。并經(jīng)生長曲線測定初步證明其確為前脂肪細(xì)胞,具有文獻(xiàn)中提出的3個典型特征:1)來自于脂肪組織,形態(tài)為梭形,胞漿內(nèi)無或很少有脂肪顆粒;2)增殖迅速,與成纖維細(xì)胞在倍增時間上接近;3)在形成單層匯合后能變?yōu)橹炯?xì)胞,胞質(zhì)內(nèi)會出現(xiàn)脂肪顆粒。同時,流式細(xì)胞儀檢測發(fā)現(xiàn)我們所分離培養(yǎng)的前脂肪細(xì)胞的CD系列抗原標(biāo)記表達(dá)特點與文獻(xiàn)報道一致。 在隨后的試驗中,我們在前脂肪細(xì)胞誘導(dǎo)分化的過程中分別加入重組腺病毒Ad-EGFP-CIDE-3和對照病毒Ad-EGFP。并在在熒光顯微鏡下觀察脂滴的形成及大小。被Ad-EGFP-CIDE-3感染的前脂肪細(xì)胞在誘導(dǎo)分化過程中較對照組細(xì)胞脂滴聚集的數(shù)量增多,脂滴的體積明顯增大,同時脂肪細(xì)胞甘油三酯合成速度明顯升高�？梢奀IDE-3與Fsp27相同均可以通過抑制細(xì)胞甘油三酯分泌促進(jìn)其積聚最終促進(jìn)脂滴的成熟。 【結(jié)論】 本研究發(fā)現(xiàn)CIDE-3蛋白定位于脂滴表面并部分位于內(nèi)質(zhì)網(wǎng)上,是一個脂滴表面蛋白。人CIDE-3蛋白通過抑制脂肪酸的氧化分解,促進(jìn)脂滴的成熟參與了脂肪細(xì)胞的分化過程。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R589.2;R363
本文編號：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R589.2;R363
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