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1.
Cells of the mouse cell line 3T3-F442A can be induced by various hormones to differentiate into adipocytes, whereas cells of 3T3-C2, a subclone of 3T3, cannot. However, transfection of DNA from uninduced 3T3-F422A cells into 3T3-C2 cells permits recovery of 3T3-C2 transfectants that differentiate into adipocytes in the presence of insulin. DNA isolated from human fat tissue, when transfected into 3T3-C2 mouse cells, also gives rise to mouse transfectants that are induced to differentiate into adipocytes by the addition of insulin. Apparently, transfection of a trans-regulatory gene (or genes) from 3T3-F442A or human fat cells into 3T3-C2 cells is sufficient to commit 3T3-C2 cells to adipocyte differentiation.  相似文献   

2.
【目的】诱导3T3-L1细胞分化为3T3-L1脂肪细胞,研究不同生物素水平对3T3-L1脂肪细胞脂肪合成相关基因转录表达的影响。【方法】利用三联诱导法将3T3-L1细胞经诱导分化为3T3-L1脂肪细胞。当3T3-L1脂肪细胞密集后分别采用0(对照组)、0.2、0.5、1 μmol/L生物素处理,分别在12 h、24 h与48 h时检测细胞上清液中PK mRNA、GLUT-4 mRNA、FAS mRNA及ACC1 mRNA相对表达量。【结果】在试验12 h时:各试验组GLUT-4、PK、ACC1 mRNA相对表达量均极显著(P＜0.01)高于对照组,1 μmol/L组与0.5 μmol/L组FAS mRNA相对表达量极显著(P＜0.01)高于对照组与0.2 μmol/L组;在试验24 h时:1 μmol/L组GLUT-4 mRNA与PK mRNA相对表达量显著(P＜0.05)高于对照组,1 μmol/L组ACC1 mRNA相对表达量极显著(P＜0.01)高于对照组,0.2 μmol/L组与0.5 μmol/L组ACC1 mRNA相对表达量显著(P＜0.05)高于对照组;在试验48 h时:1 μmol/L组GLUT-4 mRNA相对表达量显著(P＜0.05)高于对照组,1 μmol/L 组PK mRNA相对表达量极显著(P＜0.01)高于对照组,0.5 μmol/L组FAS mRNA相对表达量极显著(P＜0.05)高于1 μmol/L组,1 μmol/L组FAS mRNA相对表达量显著(P＜0.05)高于对照组,1 μmol/L组ACC1 mRNA相对表达量极显著(P＜0.01)高于其它组。【结论】生物素的添加可以提升脂肪细胞GLUT-4、PK、FAS与ACC1 mRNA相对表达量,且当以1 μmol/L浓度作用时对GLUT-4、PK与ACC1提升效果最佳,以0.5 μmol/L浓度作用时对FAS提升效果最佳。  相似文献   

3.
为了探讨蛔虫体腔液(Ascaris body cavity fluid,ABF)对小鼠3T3-L1前脂肪细胞分化过程的作用,揭示ABF对机体脂类代谢的影响.本试验采用半定量聚合酶链式反应技术(RT-PCR)检测前脂肪细胞分化相关基因过氧化物酶体增殖剂活化受体γ(Peroxisome proliferator-activated receptor γ,PPARγ)、GAPDH mRNA的表达;流式细胞技术(FCM)检测ABF对3T3-L1细胞凋亡的影响.结果表明:1 000μg/mL的ABF明显降低前脂肪细胞分化相关基因PPARγ mRNA的相对表达,与对照相比,差异极显著(P＜0.01).在3T3-L1分化过程中加入1 000 μg/mL ABF可增加细胞的凋亡.表明一定浓度的ABF对3T3-L1前脂肪细胞分化具有明显的抑制作用,并且能够诱导3T3-L1前脂肪细胞分化过程中的细胞凋亡,提示ABF能够抑制前脂肪细胞转化为脂肪细胞,可能具有调控脂类代谢以及控制高脂血症相关疾病的潜力.  相似文献   

4.
为探讨血管紧张素II（angiotensin II,AngII）对3T3-L1前脂肪细胞分化的影响,采用鸡尾酒法诱导3T3-L1前脂肪细胞分化,并用油红O染色法和还原型烟酰胺嘌呤二核苷酸（nicotinamide-adenine dinuceotid,NADH）氧化速率法检测了3T3-L1脂肪细胞分化过程中胞浆脂质的累积和甘油磷酸脱氢酶（glycerol phosphatedehydrogenase,GPDH）的活性。结果表明,100 nmol/L的AngII可显著增大3T3-L1前脂肪细胞分化过程中胞浆脂质累积和GPDH活性（P〈0.01）,外源性AngII可促进3T3-L1前脂肪细胞的分化,增加脂肪细胞中脂类合成和储存。  相似文献   

5.
Lipoprotein lipase (LPL) plays a critical role in the metabolism of plasma lipoproteins. In 3T3-L1 adipocytes, insulin elicits the rapid release of LPL through mechanisms that are independent of energy metabolism and protein synthesis. Some of the metabolic actions of insulin may be mediated by the activation of a specific phospholipase that hydrolyzes a glycosyl phosphatidylinositol (PI) molecule. The insulin-sensitive glycosyl-PI is structurally similar to the glycolipid membrane anchor of a number of proteins. LPL appears to be anchored to the 3T3-L1 cell surface by glycosyl-PI, and its rapid release by insulin may be due to activation of a glycosyl-PI-specific phospholipase C.  相似文献   

6.
[目的]鉴定水翁花提取物2,4-二羟基-6-甲氧基-3,5-二甲基查耳酮(DMC)的PPARγ配体结合活性及其特点。[方法]用GAL4嵌合体报告基因试验检测DMC的PPARγ配体结合活性;用油红O染色法检测DMC对3T3-L1前脂肪细胞分化的影响;用[3H]-2-脱氧葡萄糖摄取试验检测DMC对3T3-L1脂肪细胞葡萄糖摄取的影响;用荧光实时定量PCR检测经DMC处理的3T3-L1脂肪细胞中PPARγ靶基因的表达情况。[结果]DMC能以剂量依赖型的方式对PPARγ产生激活作用,促进脂肪细胞的分化,显著提高脂肪细胞的葡萄糖摄取率,并且提高脂肪细胞中一些PPARγ靶基因的表达量。[结论]DMC能通过激活PPARγ促进脂肪细胞的葡萄糖摄取。  相似文献   

7.
探讨了4种儿茶素单体对Na2S2O4诱导的3T3-L1细胞缺氧损伤和H2O2诱导的细胞氧化损伤的保护与修复作用。体外培养3T3-L1细胞,利用Na2S2O4诱导细胞缺氧损伤和H2O2诱导细胞氧化损伤,采用先加入诱导剂后加入儿茶素单体和先加入儿茶素单体后加入诱导剂两种处理方式,MTT法检测3T3-L1细胞存活率的变化。结果表明,先加入诱导剂可严重损伤3T3-L1细胞,再加入4种儿茶素单体均能对3T3-L1细胞进行修复,显著地提高细胞的存活率;而先加入4种儿茶素单体再加入诱导剂,细胞受损程度则显著降低,表明4种儿茶素单体对3T3-L1细胞缺氧或氧化损伤都具有较好的保护与修复作用。  相似文献   

8.
Krev-1 is an anti-oncogene that was originally identified by its ability to induce morphologic reversion of ras-transformed cells that continue to express the ras gene. The Krev-1-encoded protein is structurally related to Ras proteins. The biological activities of a series of ras-Krev-1 chimeras were studied to test the hypothesis that Krev-1 may directly interfere with a ras function. The ras-specific and Krev-1-specific amino acids immediately surrounding residues 32 to 44, which are identical between the two proteins, determined whether the protein induced cellular transformation or suppressed ras transformation. Because this region in Ras proteins has been implicated in effector function, the results suggest that Krev-1 suppresses ras-induced transformation by interfering with interaction of Ras with its effector.  相似文献   

9.
【目的】分析骨髓间充质干细胞在体外定向分化为脂肪细胞的适宜诱导条件.阻断正常干细胞的分化过程,探讨肿瘤发生机理.【方法】利用贴壁培养法获得大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),通过地塞米松、胰岛素和吲哚美辛联合诱导大鼠BMSCs,使其分化为脂肪细胞.在细胞分化过程中利用3-甲基胆蒽(3-MC)阻断大鼠BMSCs的分化过程.【结果】大鼠BMSCs分化试验显示:诱导30d后经油红O染色,细胞内可见脂肪细胞特有的红色脂滴沉淀,并且随着诱导时间的延长,油红O阳性细胞比例增加,脂滴增大,表明大鼠BMSCs已成功分化为脂肪细胞.大鼠BMSCs分化阻断试验显示:诱导30d后经油红O染色细胞内红色脂滴沉淀明显下降,表明阻断后的细胞具有脂肪细胞的特性,但分化不完全;形态学鉴定表现出:细胞接触抑制消失,细胞核异常,微核畸变率高,核型异常,表明阻断后的干细胞发生恶性转变.【结论】通过地塞米松、胰岛素和吲哚美辛的联合作用,诱导BMSCs成功向脂肪细胞分化.在BMSCs向脂肪细胞分化的过程中加入阻断剂3-MC,阻断其分化过程,表明干细胞分化过程受到阻断,细胞停止在分化的中间阶段,有转化成肿瘤细胞的趋势.  相似文献   

10.
成熟脂肪细胞中脂联素基因表达的脂肪酸应答调控   总被引：2，自引：0，他引：2  
用不同种类、不同浓度游离脂肪酸(FFA)处理体外培养的成熟3T3-L1脂肪细胞,实时荧光定量PCR和Western-blotting分析FFA处理前后脂联素基因mRNA和蛋白质水平的表达差异.结果表明:各类脂肪酸埘脂联素mRNA的表达均具有下调作用.且抑制效应呈时间、剂量依赖性;并且各种脂肪酸显著地影响脂联素蛋白的分泌作用.  相似文献   

11.
The 21-kD proteins encoded by ras oncogenes (p21Ras) are modified covalently by a palmitate attached to a cysteine residue near the carboxyl terminus. Changing cysteine at position 186 to serine in oncogenic forms produces a nonpalmitylated protein that fails to associate with membranes and does not transform NIH 3T3 cells. Nonpalmitylated p21Ras derivatives were constructed that contained myristic acid at their amino termini to determine if a different form of lipid modification could restore either membrane association or transforming activity. An activated p21Ras, altered in this way, exhibited both efficient membrane association and full transforming activity. Surprisingly, myristylated forms of normal cellular Ras were also transforming. This demonstrates that Ras must bind to membranes in order to transmit a signal for transformation, but that either myristate or palmitate can perform this role. However, the normal function of cellular Ras is diverted to transformation by myristate and therefore must be regulated ordinarily by some unique property of palmitate that myristate does not mimic. Myristylation thus represents a novel mechanism by which Ras can become transforming.  相似文献   

12.
Bone marrow mesenchymal stem cells （BMSCs） could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but also in the field of agricultural science. In an attempt to exhibit the characterization and differentiation into adipocytes and myocytes of porcine BMSCs, we isolated and purified porcine BMSCs by red blood cell lysis method and percoll gradient centrifugation. The purified cells presented a stretched fibroblast-like phenotype when adhered to the culture plate. The results of flow cytometry analysis and immunofluorescence staining demonstrated that the isolated cells were positive for mesenchymal surface markers CD29, CD44 and negative for hematopoietic markers CD45 and the adhesion molecules CD31. Cells were induced to differentiate into adipocytes with adipogenic medium containing insulin, dexamethasone, oleate and octanoate. Oil Red O staining demonstrated that the porcine BMSCs successfully differentiated to adipocytes. Moreover, the findings of real-time PCR and Western blotting indicated that the induced cells expressed adipogenic marker genes （PPAR-y, C/EBP-c~, perilipin, aP2） mRNA or proteins （PPAR-3,, perilipin, aP2）. On the other hand, porcine BMSCs were induced into myoctyes with myogenic medium supplemented with 5-azacytidine, basic fibroblast growth factor, chick embryo extract and horse serum. Morphological observation by hochest 33342 staining showed that the induced cells presented as multi-nucleus muscular tube structure. And myogenic marker genes （Myf5, desmin） mRNA or proteins （MyfS, MyoD, myogenin, desmin） were found in the induced cells. In addition, the results of immunofluorescence staining revealed that myogenic marker （Myf5, MyoD, myogenin, desmin, S-MyHC） proteins was positive in the induced cells. Above all, these results suggested that the isolated porcine BMSCs were not only consistent with the characterization of mesenchymal stem cells, but also exhibited the multipotential capacity to form adipocytes and myocytes, which provided the basis to investigate the regulation mechanism involved in the selective differentiation of porcine BMSCs.  相似文献   

13.
Second-passage rat embryo cells were transfected with a neomycin resistance gene and the activated form of the c-Ha-ras I gene, or with these two genes plus the adenovirus type 2 E1a gene. Foci of morphologically transformed cells were observed in both cases; however, the frequency of transformation was at least ten times higher with two oncogenes than with the ras gene alone. All the transformed cell lines gave rise to rapidly growing tumors when injected subcutaneously into nude mice. All but one of the cell lines transformed by the ras oncogene alone formed metastatic nodules in the lungs of animals that had been injected subcutaneously with transformed cells. When transformed cells were injected intravenously, all the ras single-gene transformants gave rise to many metastatic lung nodules. In contrast, cell lines transformed with ras and E1a did not generate metastases after subcutaneous injection and gave rise to very few metastatic lung nodules after intravenous injection. These data demonstrate that a fully malignant cell with metastatic potential, as measured in an immunodeficient animal, can be obtained from early passage embryo cells by the transfection of the ras oncogene alone.  相似文献   

14.
 【目的】探讨甘薯sporamin蛋白对3T3-L1前脂肪细胞分化与增殖的影响,为开发预防和治疗肥胖、糖尿病的保健食品提供理论依据。【方法】采用硫酸铵沉淀、离子交换、凝胶过滤层析的方法对‘55-2’甘薯中的sporamin蛋白进行分离纯化。然后,以黄连素为阳性对照,用不同浓度sporamin（0、0.025、0.125、0.250、0.500、1.000 mg?ml-1）处理3T3-L1前脂肪细胞。采用油红O染色和比色定量检测细胞内脂肪生成及细胞分化程度,以MTT法检测细胞的增殖。【结果】经离子交换、凝胶层析可纯化出高纯度的sporamin蛋白A和B（相对分子量分别为31 kD和22 kD）。与空白相比,用不同浓度的sporamin蛋白处理后,3T3-L1前脂肪细胞的分化受到明显抑制。当sporamin蛋白浓度增至0.500 mg?ml-1时,脂滴生成量明显减少,洗脱液吸光度值降至最低为0.35（P＜0.05）。此外,高浓度的sporamin蛋白能有效地抑制3T3-L1前脂肪细胞的增殖,且随着处理时间延长抑制效果更加明显(P＜0.05).【结论】甘薯sporamin蛋白能抑制3T3-L1前脂肪细胞的分化和增殖,具有潜在的减肥作用。  相似文献   

15.
【目的】构建ChREBP基因siRNA表达质粒,干扰ChREBP在原代培养猪脂肪细胞的表达,研究其在葡萄糖诱导脂肪细胞生脂中的作用。【方法】合成4对靶向ChREBP基因的siRNA寡核苷酸,分别连接于pcDNA™6.2-GW/EmGFP载体构建siRNA表达质粒,测序验证后,采用脂质体介导法转染从3日龄仔猪皮下脂肪组织分离培养的脂肪细胞,荧光定量RT-PCR检测ChREBP基因沉默效率;以葡萄糖浓度为0—20 mmol·L-1的培养液培养转染细胞48 h,测定生脂及生脂基因表达变化。【结果】筛选出了1个转染效果好、ChREBP基因沉默效率达85%的siRNA表达质粒,转染原代培养猪脂肪细胞后,细胞生脂水平及生脂基因ACC1和FAS mRNA表达比阴性对照表达质粒转染细胞和未转染细胞显著降低（P＜0.05）,且生脂水平不受葡萄糖水平的影响（P＞0.05）。【结论】构建的siRNA表达质粒能有效干扰猪脂肪细胞ChREBP表达,葡萄糖通过转录因子ChREBP调控猪脂肪细胞生脂及生脂基因表达。  相似文献   

16.
The aim of this study was to investigate the effect of different concentrations of sporamin on the differentiation and proliferation of 3T3-LI preadipocytes, providing the theoretical basis for the development of food to treat obesity and diabetes, The isolation and purification of sporamin from sweet potato species 55-2 were performed by ammonium sulphate precipitation in combination with ion-exchange and gel filtration chromatography. With berberine as a positive control, different concentrations ofsporamin （0.000, 0.125, 0.025, 0.250, 0.500, and 1.000 mg·mL^-1 were used to treat 3T3-L1 preadipocytes. Intracellular fat accumulation and the degree of adipogenesis were quantified using Oil Red O staining and colorimetry, Preadipocytes differentiation was measured by 3（4,5-dimethylthiazolyl-2-yl）-2,5-diphenyltetrazolium bromide （MTT） spectrophotometric assay. Two sporamin proteins, which were separated into sporamin A （31 kD） and sporamin B （22 kD）, could be purified by ion-exchange and gel filtration chromatography. After being treated by different concentrations of sporamin, the differentiation of 3T3-L1 preadipocytes was significantly inhibited, compared with the positive control. When the sporamin solution concentration was 0.500 mg mL-1, the accumulation of lipid droplets within the cells was significantly decreased and the optical density （OD） value of the solution from destained Oil Red O reached to 0.35, which was the lowest value （P〈 0.05）. The proliferation of 3T3-L1 preadipocytes was significantly inhibited by treating at higher sporamin concentrations. In addition, the inhibitory effect was more obvious with the prolonged treatment time （P〈 0.05）. The differentiation and proliferation of 3T3-L1 preadipocytes could be inhibited significantly by the addition of higher concentration sporamin. It was, therefore, suggested that the sporamin was potentially effective for weight loss.  相似文献   

17.
Identification of genes that function to protect cells from radiation damage is an essential step in understanding the molecular mechanisms by which mammalian cells cope with ionizing radiation. The intrinsic radiation resistance (D0) of NIH 3T3 cells was markedly and significantly increased by transformation with ras oncogenes activated by missense mutations. This radiobiologic activity appeared to be a specific consequence of the ras mutations rather than of transformation, since revertant cells that contained functional ras genes (but were no longer phenotypically transformed) retained their increased D0's.  相似文献   

18.
Several immunologically distinct isozymes of inositol phospholipid-specific phospholipase C (PLC) have been purified from bovine brain. Murine NIH 3T3 fibroblasts were found to express PLC-gamma, but the expression of PLC-beta was barely detectable by radioimmunoassay or protein immunoblot. A mixture of monoclonal antibodies was identified that neutralizes the biological activity of both endogenous and injected purified PLC-gamma. When co-injected with oncogenic Ras protein or PLC-gamma, this mixture of antibodies inhibited the induction of DNA synthesis that characteristically results from the injection of these proteins into quiescent 3T3 cells. However, when oncogenic Ras protein or PLC-gamma was co-injected with a neutralizing monoclonal antibody to Ras, only the DNA synthesis induced by the Ras protein was inhibited--that induced by PLC was unaffected. These results suggest that the Ras protein is an upstream effector of PLC activity in phosphoinositide-specific signal transduction and that PLC-gamma activity is necessary for Ras-mediated induction of DNA synthesis.  相似文献   

19.
Influx of calcium through membrane channels is an important initial step in signal transduction of growth signals. Therefore, the effects of Ras protein injection on calcium currents across the soma membrane of an identified neuron of the snail Hermissenda were examined. With the use of these post-mitotic cells, a voltage-sensitive, inward calcium current was increased 10 to 20 minutes after Harvey-ras oncoproteins were injected. The effects of oncogenic Harvey ras p21 protein (v-Ras) occurred quickly and were sustained, whereas the effects of proto-oncogenic ras protein (c-Ras) were transient. This relative potency is consistent with the activities of these oncoproteins in stimulating cell proliferation. Thus, this calcium channel may be a target for Ras action.  相似文献   

20.
Amyloid deposition in senile plaques and the cerebral vasculature is a marker of Alzheimer's disease. Whether amyloid itself contributes to the neurodegenerative process or is simply a by-product of that process is unknown. Pheochromocytoma (PC12) and fibroblast (NIH 3T3) cell lines were transfected with portions of the gene for the human amyloid precursor protein. Stable PC12 cell transfectants expressing a specific amyloid-containing fragment of the precursor protein gradually degenerated when induced to differentiate into neuronal cells with nerve growth factor. Conditioned medium from these cells was toxic to neurons in primary hippocampal cultures, and the toxic agent could be removed by immunoabsorption with an antibody directed against the amyloid polypeptide. Thus, a peptide derived from the amyloid precursor may be neurotoxic.  相似文献   

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