PPARγ2表达诱导NIH3T3成纤维细胞向脂肪细胞分化

目的应用重组逆转录病毒载体介导小鼠过氧化物酶体增殖体激活的受体γ2(mPPARγ2)基因在NIH3T3成纤维细胞中表达,并进一步研究其功能.方法从经荧光测序证实含正确mPPARγ2 cDNA序列的重组质粒pcDNA3/mPPARγ2中,双酶切下mPPARγ2全长cDNA序列,亚克隆入逆转录病毒载体pGCEN中,构建重组逆转录病毒载体pGCEN/mPPARγ2.用PA317细胞对pGCEN/mPPARγ2及pGCEN进行包装,并用G418进行PA317细胞抗性克隆筛选,收集病毒上清,感染靶细胞NIH3T3成纤维细胞.表达mPPARγ2的NIH3T3成纤维细胞在含PPARγ激活物5,8,11,14-二十碳四烯酸(ETYA)等分化介质中培养,可被诱导向脂肪细胞分化.结果构建了含mPPARγ2全长cDNA重组逆转录病毒载体pGCEN/mPPARγ2,获得了较高pGCEN/mPPARγ2及pGCEN的逆转录病毒上清.重组逆转录病毒载体可介导mPPARγ2在NIHT3T3成纤维细胞胞核中表达.油红O染色证实,表达mPPARγ2的NIHT3T3成纤维细胞在分化介质中培养分化10天后,胞浆中明显积聚了较多的中性脂肪,其细胞形态也与体内成熟脂肪细胞相似,而且这些细胞表达脂肪细胞特异性标志基因如AP2和leptin.结论本研究在体外成功地建立了由PPARγ2诱导NIH3T3成纤维细胞向脂肪细胞分化的模型,为进一步研究PPARγ2诱导脂肪细胞分化的分子机制奠定了基础.     

Blockage of PPAR8 increases the expression of-inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.     

Blockage of PPARδ increases the expression of inflammatory factors in 3T3-L1 cells stimulated with TNFα

Objective: To investigate the role of peroxisome proliferator-activated receptors δ (PPARδ) in inflammatory reaction and its possible mechanism in adipocyte. Methods :Lentivirus-mediated RNA interference (RNAi) was used to block the expression of PPARS in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α(TNFα, 20 ng/ml) for 4 h. The expression of PPAR8, nuclear factor κB (NFκB) and C reactive protein (CRP) were determined by Western blot analysis. Results:The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα, but had no effect on normal cells. Conclusion: PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.     

Verapamil inhibits 3T3-L1 preadipocyte differentiation

Objective: To investigate the effect of the calcium channel blocker verapamil on adipocyte differentiation and its mechanism of action. Methods: Preadipocytes from 3T3-L1 strain mouse embryos were cultured and differentiated into matured adipocytes in vitro. Verapamil was added to the culture medium in the concentration of 30 μmol/L on Day 0. Cell differentiation was determined by Oil Red O staining and marker gene mRNA expression was evaluated and compared by RT-PCR. The fluo-3/AM probe and laser scanning confocal microscopy were used to measure intracellular calcium concentrations. Results: ①The differentiation rate of 3T3-L1 preadipocytes exposed to verapamil was lower than that of untreated cells. ②Verapamil promoted the retention of pref-1 gene expression. Lipoprotein lipase expression in the verapamil group was significantly lower than that in the control group on Day 4, Day 6 and Day 8 (P < 0.05) and resistin expression was significantly lower than that in the control group on Day 6, Day 8 and Day 10 (P < 0.05). Fatty acid synthase expression in the verapamil group was significantly lower than that in the control group from Day 2 (P < 0.05). ③ Intracellular concentrations of calcium [Ca2+]i in the verapamil group were significantly decreased compared with those in the control group on Day 2, Day 4 and Day 6 (P < 0.05), while there was no obvious difference between the two groups on Day 0 (P > 0.05). Conclusion: In 3T3-L1 preadipocytes verapamil significantly reduced adipocyte differentiation, down-regulated the mRNA expression of three marker genes for adipocytes differentiation, and prolonged the mRNA expression of an inhibitor of differentiation. The inhibitory effect of verapamil on differentiation may involve its role as a blocker of calcium influx in adipocytes.     

Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes

The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study. 3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin. Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR. Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining. The mRNA levels of peroxisome proliferator-activated receptor γ （PPARγ） and CAAT/enhancer binding protein （C/EBPa） in the cells at different differentiation stages were detected by RT-PCR. The results showed that ghrelin at concentrations of 10^-7 to 10^-15 mol/L could significantly promote preadipocyte proliferation （P〈0.05）, with the most pronounced effect observed at 10^-11 mol/L （P〈0.01）. Treatment of 3T3-L1 preadipocytes with ghrelin significantly increased the mRNA levels of c-myc and thymidine kinase （P〈0.01）. Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin. Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Ghrelin significantly increased the mRNA levels of PPAR7 and C/EBPα during the differentiation, when compared with control group （P〈0.05）. The mRNA levels of PPARγ and C/EBPα were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin. There were significant difference in the mRNA levels of PPARγ and C/EBPu on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes （P〈0.01）. In conclusion, ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARγ and C/EBPα and therefore enhance the sensitivity of adipocytes against insulin.     

Effects of secretive bone morphogenetic protein 2 induced by gene transfection on the biological changes of NIH3T3 cells

Background Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are powerful regulators of cartilage and bone formation. This study investigated the biological changes of NIH3T3 cells incubated with secretive BMP2 that was induced by gene transfection through transwell. Methods Eukaryonic expression vector (pcDNA3.1-B2) was transfered into NIH3T3 cells with Sofast&#8482;,a positive compound transfection agent. The positive cell clones were selected with G418. The cytoplasmic and extracellular expressions of BMP2 were determined by immunohistochemical stain and enzyme-linked immunosorbent assay. NIH3T3 cells were co-cultured with hBMP2 gene transfecting cells through transwell, and the ultrastructure, alkaline phosphatase activity and the expression of osteocalcin (the marker of osteogenetic differentiation) changes were observed. Results There were cytoplasmic and extracellular expressions of BMP2 in transfecting NIH3T3 cells. The ultrastructural changes, the high activity of alkaline phosphatase and the positive stain of osteocalcin suggested the osteogenetic differentiation tendency of NIH3T3 cells co-cultured with transfecting NIH3T3 cells. Conclusion Secretive BMP2 that is induced by gene transfection could promote the osteogenetic differentiation of fibroblast cells.     

Construction,Expression and in vitro Biological Effects of Idiotype Ig Fab Fragment of B-Chronic Lymphocytic Leukemia

The purpose of this study was to construct expression vectors of idiotype （Id） SmIg in patients with B-chronic lymphocytic leukemia and to express them in E.coli to obtain recombinant Id, and to investigate the effect of the protein on the proliferation and secretion of IL-2 and IFN-γ of stimulated peripheral blood mononuclear cells （PBMC） in vitro. Light chain gene and Fd fragment of heavy chain gene were inserted into fd-tet-DOG2 vector to construct fd-tet-DOG2-Fab. Fab gene was further cloned into expression vector pHEN2 to construct the soluble expression vector pHEN2-Fab. After induction by IPTG, Fab protein was purified by Ni-NTA-chromatography. MTT was used to determine the effects of purified protein on the proliferation of stimulated PBMC in vitro and the concentrations of IL-2 and IFN-γ in the culture supernatants were detected by ELISA. The results showed that recombinant pHEN2-Fab expression vector was constructed successfully. Fab protein was expressed in positive clone after induced by IPTG and two specific bands at 24-25 kD position were observed by SDS-PAGE electrophoresis. Proliferation of PBMC could be induced by purified Fab and the concentrations of IL-2 and IFN-γ in culture supernatants were increased significantly af- ter induction. It was suggested that the expression vector of SmIg Fab fragment was constructed suc- cessfully, and expressed and secreted from E. coli. The Fab protein could induce proliferation of PBMC and promote secretion of IL-2 and IFN-γ.     

Construction of eukaryotic expression vector encoding ATP synthase lipid-binding protein-like protein gene of Sj and its expression in HeLa cells

Objective： To clone and construct the recombinant plasmid containing ATP synthase lipid-binding protein-like protein gene of Schistosomajaponicum,（SjAslp） and transfer it into mammalian cells to express the objective protein. Methods： By polymerase chain reaction （PCR） technique, SjAslp was amplified from the constructed recombinant plasmid pBCSK＋/SjAslp, and inserted into cloning vector pUCm-T. Then, SjAslp was subcloned into an eukaryotic expression vector pcDNA3.1（＋）. After identifying it by PCR, restrictive enzymes digestion and DNA sequencing, the recombinant plasmid was transfected into HeLa cells using electroporation, and the expression of the recombinant protein was analyzed by immunocytochemical assay. Results： The specific gene fragment of 558 bp was successfully amplified. The DNA vaccine of SjAslp was successfully constructed. Immunocytochemical assay showed that SjAslp was expressed in the cytoplasm of HeLa cells. Conclusion： SjAslp gene can be expressed in eukaryotic system, which lays the foundation for development of the SjAslp DNA vaccine against schitosomiasis.     

绿色荧光蛋白基因在脂肪细胞分化研究中的应用

目的建立人脂肪细胞分化的绿色荧光蛋白活体检测动态模型，为进一步进行脂肪细胞分化及其相关基因表达研究奠定基础。方法用PCR法从培养的人前脂肪细胞总DNA中扩增出过氧化物体酶增殖物激活受体γ2（PPARγ2）基因启动子．将该启动子插入真核细胞表达载体pEGFP-1后转染3T3成纤维细胞及人前脂肪细胞，并诱导脂肪细胞分化，结果在胰岛素、地塞米松及30异丁基-1-甲基黄嘌呤诱导下，人前脂肪细胞分化为成熟脂肪细胞时表达绿色荧光蛋白基因，而3T3细胞则不能防导为脂肪细胞及表达绿色荧光蛋白基因。结论脂肪组织特异表达的PPARγ2基因启动子与绿色荧光蛋白基因构成的重组基因转化人前脂肪细胞后，能够用来动态活体检测脂防细胞分化状态。这一模型的建立为脂肪细胞分化调控的分子机制研究提供一个更加方便的检测途径。     

mIL-12重组逆转录病毒载体的构建及包装细胞系的建立

目的：构建 mIL-12重组逆转录病毒载体。方法：将 mIL-12 DNA克隆到载体pLEGFP,用该重组质粒转染PA317包装细胞获得重组逆转录病毒。结果：重组质粒pLEGFP-mIL12酶切及PCR产物在2 290 bp区域见强荧光条带与mIL-12基因大小相符;转染PA317细胞后的上清感染NIH3T3细胞后,经共聚焦显微镜能观察到融合蛋白的表达。结论：包装细胞上清中有含mIL-12 DNA的重组逆转录病毒,mIL-12重组逆转录病毒可感染NIH3T3细胞并在细胞中进行有效的复制。     

小鼠Wnt-3a基因在NIH3T3细胞中的表达及其活性的初步分析

目的:表达具备生物活性的重组小鼠Wnt-3a信号蛋白.方法:应用脂质体转染试剂将重组真核表达载体pSecTag2/Hygro B-Wnt3a转染并筛选稳定表达的NIH3T3细胞,Western Blot鉴定重组Wnt-3a蛋白的表达,并对Wnt3a/NIH3T3细胞的融合密度及抗凋亡能力给予检测.结果:Wnt-3a信号蛋白在Wnt3a/NIH3T3细胞中获得稳定表达,Wnt-3a信号蛋白能够明显提高NIH3T3细胞的融合密度及抗凋亡能力.结论:在NIH3T3细胞中表达的重组Wnt-3a信号蛋白具备生物活性.     

mTOR信号通路中上游调控蛋白Rheb在脂肪细胞分化中的作用

目的 观察mTOR信号通路中上游调控蛋白Rheb对脂肪细胞分化的影响。方法 构建Rheb (Ras homolog enriched in brain)的重组质粒(pCAG-Insulator-Rheb),并将其注射到B6小鼠的胚胎内,产生全身高表达Rheb的转基因小鼠。提取怀孕雌鼠第13.5天的胚胎成纤维细胞(mouse embryonic fibroblast cells, MEFs),PCR鉴定基因型之后进行成脂诱导分化。通过检测分化第12天MEFs内三酰甘油的含量、油红O染色的结果、实时定量PCR方法检测脂肪细胞特异性转录因子PPARγ和C/EBPα的表达情况,来观察Rheb在脂肪细胞分化过程中的作用。结果 成功构建了全身高表达Rheb的转基因小鼠模型,并且检测到在高表达Rheb后,可以促进MEFs内脂滴的生成,三酰甘油含量增高,油红O染色有明显区别,并且脂肪细胞特异性转录因子PPARγ和C/EBPα的表达量均升高。结论 Rheb过表达以后可以促进脂肪细胞的分化。     

重组白细胞介素13对成纤维细胞增殖的影响

目的：观察重组白细胞介素13（rIL-13）对NIH3T3细胞增殖作用的影响，探讨肺纤维化的发病机制。方法：NIH313细胞分为rIL-13组和不含rIL-13的对照组，rIL-13组加入不同浓度的rIL-13，作用24及48h。透射电镜观察313细胞的超微结构，Hoechst试剂盒观察细胞DNA形态变化，噻唑蓝比色试验（MTT法）测定细胞增殖率。结果：rIL-13呈剂量依赖方式刺激313细胞细胞生长。经rIL-1380ng／ml孵育的细胞DNA合成增加，细胞核增大，细胞质中可见较多核糖体及线粒体。rIL-13组细胞增殖率增加，与对照组比较有显著性差异（P〈0．05）。结论：rIL-13可能通过促进成纤维细胞增殖，在肺纤维化的发病机制中发挥重要作用。     

G_0S_2基因参与调节鼠骨髓基质细胞向脂肪细胞分化

目的:探讨G_0S_2在调节鼠骨髓基质细胞向脂肪细胞分化过程中的作用.方法:利用原代培养小鼠骨髓基质细胞(Marrowstrom cell,MSC),脂质体转染法将表达质粒pCAG-PPARγ2及含报告基因的pGL_2-COL_1A质粒共转染至MSC,G418筛选,RT-PCR检测PPARγ及G0S2mRNA表达,免疫组化检测PPARγ的表达,westen blot检测G_0S_2的表达,油红O染色检测细胞内脂滴.结果:MSC在未分化状态下不表达G_0S_2,经PPARγ基因转染后,G_0S_2开始表达,转染细胞最终分化为脂肪细胞.结论:PPARγ通过调节G_0S_2启动子调节后者表达,进而调节MSC向脂肪细胞分化,G_0S_2基因参与调节MSC向脂肪细胞分化.     

Galectin-3重组真核表达载体的构建及其在NIH/3T3细胞中的表达

目的:构建半乳糖凝集素-3(Gal-3)的真核表达载体,在NIH/3T3细胞中表达,并检测其表达?方法:通过DNA重组技术和PCR方法从人肿瘤细胞克隆Gal-3基因,插入真核表达载体pEGFP-N1中,通过酶切和测序鉴定重组载体的正确性;采用脂质体转染技术将重组质粒pEGFP-Gal-3瞬时转染NIH/3T3细胞,经荧光和Western blot方法检测Gal-3表达,MTT法检测Gal-3对NIH/3T3细胞增殖的影响?结果:限制性内切酶鉴定和核酸序列测序证实成功构建含Gal-3的重组真核表达载体pEGFP-Gal-3?以重组质粒瞬时转染NIH/3T3细胞,检测到Gal-3蛋白表达,并证实Gal-3蛋白促进NIH/3T3细胞增殖?结论:成功构建的pEGFP-Gal-3真核表达载体在小鼠NIH/3T3细胞中成功表达,并促进NIH/3T3细胞增殖?     

3T3-L1前脂肪细胞分化过程中miRNAs表达谱的变化

目的探讨3T3-L1前脂肪细胞分化过程中微小RNA(miRNAs)表达谱的变化。方法3T3-Ll前脂肪细胞培养和诱导分化,通过形态学观察和RT-PCR方法,判断3T3-Ll前脂肪细胞是否分化成脂肪细胞。运用miRNAs芯片技术检测3T3-Ll前脂肪细胞和脂肪细胞中差异表达miRNAs。结果3T3-L1前脂肪细胞呈现成纤维状,诱导分化的第9天,细胞变成球形,内含许多脂滴;过氧化物酶体增殖物激活受体r2(PPARr2)和脂肪性脂肪酸结合蛋白(aP2)的表达显著增加。3T3-L1前脂肪细胞分化成脂肪细胞上调的miRNAs有26个,下调的miRNAs有2个。结论3T3-L1前脂肪细胞分化过程中存在miRNAs表达谱的变化。     

供转导PDGF-A基因的高滴度逆转录病毒的制备

目的制备高滴度供转导血小板衍生生长因子Ａ链（ＰＤＧＦ－Ａ）基因的重组逆转录病毒,为体内、外深入研究该基因表达产物自分泌和旁分泌的生物学功能奠定基础。方法将人ＰＤＧＦ－ＡｃＤＮＡ片段插入含巨细胞病毒（ＣＭＶ）启动子序列的逆转录病毒ＧＩＮａ载体的多克隆位点中,将构建的重组质粒导入病毒包装细胞ＰＡ３１７中,经Ｇ４１８筛选并扩增,以病毒液感染ＮＩＨ３Ｔ３细胞,测病毒滴度,选择出抗性克隆,进行免疫组织化学检测,表达产物经ＳＤＳ－ＰＡＧＥ电泳、Ｗｅｓｔｅｒｎｂｌｏｔ分析和３Ｈ－ＴｄＲ掺入法测生物学活性。结果病毒滴度最高为１．４×１０５ＣＦＵ／ｍｌ;免疫荧光染色法和ＳＤＳ－ＰＡＧＥ电泳、Ｗｅｓｔｅｒｎｂｌｏｔ证实ＰＤＧＦ－ＡＡ的表达;抗性细胞条件培养基的细胞活性高达５１．２Ｕ／（１０６·ｄ）。结论供转导ＰＤＧＦ－Ａ基因的高滴度逆转录病毒得以制备,该基因获得有效表达,表达产物具有明显的致丝裂活性。