重组pcDNA3.1-hBMP-2转染兔脂肪干细胞体外诱导的成骨分化

背景：骨形态发生蛋白2具有很强的诱导干细胞成骨活性。 目的：构建人骨形态发生蛋白2基因真核表达载体,探讨其转染脂肪干细胞后的成骨效果。 方法：通过噬菌斑原位杂交筛选人混合细胞cDNA文库获得人骨形态发生蛋白2基因,与真核表达载体pcDNA3.1-连接,构建重组质粒pcDNA3.1-hBMP-2。利用脂质体Lipofectamine™ 2000分别介导人骨形态发生蛋白2 基因、EGFP基因转染第4代脂肪干细胞,并经G418进行筛选。 结果与结论：酶切鉴定及DNA测序结果证实重组质粒pcDNA3.1-hBMP-2构建成功。经计算脂质体介导的脂肪干细胞瞬时转染率为(18.0±0.42)%,并经过G418筛选后获得了稳定转染的细胞。细胞生长曲线表明转染后对脂肪干细胞生长、增殖无明显影响。ELISA检测发现人骨形态发生蛋白2组的人骨形态发生蛋白2因子表达量均高于EGFP组及未转染组,且能够稳定表达。经人骨形态发生蛋白2基因转染的脂肪干细胞的Ⅰ型胶原含量、碱性磷酸酶活性以及钙结节数目均比EGFP组及未转染组有明显升高。     

Glucocorticoid‐induced differentiation of primary cultured bone marrow mesenchymal cells into adipocytes is antagonized by exogenous Runx2

Lin L, Dai S‐Dong, Fan G‐Yu. Glucocorticoid‐induced differentiation of primary cultured bone marrow mesenchymal cells into adipocytes is antagonized by exogenous Runx2. APMIS 2010; 118: 595–605. Long‐term clinical use of glucocorticoids often causes the serious side effect of non‐traumatic avascular osteonecrosis. The aim of this study was to examine the effects and mechanisms of a glucocorticoid, dexamethasone (Dex), on differentiation of primary cultured rat bone marrow mesenchymal cells (BMCs). We also tried to block the inhibitory effects of Dex on osteoblast differentiation. Adipocyte markers (peroxisome proliferator‐activated receptorγ‐2 and aP2) were increased in response to Dex treatment in a dose‐ and time‐dependent manner, while osteoblastic markers [Runx2, COL 1, osterix, alkaline phosphatase (ALP) and OC] were down‐regulated, consistent with ALP and osteocalcin promoter activity. To validate the effects of Runx2 on the expression of osteogenesis and adipocyte genes, pCMV/Flag‐Runx2 was transfected into BMCs, and relevant markers were detected after 10^?7 M Dex treatment for 48 h. The results indicated that Dex treatment induced adipogenic differentiation and suppressed proliferation. No significant difference was detected in expressions of these genes between Runx2‐transfected cells and Dex‐treated BMCs. These data suggest that Dex primarily induced adipocyte differentiation of BMCs. Exogenous Runx2 can antagonize the effect of Dex on osteoblast differentiation.     

resistin基因过表达影响3T3-L1脂肪细胞的脂质代谢

目的观察resistin基因过表达对3T3-L1脂肪细胞的脂质代谢、糖代谢的影响。方法构建大鼠resistin真核表达载体并转染3T3-L1前体脂肪细胞,获得稳定表达resistin基因的细胞株;采用油红O染色,观察脂肪细胞分化及脂质积聚情况;采用逆转录PCR技术,检测脂肪细胞分化标志基因及葡萄糖转运体4（glucose transporter4,GLUT4）基因表达变化;采用全自动生化仪比色法,检测脂肪细胞内甘油三酯（triglyceride,TG）、游离脂肪酸（free fatty acids,FFAs）的含量变化。结果（1）resistin基因过表达脂肪细胞中,脂滴出现时间提前,且细胞内布满了小而多的圆形脂滴;（2）resistin基因过表达脂肪细胞中,分化中、晚期标志基因C/EBPα、FAS的mRNA表达水平明显上调,分化早期标志基因Pref-1的表达则明显下调;（3）re-sistin基因过表达脂肪细胞中,胞质内TG、FFAs含量均显著增加;（4）resistin基因过表达脂肪细胞中,分化第2、4、8d的GLUT4基因mRNA表达水平间无显著变化,与正常脂肪细胞中的表达水平差异也无统计学意义。结论resistin基因过表达能够显著干扰3T3-L1脂肪细胞的脂质代谢,有助于肥胖和胰岛素抵抗的发生,而并不影响GLUT4基因的表达。     

肿瘤坏死因子α干预小鼠成骨细胞cbfa1/runx2基因的表达

背景：肿瘤坏死因子α可降低牙周膜纤维细胞碱性磷酸酶的活性,抑制牙周膜纤维细胞向成骨细胞的功能转化。 目的：观察肿瘤坏死因子α对小鼠成骨细胞生长及cbfa1/runx2基因表达的影响。 方法：取生长良好的小鼠成骨细胞系MC3T3/E1细胞,分别以20,40,60,80 μg/L的肿瘤坏死因子α进行干预,以正常培养的细胞作为对照。采用RT-PCR法检测MC3T3/E1细胞cbfa1/runx2 mRNA的表达;PNPP法测定碱性磷酸酶活性;MTT法检测细胞活力。 结果与结论：正常培养的MC3T3/E1细胞cbfa1/runx2 mRNA呈阳性表达,随着肿瘤坏死因子α浓度的增高,其表达水平逐渐下降。同时MC3T3/E1细胞活力和碱性磷酸酶活性也随肿瘤坏死因子α浓度的增高而下降。提示肿瘤坏死因子α可抑制MC3T3/E1细胞生长,而cbfa1/runx2可能参与了成骨细胞的分化过程。     

Tet-On系统调控的人骨形态发生蛋白2基因的表达

目的 在骨髓间充质干细胞(BMSC)水平上构建调控表达人骨形态发生蛋白2(hBMP-2)基因的Tet-On系统,在低剂量强力霉素(DOX)诱导下测定其表达水平.方法 将hBMP-2的cDNA亚克隆至穿梭载体pTRE-Shuttle2,再定向克隆到腺病毒,构建重组的Adeno-X-hBMP-2载体后,将线性化后的Adeno-X-hBMP-2与携带Tet-On转录活化因子的腺病毒载体BD Adeno-X Tet-On virus Stock分别转染人胚肾293(HEK293)细胞扩增,再共转染小鼠BMSC.在DOX诱导下用RT-PCR检测hBMP-2 mRNA的表达,并用ELISA检测DOX诱导浓度与hBMP-2蛋白表达量之间的关系.结果 在BMSC水平上成功构建能诱导表达hBMP-2的Tet-On调控系统,RT-PCR可以检测到hBMP-2 mRNA显著表达;ELISA检测到浓度为0.01 mg/L的DOX,hBMP-2含量为(165.677±0.008)ng/L;高于该浓度时,hBMP-2分泌量对DOX有浓度依赖性(R2=0.892 4,P<0.05).结论 表达hBMP-2的Tet-On调控系统在低剂量DOX诱导后能够显著表达hBMP-2,并在DOX>0.01 mg/L时有浓度依赖性.     

Interleukin-11 induces osteoblast differentiation and acts synergistically with bone morphogenetic protein-2 in C3H10T1/2 cells.

Interleukin-11 (IL-11) is a pleiotropic cytokine that supports various types of hematopoietic cell growth and is involved in bone resorption. We report here the involvement of recombinant human IL-11 (rHuIL-11) in osteoblast differentiation in mouse mesenchymal progenitor cells, C3H10T1/2. rHuIL-11 alone increased alkaline phosphatase (ALP) activity and upregulated expression levels of osteocalcin (OC), bone sialo protein (BSP), and parathyroid hormone receptor (PTHR) mRNA. rHuIL-11 had no effect on expression of type II collagen, peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), adipocyte fatty acid-binding protein P2 (aP2), and myogenic MyoD protein (MyoD). Recombinant human bone morphogenetic protein (rHuBMP)-2 increased ALP activity and mRNA expression of these genes except for MyoD. The expression patterns of ALP activity and osteoblast-specific or chondrocyte-specific genes suggest that rHuIL-11 may be involved in early differentiation of osteoblasts at a step earlier than that which is affected by rHuBMP-2. In support of this hypothesis, combined treatment with rHuIL-11 and rHuBMP-2 synergistically increased ALP activity and mRNA expression of OC and type II collagen, rHuIL-11 also abrogated the increased levels of PPAR-gamma2, aP2 mRNA caused by rHuBMP-2. Our results suggest that rHuIL-11 alone and in combination with rHuBMP-2 can induce osteoblastic differentiation of progenitor cells and plays an important role in osteogenesis.     

hBMP-2基因转染骨髓基质细胞及效果检测

目的　通过观察转染hBMP 2基因在骨髓基质细胞生物学性状变化和检测hBMP 2基因在受体细胞中表达水平 ,探讨骨髓基质细胞作为hBMP 2基因受体细胞的可行性。方法　应用脂质体介导的方法将携带hBMP 2基因的重组载体PcDNA3 hBMP2导入体外培养的骨髓基质细胞中 ,用G4 18筛选获得阳性细胞 ,分别应用PCR技术、原位杂交技术和ELISA检测目的基因DNA、mRNA和蛋白质的存在与表达状况 ,并绘制细胞的生长曲线。结果　PCR方法显示实验组可见约 36 0bp的基因条带 ,原位杂交方法显示实验组约有 15～ 2 0 %的阳性细胞 ,ELISA方法显示培养第 1～ 4天时hBMP 2的含量为 37ng/3× 10 5细胞 ,培养第 11～14天和第 2 5～ 2 8天均约 5 4ng/3× 10 5细胞。转染后阳性细胞的倍增时间和生长时间没有因为目的基因的导入而改变 ,与正常培养的细胞相比差异不显著。结论　骨髓基质细胞可以作为hBMP 2基因的受体细胞 ,基因表达可分泌hBMP 2蛋白质 ,可作为骨组织工程学中的种子细胞。     

SHP-2 positively regulates adipogenic differentiation in 3T3-L1 cells

The Src homology domain 2 (SH2)-containing tyrosine phosphatase SHP-2 has been implicated in the regulation of proliferation and differentiation in various cell types. Here, we investigated the ability of SHP-2 to mediate insulin-induced adipogenic differentiation of mouse 3T3-L1 cells. We found that the expression of SHP-2 was increased along with adipogenic differentiation. Overexpression of wild-type SHP-2 in 3T3-L1 cells resulted in enhanced adipocyte differentiation. Furthermore, insulin-stimulated adipogenic differentiation of 3T3-L1 cells was abolished by down-regulating SHP-2 expression using short interfering RNA. These results suggest that SHP-2 is a positive effector in signal transduction pathways necessary for adipocyte differentiation. In SHP-2 knockdown cells, the expression of peroxisome proliferator-activated receptor gamma, a master regulator of adipogenesis, was entirely suppressed even in the late phase of differentiation, whereas the expression level of C/EBPdelta was unchanged. These results highlight a novel role of SHP-2 in the signal transduction pathways regulating adipocyte differentiation.     

A Novel Semisynthetic Molecule Icaritin Stimulates Osteogenic Differentiation and Inhibits Adipogenesis of Mesenchymal Stem Cells

Background We previously reported that the constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common nuclear stem) exerted beneficial effects on the bone, including promoting bone formation and inhibiting bone marrow fat deposition. Recent in vivo study showed that Icaritin was a common metabolite of these constitutional flavonoid glycosides, indicating that Icaritin is a bioactive compound. The present study was designed to investigate whether Icaritin could promote osteogenic differentiation and suppress adipogenic differentiation of marrow mesenchymal stem cells (MSCs).Methods Primary MSCs were harvested from adult mice and exposed to Icaritin to evaluate whether it could promote osteogenesis and suppress adipogenesis using the following assays: determination of alkaline phosphatase (ALP) activity and mineralization; mRNA expression of osteogenic differentiation marker Runx2; osteocalcin and bone sialoprotein (BSP) by RT-PCR; quantification of adipocyte-like cells by Oil Red O staining assay and mRNA expression for adipogenic differentiation markers peroxisome proliferator-activated receptor gamma (PPARγ); adipocyte fatty acid binding protein (aP2) and lipoprotein lipase (LPL) by RT-PCR. For the underlying mechanism, glycogen synthase kinase-3beta (GSK3β) and β-catenin were also explored by western blotting.Results Icaritin promoted osteogenic differentiation and maturation of MSCs as indicated by increased mRNA expression for Runx2, osteocalcin and BSP, and enhanced ALP activity and mineralization; Icaritin inhibited adipogenic differentiation, as indicated by decreased mRNA expression for PPARγ, LPL, aP2, and suppressed formation of adipocyte-like cells; Icaritin inactivated GSK3β and suppressed PPARγ expression when promoting osteogenesis and suppressing adipogenesis of MSCs.Conclusion This was the first study demonstrating that the novel semisynthetic molecule Icaritin could stimulate osteogenic differentiation and inhibit adipogenesis of MSCs, which was associated with the suppression of GSK3β and PPARγ.     

吡格列酮对前脂肪细胞分化及GILZ蛋白表达的影响

目的:探讨吡格列酮在3T3-L1前脂肪细胞分化及糖皮质激素诱导亮氨酸拉链(GILZ)蛋白表达调节方面的作用。方法:形态观察3T3-L1细胞分化过程,不同浓度吡格列酮(1×10~(-4)mmol/L~1×10~(-2)mmol/L)处理细胞48 h,然后于分化的第2、4、6天用油红O染色测定细胞甘油三酯相对含量,实时荧光定量PCR法检测过氧化物增殖活化受体(PPAR)γ2和脂蛋白酶(LPL)的mRNA表达。不同浓度药物处理细胞48 h后,Western blot检测GILZ蛋白表达。结果:油红O法显示甘油三酯相对含量随药物处理浓度的增加而增加,与对照组比,1×10~(-3)mmol/L和1×10~(-2)mmol/L组甘油三酯含量显著性增加(P0.05)。实时荧光定量PCR检测显示PPARγ2、LPL的mRNA表达也是随着药物处理浓度的增加而增加。与对照组比,吡格列酮高于1×10~(-3)mmol/L浓度时,PPARγ2、LPL的mRNA表达显著性增加(P0.01)。Western blot显示GILZ蛋白表达随着药物处理浓度的增加而降低。结论:吡格列酮可下调GILZ表达,上调PPARγ2及下游LPL的表达。     

Use of a PPAR gamma-specific monoclonal antibody to demonstrate thiazolidinediones induce PPAR gamma receptor expression in vitro.

Troglitazone and rosiglitazone (BRL49653), members of the thiazolidinedione (TZD) class of antidiabetic drugs, are peroxisome proliferator-activated receptor gamma (PPARgamma) ligands that induce adipocyte differentiation and increase the expression of PPARgamma protein. Here, we report the characterization of a PPARgamma specific monoclonal antibody (MAb), PgammaA53.25, and its use to monitor PPARgamma expression in the noncommitted pluripotent murine mesenchymal stem cell line, C3H10T1/2, treated with TZDs. MAb PgammaA53.25 was raised against a region in the N-terminal domain of human PPARgamma shared by splice variants PPARgamma1 and PPARgamma2. It recognizes immunizing antigen in enzyme-linked immunoadsorbent assay (ELISA), and does not cross-react with the N-terminal domains of PPARalpha or PPARdelta. In Western blotting, PgammaA53.25 reacts with the immunizing antigen as well as distinct protein bands corresponding to the molecular weight of full length PPARgamma from C3H10T1/2 cells and rat tissue lysates. In fluorescent microscopy, PgammaA53.25 immunostains nuclei of C3H10T1/2 cells treated with PPARgamma ligands. The fluorescence intensity of the treated cells is TZD dose-dependent, and correlates with lipid accumulation consistent with adipogenesis. Based on these results, we propose that MAb PgammaA53.25 will be a useful tool for elucidating the role of PPARgamma in fatty acid metabolism and adipocyte differentiation.     

PcDNA3-hBMP2转染骨髓基质细胞及其稳定表达

目的：通过观察转染hBMP-2基因的骨髓基质细胞的生长特性的变化和检测目的基因在受体细胞中表达，探讨骨髓基质细胞用作hBMP-2基因的受体细胞的可能性。方法：在脂质体介导下将hBMP-2基因导入体外培养的骨髓基质细胞中，用G418筛选获得阳性细胞，分别应用原位杂交技术和酶联免疫吸附方法检测目的基因在受体细胞内的存在与表达。结果：在mRNA水平可检测到目的基因在阳性细胞中进行了转录，在培养基中，用ELISA的方法能检测到目的基因在骨髓基质细胞中进行了表达，并分泌到培养基中。从细胞的生长曲线上，可知细胞的倍增时间并没有因为目的基因的导入而改变，其生长时间也与正常的细胞相近。结论：骨髓基质细胞可以用作hBMP-2基因的受体细胞，表达并分泌hBMP-2蛋白质，也可作为骨组织工程学中的种子细胞。     

Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro.

Bone morphogenetic protein-2 (BMP-2) delivered in a suitable implantable matrix has the potential to repair local skeletal defects by inducing new bone formation from undifferentiated pluripotent stem cells resident in host tissue. In this study, we examined in vitro the potential of a derivatized hyaluronic acid (Hyaff-11) scaffold as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) in bone and cartilage repair therapies. Hyaff-11 scaffolds were fabricated using a phase inversion/particulate leaching method and soak-loaded with rhBMP-2. In vitro release kinetics of rhBMP-2, demonstrated using enzyme-linked immunosorbant assay and alkaline phosphatase (ALP) assay revealed a slow, sustained rhBMP-2 release during 28 days, with a cumulative release of 31.82% of the initial rhBMP-2 loaded. rhBMP-2 was released in bioactive form as demonstrated by ALP induction of pluripotent cell line, C3H10T1/2 (T1/2), down the osteoblast lineage when incubated with the release supernatants. rhBMP-2 retention in Hyaff-11 scaffolds was greater than that from collagen gels, which released most of the initially loaded rhBMP-2 by 14 days. rhBMP-2-loaded Hyaff-11 scaffolds were also seeded with T1/2 cells and evaluated at 3, 7, 14, and 28 days for viability and expression of osteoblast phenotype. Cells remained viable throughout the study and expressed a time- and dose-dependent ALP and osteocalcin expression in the rhBMP-2 groups. Based on these observations, Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBMP-2, release low levels of bioactive rhBMP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.     

INSIG2稳定表达细胞系的建立及该基因对脂肪代谢的影响

目的 建立胰岛紊诱导基因2(INSIG2)稳定表达细胞系,观察过表达INSIG2后对脂肪代谢的影响.方法 构建INSIG2真核表达质粒,经脂质体转染313-L1细胞,RT-PCR、免疫细胞化学法鉴定INSIG2阳性细胞株及检测胰岛素诱导基因1(INSIG1)、脂肪酸合成酶(FAS)mRNA表达;ELISA检测细胞培养液中游离脂肪酸(FFA)的含量;油红"O"染色检测脂肪细胞的分化.结果 pcDNA3.1( )-INSIG2成功转染3T3-L1细胞,过表达INSIG2后,INSIG1、FAS mRNA表达均下调,细胞培养液中游离脂肪酸含量降低,脂肪细胞分化明显抑制.结论 获得了INSIG2稳定表达细胞系,过表达INSIG2对脂肪代谢具有抑制作用.     

以PPARγ2为靶点的传统中药细胞学筛选方法的建立和应用

目的构建含人PPARγ2基因启动子的荧光素酶表达质粒,通过建立稳定转染该质粒的细胞系,建立传统中药细胞学筛选方法。方法首先以p GL3-Basic-Luc和p GL3-Enhancer-Luc为载体构建含不同长度人PPARγ2基因启动子序列的荧光素酶表达质粒。脂质体转染,建立稳定转染上述质粒的3T3-L1细胞系。选取红花和茜草等28种可能具有减肥作用的中药制备成中药水溶性提取物(ECMH),观察不同浓度ECMH对3T3-L1细胞中荧光素酶表达的影响。结果 p GL3-Enhancer-PPARγ2 625 bp-Luc质粒稳定转染的细胞荧光素酶表达最高,是本底的200倍左右。故采用此细胞系进行传统中药筛选,红花ECMH在10、100和1 000μg/m L时均可使3T3-L1细胞中荧光素酶的表达明显增加,分别为对照组的1.63倍、1.90倍和2.30倍(P0.05)。在10~1 000μg/m L荷叶、茜草ECMH也均能促进3T3-L1细胞中荧光素酶的表达,在浓度为1 000μg/m L时荷叶和茜草对3T3-L1细胞中荧光素酶的表达作用最强,分别为对照组的2.03倍(P0.01)和2.00倍(P0.01)。结论成功构建p GL3-Enhancer-PPARγ2625 bp-Luc质粒,并建立稳定转染的3T3-L1细胞系。红花、荷叶和茜草的ECMH能明显促进3T3-L1细胞中人PPARγ2基因启动子的活性,它们可能成为未来潜在的减肥药物。     

Stochastic Modeling of Adipogenesis in 3T3-L1 Cultures to Determine Probabilities of Events in the Cell’s Life Cycle

3T3-L1 preadipocytes are often being used in research of adipose-related diseases such as obesity, insulin resistance, and hyperlipidemia. We developed a stochastic model that simulated differentiation of four 3T3-L1 culture conditions distinct by the insulin concentration in the differentiation medium (2.5, 5, 7.5, or 10 μg/mL). The model simulated culture behavior and the accumulation of lipid droplets in the maturing cells from the day of induction of differentiation through 28 days after that. The cellular processes including cell adhesion, mitosis, growing after undergoing mitosis, commitment to the adipocyte lineage, and apoptosis were referred to as stochastic events in the modeling. By minimizing the error between our model and experimental results, we found that the probability for becoming committed to the adipocyte lineage in a single division and the probability for growing after undergoing mitosis were 0.02 and 0.8, respectively, regardless of the insulin concentration. The probability for undergoing mitosis was equal to 0.2 and 0.4 in cultures that had insulin concentrations of 2.5 and 5–10 μg/mL in the differentiation medium, respectively; hence the insulin concentration affected the probability for mitosis in the 3T3-L1 cells. The model and resulted probabilities now allow quantitative and visual predictions of adipogenesis in 3T3-L1 cultures, toward computational design of cell culturing protocols.     

Generation of novel adipocyte monolayer cultures from embryonic stem cells

Here we show a simplified and improved method to produce large quantities of evenly distributed monolayer cultures that display major characteristics of adipocytes. These cultures are applicable for quantitative analysis for biochemical and molecular events in adipogenesis during development and may provide a useful system for high-throughput drug screening assays of antiobesity drugs. In our method, we treated embryoid bodies (EBs) with all-trans retinoic acid (ATRA) for 3 days, 1 day after they attached to the gelatin-coated culture plates without further transfer. The cells were maintained in insulin and trioiodothyronine (T(3))-containing medium until day 12, when they were dispersed by enzymatic digestion and replated onto multiple culture plates. Two days later, adipocyte induction factors were added for 6 days and examined 6 days later. The amount of lipid droplet-laden adipocytes in the culture reached approximately 80%, with a nearly five-fold increase in GPDH activity. The cells expressed high levels of adipose-specific proteins (adipocyte markers), including PPARgamma2, ALBP, LPL, HSL, perilipin, and DGAT1. The adipocytes are functionally active, as evidenced by their response to lipolytic agents, such as forskolin, Bt2-cAMP, and isoproterenol, with more than 20-fold increases in glycerol release.