骨髓脂肪细胞向成骨细胞的转分化

背景：骨髓脂肪细胞、成骨细胞共同来源于骨髓基质细胞,二者存在基因同源性,在一定的条件下可以相互转分化。 目的：观察骨髓细胞源性脂肪细胞在成骨诱导分化培养条件下转分化为成骨细胞的活性,探索股骨头坏死细胞水平治疗的新途径。 方法：将前脂肪细胞3T3-L1分别进行成骨诱导培养和成脂诱导培养。培养后不同时间点观察细胞形态的变化;并于诱导培养后5,21 d分别进行实时定量-聚合酶链反应检测成骨、成脂分化过程中,细胞中Runt相关基因转录因子2、氧化物增殖体激活物受体γ2、骨钙素和Ⅰ型胶原mRNA表达。并于成骨、成脂培养21 d后采用Wertern-blot法检测相关蛋白的表达。培养细胞爬片,分别进行碱性磷酸酶、钙结节茜素红、油红O染色,观察3T3-L1成骨转分化情况以及成骨细胞活性表达。 结果与结论：3T3-L1在成骨诱导培养5 d后,细胞由圆形逐渐演变成纺锤形和梭形;实时定量-聚合酶链反应检测结果与对照组相比,氧化物增殖体激活物受体γ2 mRNA表达减弱,而Runt相关基因转录因子2、骨钙素和Ⅰ型胶原mRNA表达微量增强。至21 d时,这种表达改变更加明显;Western-blot显示,Runt相关基因转录因子2、骨钙素和Ⅰ型胶原蛋白量增加,而氧化物增殖体激活物受体γ2微量甚至无表达;碱性磷酸酶染色阳性表达较多,茜素红钙结节染色可见多个散在分布的钙结节;油红O染色微量脂滴。提示小鼠骨髓基质细胞源性前脂肪细胞3T3-L1在成骨诱导培养下,可在一定程度上转分化为有生物活性的成骨细胞;小鼠骨髓基质细胞的脂肪细胞和成骨细胞二者之间存在着可塑性。     

miR-202通过抑制PGC1β表达促进3T3-L1前脂肪细胞分化

目的观察miR-202对3T3-L1前脂肪细胞分化的影响及可能的机制。方法通过慢病毒感染构建稳定表达AMO-miR-202和乱序对照miRNA细胞系,随后诱导分化。至分化的第9天,油红O染色观察细胞内脂滴的情况;RT-PCR检测过氧化物酶体激活增殖受体γ2(PPARγ2)和a P2的基因表达;Western blot检测PPARγ2、a P2和miR-202靶基因PPARγ辅助活化因子1β(PGC1β)蛋白表达。结果经293T细胞慢病毒包装AMO-miR-202、乱序对照miRNA,荧光显微镜下可见约80%~90%荧光细胞;将上述2组病毒液分别感染3T3-L1前脂肪细胞后,可见约70%~80%荧光细胞。AMO-miR-202组细胞内脂滴及PPARγ2和a P2的mRNA表达显著低于乱序对照组和对照组(P<0.05)。与乱序对照组和对照组相比,AMO-miR-202组PGC1β蛋白表达显著增加(P<0.05),PPARγ2和a P2蛋白表达显著降低(P<0.01),而乱序对照组和脂肪细胞组上述指标无明显差异。结论 miR-202可能通过抑制PGC1β、提高PPARγ2和a P2的表达促进3T3-L1前脂肪细胞分化。     

无血清原代培养人前脂肪细胞并诱导分化

目的无血清原代培养人前脂肪细胞并诱导其分化为成熟脂肪细胞。方法采用胶原酶消化法分离并原代培养人前脂肪细胞,通过对细胞形态学的观察、油红O染色,以及脂肪细胞标志性酶G-3-PDH活性的测定对细胞进行鉴定。结果摸索出无血清培养并诱导人前脂肪细胞分化为成熟脂肪细胞的条件。在无血清的基础培养基中前脂肪细胞能够维持增殖。无血清分化培养基培养4d后细胞形态逐渐变圆,并出现球性脂滴,脂滴的数量逐渐增多至分化培养的第21天到达顶峰。结论在无血清培养的状态下成功诱导前脂肪细胞向脂肪细胞的分化,以作为激素或细胞因子对前脂肪细胞增殖或分化影响的研究基础。     

甘露聚糖结合凝集素(MBL)对3T3-L1前脂肪细胞成脂分化的调节作用及机制

目的研究甘露聚糖结合凝集素(mannan-binding lectin,MBL)对3T3-L1前脂肪细胞成脂分化的调节及其机制。方法体外诱导3T3-L1前脂肪细胞成脂分化,同时给予不同浓度的MBL(0、1、10、20μg/ml)干预。CCK-8法检测细胞增殖能力变化,油红O染色和细胞内甘油三酯含量测定法分析脂质积累情况。Western blot及qRT-PCR检测脂肪细胞成脂分化相关因子PPARγ及C/EBPα的蛋白质及mRNA表达水平。Western blot分析脂肪合成调控信号分子Akt的表达及磷酸化。结果实验组各浓度MBL(0、1、10、20μg/ml)对3T3-L1前脂肪细胞增殖都无影响。3T3-L1前脂肪细胞诱导分化3 d,甘油三酯检测发现MBL处理组细胞内甘油三酯水平下降,并呈剂量依赖关系;油红O染色结果进一步显示,MBL处理组的脂滴数量显著减少,吸光度值也显著降低,同样呈现浓度依赖关系。Western blot及qRT-PCR检测结果证实,MBL处理组PPARγ和C/EBPα的蛋白质及mRNA表达水平均显著下降,呈剂量依赖性。在MBL干预下,Akt的磷酸化水平也明显下调。结论MBL通过Akt信号通路调控3T3-L1前脂肪细胞的成脂分化。     

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。     

脂肪细胞在肝细胞胰岛素耐受过程中的作用

目的研究脂肪细胞在不同分化阶段对肝细胞胰岛素抵抗的影响。方法体外诱导3T3-L1脂肪前体细胞分化,细胞内脂滴增加,逐步分化成脂肪细胞。采用不同分化阶段脂肪细胞(未分化0d、中期分化4d、接近完全分化8d)与原代肝细胞共培养。Western印迹法检测共培养后肝细胞内胰岛素信号通路的反应性;葡萄糖同位素标记方法检测肝细胞糖原合成能力。结果以未共培养的肝细胞为对照组,共培养后肝细胞内胰岛素受体底物-2酪氨酸磷酸化(Tyr612)(pIRS-2)水平及Akt磷酸化(Ser473)(pAkt)水平均显著下调;肝糖原合成能力明显降低;与较成熟脂肪细胞共培养后,肝细胞pIRS-2及pAkt水平与其他分化阶段组共培养比较下调明显,肝糖原合成能力随着脂肪细胞的成熟而明显降低。结论脂肪细胞可能诱导肝细胞发生胰岛素抵抗,肝细胞胰岛素信号通路的阻滞程度与脂肪细胞的分化程度呈正相关。     

SSAO介导的氧化脱氨反应对3T3-L1脂肪细胞的氧化应激作用

目的:观察氨基脲敏感型胺氧化酶(SSAO)催化其底物甲胺(MA)和苯甲胺(BZA)的氧化脱氨反应对成熟3T3-L1脂肪细胞的氧化应激作用。方法:3T3-L1前脂肪细胞诱导分化为成熟脂肪细胞;高效液相色谱法检测不同分化天数下细胞SSAO活性的变化;MTT法检测不同浓度的MA或BZA对细胞活力的影响;荧光探针方法检测细胞在药物作用下产生的活性氧;以0.5 mmol/L MA或BZA处理成熟脂肪细胞或未经诱导分化的前脂肪细胞4 h,观察细胞产生的甲醛(FA)、苯甲醛(BZ)、脂质过氧化指标丙二醛(MDA)、总超氧化物歧化酶(T-SOD)和谷胱甘肽(GSH)的变化。结果:SSAO活性随细胞分化天数的增加而增加,并于第8天达到高峰;不同浓度MA或BZA作用细胞4 h对细胞活力无显著影响(P0.05);0.5 mmol/L MA或BZA孵育后,细胞产生的活性氧增高,约为阴性对照组的3~4倍,差异有统计学意义(P0.05);在成熟脂肪细胞中,MDA的含量增加,而T-SOD和GSH的活性和含量减少,与阴性对照组相比,差别有统计学意义(P0.05);MA和BZA作用未经诱导分化的前脂肪细胞,其MDA和SOD和GSH的变化不明显,差别无统计学意义(P0.05)。结论:SSAO可能通过其介导的氧化脱氨反应引起3T3-L1成熟脂肪细胞产生氧化应激。     

甘氨酸对3T3-L1脂肪细胞胰岛素抵抗的影响

目的：探讨甘氨酸对3T3-L1脂肪细胞胰岛素抵抗的影响及其机制。方法:诱导分化3T3-L1脂肪前体细胞为成熟的脂肪细胞，肿瘤坏死因子α（TNF-α）诱导建立脂肪细胞的胰岛素抵抗模型，以罗格列酮为阳性对照，观察甘氨酸干预后胰岛素受体底物-1（IRS-1）和过氧化物酶体增殖物激活受体γ（PPARγ）基因的表达。结果:正常对照组脂肪细胞IRS-1mRNA和PPARγ mRNA的表达最强；TNF-α组IRS-1mRNA和PPARγ mRNA表达显著低于正常对照组；TNF-α加罗格列酮组与TNF-α加甘氨酸组相似，IRS-1mRNA和PPARγ mRNA表达显著高于TNF-α组。结论:甘氨酸对TNF-α诱导的3T3-L1脂肪细胞胰岛素抵抗具有抑制作用，其机制与其增强IRS-1、PPARγ基因的表达有关。     

性激素对3T3-L1前脂肪细胞和脂肪细胞胰岛素敏感性的影响

目的观察不同浓度雌、雄激素对3T3-L1前脂肪细胞葡萄糖转运的影响,探讨性激素在胰岛素抵抗形成中的意义。方法体外培养3T3-L1前脂肪细胞,并诱导其分化成熟,利用2-脱氧-[3H]-D-葡萄糖掺入法,研究不同浓度的17β雌二醇、睾酮对胰岛素刺激的前脂肪细胞和脂肪细胞葡萄糖摄取能力的影响。结果10-8mol/L的17β雌二醇即能够抑制3T3-L1前脂肪细胞胰岛素刺激状态下的葡萄糖转运,且呈现明显的浓度依赖性抑制;而睾酮为10-8mol/L时3T3-L1前脂肪细胞胰岛素刺激状态下的葡萄糖转运并无明显影响,在浓度达到10-7mol/L开始出现抑制效应,浓度越高抑制效应越明显。结论性激素可以调节3T3-L1前脂肪细胞的胰岛素敏感性。     

干扰3T3-L1脂肪细胞生长激素受体对生长激素诱导的细胞炎症因子表达和分泌的影响

目的:探讨干扰3T3-L1脂肪细胞生长激素受体(GHR)对生长激素(GH)诱导的脂肪细胞核因子κB(NF-κB)激活及炎症细胞因子mRNA表达和分泌的影响。方法:采用RNA干扰技术抑制3T3-L1脂肪细胞中GHR的表达;Western blot检测GHR的蛋白表达;双萤光素酶报告基因系统分析GHR对GH激活的3T3-L1脂肪细胞NF-κB转录活性的影响;real-time RT-PCR和ELISA技术检测GHR对GH诱导的3T3-L1脂肪细胞炎症因子mRNA表达和分泌的影响。结果:干扰3T3-L1脂肪细胞GHR的表达能够显著抑制生长激素诱导的细胞NF-κB的激活,并减少TNF-α、IL-1β和IL-6等炎症细胞因子的mRNA表达和分泌。结论:干扰3T3-L1脂肪细胞GHR可抑制GH诱导的炎症细胞因子表达和分泌。     

3-Hydroxy-3-Methylglutaric Aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglecemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

3-Hydroxy-3-methylglutaric aciduria

3-Hydroxy-3-methylglutaric aciduria was found in a newborn infant whose parents are first cousins. The patient presented at 5 days of life with hyperammonemia, hypoglycemia, and metabolic acidosis. There was no ketonuria. Diagnosis was made by analysis of the pattern of organic acids excreted in the urine. A profound deficiency in activity of 3-hydroxy-3-methylglutaryl-coenzyme A lyase was found in cultured skin fibroblasts. The parents had intermediate levels of enzyme activity.     

Fetal thymocyte potential for T cell receptor Vγ3-Jγ1 junctional modification

Junctional modifications of T cell receptor (TcR) and immunoglobulin (Ig) gene joining regions provide great diversity to respective protein repertoires. The addition of non-germ-line-encoded nucleotides (N-regions) in the V-Jγ junction is one such modification which is developmentally regulated, rarely evident in the fetal animal, but common in the adult. A question has recently arisen as to whether developmentally patterned N-region additions in V-Jγ joins are a reflection of T cell progenitors which are committed to particular types of rearrangement prior to the event, or of changing environmental influences on uncommitted cell populations. To address this question with regard to theVγ3-Jγ1 join, T cells were examined in the fetal thymic organ culture (FTOC), a system with which the environment of early progenitor cells could be deliberately altered. At various times following FTOC initiation, cells were isolated for examination by the polymerase chain reaction, cloning and sequencing. Vγ3-Jγl sequences within genomic DNA as well as cDNA were evaluated. Data from these studies revealed frequent N-region additions within V-Jγ joins among day 14 fetal thymocyte populations, a situation dissimilar from that in vivo. Also dissimilar from the in vivo situation was the degree of exonuclease activity evident in FTOC. The canonical Vγ3-Jγl join (a frequent junction lacking N-region addition) was recognized in all experiments, but was least common among DNA versus cDNA sequences. Results illustrate that early progenitor cell populations are not programmed to exclude junctional modifications from Vγ3-Jγ1 joins.     

γδ cells involved in contact sensitivity preferentially rearrange the Vγ3 region and require interleukin-7

Ptak and Askenase showed that both αβ and γδ cells are required for transfer of contact sensitivity (CS). This study confirms that day 4 immune cells depleted of γδ cells fail to transfer CS to trinitrochlorobenzene (TNP-Cl) systemically and demonstrates that administration of anti-γδ monoclonal antibodies (mAb) in vivo abolishes the CS reaction. Moreover, γδ cells accumulate at the antigen challenge site: these cells have the unusual phenotype CD8α⁺, CD8β^-, IL-4 R⁺ which we suggest is due to their state of activation. Following immunization with contact sensitizer on the skin, the absolute number of γδ cells increases in the regional lymph nodes with a peak at 4 days. Of the γδ cells, 80%, both in the lymph nodes of TNP-Cl-immune mice and accumulating at the antigen challenge site are Vγ3⁺. The γδ cells expressing Vγ3, which is characteristic of dendritic epithelial T cells (DETC), obtained 4 days after sensitization, proliferate in response to interleukin (IL)-7, but only poorly to IL-2 and IL-4. They also respond to concanavalin A and immobilized anti-γδ mAb, but not to haptens or heat-shocked syngeneic spleen cells. Furthermore, injection of mice with mAb to IL-7 inhibits accumulation of Vγ3⁺ cells both in the lymph nodes after skin sensitization and at the antigen-challenge site. Altogether, these results strongly support the view that DETC are related to, or the original source of, the γδ cells found in the lymph node after skin sensitization and at the site of challenge, and that IL-7 is implicated in these phenomena.     

Hypoxia Increases Serum Amyloid A3 (SAA3) in Differentiated 3T3-L1 Adipocytes

Hypoxia has been implicated as a possible cause of adipose tissue inflammation. Furthermore, the acute phase protein serum amyloid A (SAA) has been associated with the modulation of the adipogenic process, and it is well-known that obese individuals have increased levels of SAA. The effect of hypoxia in the expression and production of SAA was examined in murine 3T3-L1 adipocytes. Hypoxia leads to a substantial increase in SAA3 mRNA and protein level, apparently in a time-dependent manner (threefold in 48 h), in fully differentiated 3T3-L1, followed by reestablishment of gene expression to basal levels after 24 h of reoxygenation. Hypoxia-induced SAA may be one of the key molecules to the development of the inflammatory response in adipose tissue.     

γδ cells regulate autoimmunity

γδ cells are attractive candidates for mediators of autoimmune disease. They can expand in germ-free mice, probably through recognition of autoantigens, and γδ-cell-deficient mice, unlike mice deficient in αβ T cells or B cells, show no severe defects in the immune response to foreign antigen challenge. A capacity of γδ cells to effect or regulate tissue damage is also plausible, given their ready localization to tissues, and their myriad of effector functions. Added to this, attempts to reconstruct the physiological course of autoimmune diseases with only autoreactive αβ T cells seem invariably to fall short for lack of other unidentified players. γδ cells and their putative ligands have been linked to autoimmune conditions, and recent experiments confirm that γδ cells play a significant role in autoimmune disease in vivo.     

Assembly of the TCR/CD3 complex: CD3ϵ/δ and CD3ϵ/γ dimers associate indistinctly with both TCRα and TCRβ chains. Evidence for a double TCR heterodimer model

The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3ϵ with either CD3γ or CD3δ. These dimers assemble with TCRα and TCRβ chains, and finally, the CD3ζ homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3ϵ/CD3γ dimer interacts exclusively with TCRβ and the CD3ϵ/CD3δ dimer with TCRα to form a complex with a single TCRα/β heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3ϵ/CD3γ and CD3ϵ/CD3δ dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCRα-negative T cell line MOLT-4 in which TCRβ is found separately associated with CD3ϵ/CD3γ and with CD3ϵ/CD3δ dimers. Indirectly, our results support a model of stoichiometry in which two TCRα/β heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3γ and anti-CD3δ antibodies from 1 % NP40 and 1 % Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3ζ homodimer. This suggests that CD3ζ mediates the interaction between both TCRα/β heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCRβ that impairs the association of CD3ζ. In this mutant cell line, unlike a control line with wild-type TCRβ, the CD3γ- and CD3δ-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-α/β heterodimers form two hemicomplexes containing either CD3ϵ/γ or CD3ϵ/δ dimers which become associated via the CD3ζ homodimer.