Cbfα1、PPARγ在骨髓间质干细胞定向分化中的作用及对骨质疏松症的影响的研究

目的研究转录因子Cbfα1、PPARγ对骨髓间质干细胞向成骨细胞和脂肪细胞分化的调控,探讨骨质疏松症的发病机制。方法塑料包埋椎骨组织,通过Goldner′s Massion Triehrome染色,通过骨量测量,分析骨密度正常组、低骨量组和骨质疏松组椎骨骨组织形态计量学参数的变化;采用免疫组织化学的方法,检测不同组成骨细胞中Cbfα1、PPARγ蛋白阳性表达率在三组中的差异及其与骨计量学参数的相关性。结果骨质疏松组与正常组相比,成骨细胞中Cbfα1蛋白的表达明显下调(P<0.05),与骨小梁间隔呈负相关,与骨小梁数目、骨小梁占全部骨小梁体积呈正相关;基质细胞中PPARγ蛋白的表达明显增强(P<0.05),与骨小梁间隔呈正相关,与骨小梁数目、骨小梁占全部骨小梁体积呈负相关。结论Cbfα1、PPARγ蛋白具有使骨髓间质干细胞分别向成骨细胞和脂肪细胞分化的潜能。两者表达水平的失衡导致成骨细胞生成减少,脂肪细胞生成增多,骨吸收增强,骨量丢失增加,这可能是骨质疏松发病的又一重要机制。     

Epigenetic changes in mesenchymal stem cells differentiation

Epigenetic factors are known to play a major role in determining stem cell fate and differentiation. Mesenchymal stem cells are the most studied population of stem cells due to their important applications in experimental biology and regenerative medicine. After a brief overview on mesenchymal stem cells, this review aims to highlight the role of epigenetic changes on mesenchymal stem cells biology and differentiation protocols with a focus on osteocytic, chondrocytic and adipocytic differentiation. Chromatin remodeling, DNA methylation, histone modifications and miRNA expression will be investigated. The impact of epigenetics on transdifferentiation of mesenchymal stem cells will also be discussed. Indeed, epigenetic modulation appears to constitute a promising experimental target in stem cell basic and translational research.     

不同浓度细胞松弛素D对人脂肪源干细胞分化能力的影响

目的　观察不同浓度细胞松弛素D（Cyto D）对人脂肪源干细胞（hASC）成骨分化和成脂分化能力的影响。 方法　在生长培养基、成脂诱导培养基及成骨诱导培养基中分别加入0.05、0.1、0.2 μg/ml的Cyto D处理hASC,干扰肌动蛋白的延长,并在处理的第1、4、7 d时分别进行油红O染色、ALP染色及CCK8等实验检测细胞性能的改变。 结果　7 d时实验组0.2 μg/ml的存活率相比于对照组下降了1/3。Cyto D能抑制hASCs的存活和增殖,且浓度越大细胞的存活率越低。单纯的Cyto D既能促使hASC胞内脂滴形成,又能改变细胞内成骨的基础状态。Cyto D与成脂诱导液联合使用后能极大的促进成脂,且不同的浓度具有不同的成脂效果,低浓度使脂滴数量增加,高浓度促进脂滴成熟;与成骨诱导液联合使用后Cyto D用药浓度和成骨效果成反比。 结论　细胞松弛素D能增强成骨或成脂诱导液效果,且低浓度细胞松弛素D能够促进成骨,低浓度和高浓度细胞松弛素D都能促进成脂。本实验探究了细胞松弛素D引起的肌动蛋白解聚对脂肪源干细胞的分化能力影响,为研究脂肪源干细胞的分化机制提供了重要生物学信息。     

Characterization of human adipose tissue‐derived stem cells with enhanced angiogenic and adipogenic properties

Autologous fat grafting is a popular method for soft tissue reconstructions but graft survival remains highly unpredictable. Supplementation of the graft with the stromal vascular fraction (SVF) or cultured adipose tissue‐derived stem cells (ASCs) can enhance graft viability. In this study we have examined the phenotypic properties of a selected population of cells isolated from ASCs, with a view to determining their suitability for transplantation into grafts. ASCs were isolated from the SVF of human abdominal fat (n  = 8 female patients) and CD146⁺ cells were selected using immunomagnetic beads. The angiogenic and adipogenic properties of the positively selected cells were compared with the negative fraction. CD146⁺ cells expressed the immunophenotypic characteristics of pericytes. With prolonged in vitro expansion, CD146^? cells exhibited increased population doubling times and morphological signs of senescence, whereas CD146⁺ cells did not. CD146⁺ cells expressed higher levels of the angiogenic molecules VEGF‐A, angiopoietin‐1 and FGF‐1. Conditioned medium taken from CD146⁺ cells significantly increased formation of in vitro endothelial cell tube networks, whereas CD146^? cells did not. CD146⁺ cells could be differentiated into adipocytes in greater numbers than CD146^? cells. Consistent with this, differentiated CD146⁺ cells expressed higher levels of the adipocyte markers adiponectin and leptin. These results suggest that CD146⁺ cells selected from a heterogeneous mix of ASCs have more favourable angiogenic and adipogenic properties, which might provide significant benefits for reconstructive and tissue‐engineering applications. Copyright © 2016 John Wiley & Sons, Ltd.     

Changes in UCP2, PPARγ2, and C/EBPα Gene Expression Induced by a Neuropeptide Y (NPY) Related Receptor Antagonist in Overweight Rats

Neuropeptide Y (NPY), a peptide released by nervous cells, appears to contribute to adiposity regulation by increasing food intake and inhibiting lipolysis. New NPY receptor related antagonists such as S.A.0204 are being developed as potential anti-obesity drugs affecting adipocyte lipid metabolism and thermogenesis. In this sense, those animals fed on a high-energy yielding (cafeteria) diet decreased body fat weight as compared to overweight controls, when they were administered with S.A.0204, and increased body temperature, which statistically correlated with high UCP2 mRNA expression levels in white adipose tissue. In addition, the in vivo NPY-antagonist administration was able to prevent white adipose tissue growth in animals fed the cafeteria (high-fat) diet by impairing PPARγ and C/EBPα mRNA expression in white fat cells. In summary, this novel NPY related-antagonist S.A.0204 may regulate body fat deposition by affecting both energy dissipation and white adipose tissue deposition, representing a potential new pharmacological strategy for obesity management.     

人血小板裂解液对骨髓间质干细胞成骨成脂分化的影响

目的探讨人血小板裂解液(HPL)体外培养人骨髓间质干细胞(MSCs)及其对MSCs成骨和成脂分化能力的影响。方法采用贴壁法分离培养3株人骨髓来源MSCs,每株细胞在培养时平行分为2个实验组,分别用含10%胎牛血清的低糖培养基和含7.5%HPL的低糖培养基培养,均培养MSCs至第5代,分别用成骨及成脂诱导液对每组MSCs做成骨、成脂分化诱导,诱导成功后分别用茜素红、油红O对成骨和成脂分化结果染色鉴定,并进一步对成骨、成脂诱导结果做定量分析:成骨分化染色后洗脱茜素红在562nm波长下测定OD值并对每个培养孔细胞做蛋白定量,以茜素红OD值与蛋白定量值的比值作为成骨分化的定量值;成脂分化染色后用异丙醇洗脱油红O,在510nm波长下测定OD值,以之作为成脂分化的定量值。对所得的结果做统计学分析。结果对第5代的MSCs做成骨诱导12d后,光镜下可见致密结节形成,茜素红染色及定量鉴定结果显示2种培养液培养的MSCs均能成功向成骨细胞分化,而7.5%HPL培养的MSCs成骨能力比10%FBS培养的MSCs强,定量鉴定结果分别为16.548±4.397、4.151±5.631(P<0.05)。成脂诱导12d后,细胞内有明显脂滴形成,油红O染色及定量分析结果显示MSCs成功向脂肪细胞分化,但7.5%HPL培养的MSCs成脂能力与10%FBS培养的MSCs相比较弱,定量鉴定结果分别为0.239±0.030、0.497±0.105(P<0.05)。结论 HPL可以促进MSCs成骨分化,抑制其成脂分化。     

Constituents from Terminalia species increase PPARα and PPARγ levels and stimulate glucose uptake without enhancing adipocyte differentiation

Ethnopharmacological relevance

The fruits of Terminalia bellerica Roxb. (Combretaceae) and T. chebula Retz. (Combretaceae) are important components of triphala, a popular Ayurvedic formulation, for treating diabetes in Indian traditional medicine.

Aim of the study

The aim of this study was to evaluate the effects of the constituents of T. bellerica and T. chebula fruit extracts on PPARα and PPARγ signaling/expression, cellular glucose uptake and adipogenesis.

Materials and methods

PPARα and PPARγ signaling and expression (luciferase assay and western blot) and the insulin-stimulated uptake of 2-NBDG were determined in HepG2 cells. The effects on adipogenesis were determined in 3T3-L1 cells by Oil red O staining and measurement of lipid content by AdipoRed reagent.

Results

Out of the 20 compounds, two ellagitannins, chebulagic acid (1) and corilagin (2), and three gallotannins, 2,3,6-tri-O-galloyl-β-d-glucose (3), 1,2,3,6-tetra-O-galloyl-β-d-glucose (4), and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (5), showed the enhancement of PPARα and/or PPARγ signaling. Two of the gallotannins (4 and 5) also increased PPARα and PPARγ protein expression, while all three (3–5) enhanced insulin-stimulated glucose uptake into HepG2 cells. Compound 1,2,3,6-tetra-O-galloyl-β-d-glucose (4) was the most potent in increasing cellular glucose uptake (9.92-fold increase at 50 μM). In the test for adipogenesis, 3–5 did not enhance the differentiation of 3T3-L1 preadipocytes but inhibited the adipogenic effect of rosiglitazone.

Conclusion

Three gallotannins (3–5) from Terminalia fruits acting as enhancers of both PPARα and PPARγ signaling increased insulin-stimulated glucose uptake without inducing the adipogenesis, with 1,2,3,6-tetra-O-galloyl-β-d-glucose (4) being the most effective in stimulating glucose uptake and 1,2,3,4,6-penta-O-galloyl-β-d-glucose (5) being most effective in increasing PPAR protein expression.     

去势大鼠骨量丢失过程中骨髓间充质干细胞的增殖分化功能

目的: 研究骨髓间充质干细胞(BMSCs)增殖分化功能在去势大鼠骨质疏松发病过程中对骨量丢失的作用。方法: 选用10周龄健康雌性SD大鼠行双侧卵巢切除术(OVX),建立骨质疏松症(OP)的动物模型;选用同一批次、周龄相同、体重相近的健康雌性SD大鼠行双侧卵巢附近脂肪组织部分切除术,建立假手术(sham)组,假手术大鼠组(sham group)。采用离心法、贴壁法和有限稀释法分离、培养、纯化大鼠BMSCs,体外培养传至3~4代后用于实验:流式细胞术进行BMSCs表型鉴定;克隆形成实验检测BMSCs增殖状况;MTT法测定BMSCs生长曲线;成脂诱导后脂滴油红O染色法检测比较2组大鼠BMSCs成脂能力;成骨诱导后钙化结节茜素红染色法检测比较2组大鼠BMSCs成骨能力;RT-PCR法检测大鼠BMSCs成骨相关蛋白Runx2、骨钙素(OCN)和骨桥蛋白(OPN)mRNA的表达。结果: 与sham组大鼠BMSCs相比,OVX组大鼠BMSCs克隆形成能力减弱,增殖能力降低,成脂向分化增强,成骨向分化减弱(P<0.05)。结论: 去卵巢骨质疏松大鼠BMSCs增殖及成骨分化减弱,成脂分化增强;这导致去势大鼠快速的骨量丢失,在去势大鼠OP发病过程中发挥着重要作用。     

分泌型卷曲相关蛋白/β-联蛋白信号通路对脂肪形成的影响

目的:探讨分泌型卷曲相关蛋白(sFRP5)和经典Wnt信号通路在脂肪细胞形成中的作用及机制。方法:诱导3T3-L1前体脂肪细胞分化,用过表达sFRP5的携带绿色荧光蛋白基因的重组腺病毒(Ad-sFRP5-GFP)感染3T3-L1细胞并诱导成熟,利用实时荧光定量PCR检测经典Wnt下游靶基因和脂肪分化相关基因mRNA水平的变化。3T3-L1细胞分化成熟后,进行油红染色,观察过表达sFRP5对脂滴形成的影响;提取核浆蛋白,用蛋白质印迹法检测sFRP5对β-联蛋白核转位的影响。结果:3T3-L1细胞分化成熟后期,sFRP5、CCAAT增强子结合蛋白α(C/EBPα)、过氧化物酶体增殖物活化受体γ2(PPARγ2)表达均显著增加,同时,细胞周期蛋白D1表达显著下调。而诱导分化成熟后,过表达sFRP5的3T3-L1细胞周期蛋白D1无显著改变。与空载对照组相比,过表达sFRP5的3T3-L1细胞脂滴形成无明显变化。给予3T3-L1细胞重组小鼠sFRP5直接刺激或感染Ad-sFRP5-GFP,均未明显改变β-联蛋白核转位。结论:sFRP5的表达随着脂肪细胞分化而显著增加,但不影响体外脂肪细胞分化,sFRP5在体外不直接通过拮抗Wnt/β-联信号通路的方式发挥作用。