骨髓脂肪细胞对成骨细胞和破骨细胞功能的影响

发布时间：2018-05-04 00:38

本文选题：BMSCs + 脂肪细胞　；参考：《石河子大学》2016年硕士论文

【摘要】：目的观察不同时期骨髓间充质干细胞来源的脂肪细胞内分泌功能的差异;采用细胞共培养观察不同大小脂肪细胞对成骨和破骨细胞诱导分化的影响;观察不同浓度脂肪酸对成骨细胞诱导分化的影响。方法1.各型细胞诱导分化及鉴定:选取4周龄Wistar雄性大鼠,分离骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs),分别向脂肪细胞和成骨细胞方向诱导分化;油红O染色鉴定成熟脂肪细胞,茜素红染色鉴定成骨细胞;收集Wistar大鼠全骨髓液,诱导分化为破骨细胞,抗酒石酸酸性磷酸酶染色鉴定。2.BMSCs来源的脂肪细胞与成骨细胞、破骨细胞共培养:1)BMSCs来源的脂肪细胞成熟后继续诱导分化,每隔2 d收集一次脂肪细胞培养液上清,-20℃冻存(采样时间点:21 d、24 d、27 d、30 d、33 d、36 d、39 d、42 d、45d、48 d、50d),所有样本收集完成后监测炎症细胞因子;同时观察脂肪细胞体积和数量,细胞体积用标尺测量,细胞数量在400×显微镜下计数6个视野,取平均值。综合以上监测数据,确定共培养的时间点。2)脂肪细胞诱导剂诱导P3代BMSCs向脂肪细胞方向分化,分别诱导到第21 d和40 d进行共培养。3)非直接接触共培养:更换脂肪培养基为脂肪维持培养基,每隔一天分别将脂肪细胞培养皿中的培养基吸出并在其中添加成骨细胞诱导剂/破骨诱导剂,进行成骨细胞/破骨细胞的诱导。4)直接接触共培养:分别在培养皿中刮去约10 cm2的脂肪细胞,在其中接种新鲜的BMSCs,培养基更换为成骨细胞诱导剂加脂肪细胞维持培养液,向成骨细胞方诱导分化;将加有完全培养基的骨髓细胞于细胞培养箱孵育24小时,收集非贴壁细胞,分别在培养皿中刮去约10 cm2的脂肪细胞,在其中接种非贴壁细胞,培养基更换为破骨细胞诱导剂加脂肪细胞维持培养液,向破骨细胞方向诱导分化。5)培养基中炎症细胞因子及骨代谢因子表达水平的检测。运用ELISA方法检测炎症因子运用RT-PCR方法检测骨代谢因子。3.不同浓度脂肪酸对成骨细胞分化的影响:Wistar大鼠P3代BMSCs向成骨细胞诱导分化,在成骨诱导培养基中加入不同浓度(25m M、50 m M、100 m M和200 m M)油酸和棕榈酸,观察两种脂肪酸对成骨细胞分化的影响。结果1.Wistar大鼠BMSCs 15 d左右培养成功,传三代后其表面分子标志物CD29和CD44呈阳性。P3代BMSCs在脂肪诱导培养基作用下21 d分化为脂肪细胞,油红O染色阳性;在成骨诱导培养基作用下21 d分化为成骨细胞,茜素红染色阳性。Wistar大鼠全骨髓液在破骨细胞培养基作用下28 d分化为破骨细胞,抗酒石酸酸性磷酸酶染色阳性。2.脂肪细胞成熟后,继续诱导分化,第21 d脂肪细胞数量显著高于第40 d(P0.001),但体积显著低于第40 d脂肪细胞(P0.05)。随着诱导时间增加,脂肪细胞分泌TG、TNF-α及IL-6表达量呈上升趋势,诱导至40 d表达量均显著高于第21 d(P0.01),在40 d-50 d基本保持水平不变。3.在非直接接触和直接接触共培养过程中,诱导至21 d的脂肪细胞与成骨细胞共培养组钙结节数量比阳性对照组显著增加(P0.001),诱导至40 d的脂肪细胞与成骨细胞共培养组钙结节数量比阳性对照组显著减少(P0.05)。4.诱导至21 d脂肪细胞共培养组比诱导至40 d脂肪细胞共培养组成骨细胞的OPG表达量显著增高(P0.001),RANKL的表达量显著降低(P0.001)。5.在成骨诱导培养基中加入不同浓度油酸(25m M、50m M、100m M、200m M),BMSCs多数分化为脂肪细胞,且随着浓度的增加,脂肪细胞的数量和体积都增加,但未观察到有成骨细胞分化。不同浓度棕榈酸均对细胞有毒性作用,细胞死亡。200m M油酸可作为诱导剂直接诱导骨髓间充质干细胞分化为脂肪细胞,此方法诱导的脂肪细胞数量比传统方法显著增多(P0.01),并且体积显著增大(P0.01)。结论1.骨髓腔内小脂肪细胞与成骨细胞共培养,可上调OPG的表达,促进成骨细胞形成。2.骨髓腔内大脂肪细胞与成骨细胞共培养,可下调OPG的表达,抑制成骨细胞形成。
[Abstract]:Objective To observe the differences in the endocrine function of adipocyte derived from bone marrow mesenchymal stem cells in different periods; to observe the effect of different adipocytes on the induction of differentiation of osteoblasts and osteoclasts by cell co culture, and to observe the effect of different concentrations of fatty acids on the induced differentiation of osteoblasts. Method 1. the differentiation and identification of different types of cells were induced. 4 weeks old Wistar male rats were selected to separate bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) and differentiate into adipocytes and osteoblasts respectively. Oil red O staining was used to identify mature adipocytes and alizarin red staining was used to identify osteoblasts, and the whole bone marrow fluid of Wistar rats was collected to differentiate into osteoclast and anti alcohol. Stone acid acid phosphatase staining identified.2.BMSCs derived adipocytes and osteoblasts, osteoclasts and osteoclasts co culture: 1) BMSCs derived adipocytes continued to induce differentiation after maturation, and collected a fat cell culture supernatant every 2 D intervals, and -20 C (sampling time points: 21 d, 24 D, 27 D, 30 d, 33 D, 36 D, 39 D, 42 d, 48, 42 d, 48), all samples The volume and quantity of adipocyte were monitored at the same time, and the volume and quantity of adipocyte were observed. The cell volume was measured by a scale. The number of cells was counted in 6 fields under 400 x microscope. The average value was taken. The time point.2 of the co culture was determined by the comprehensive monitoring data. The direction of the adipocyte inducer was induced to differentiate into the adipocytes in the P3 generation BMSCs, respectively. Induction of twenty-first D and 40 d for co culture.3) non direct contact co culture: replacement of fat medium as fat maintenance medium, sucking out culture medium in adipocyte culture dish every other day and adding osteoblast inducer / osteoclast inducer and inducing osteoblast / osteoclast induced.4) direct contact co culture. Do not scrape about 10 cm2 of adipocytes in a Petri dish, inoculate the fresh BMSCs, replace the medium with the osteoblast inducer and maintain the adipocyte culture medium, induce the differentiation to the osteoblast side, and incubate the bone marrow cells with the complete medium in the cell culture box for 24 hours, collect the non adherent cells, and scrape them in the culture dish. About 10 cm2 of adipocytes, in which non adherent cells were inoculated, the culture medium was replaced by osteoclast inducer and adipocyte maintenance medium, and the expression level of inflammatory cytokines and bone metabolic factors in the medium of osteoclast induced differentiation.5) was detected. ELISA method was used to detect the inflammatory factors by using RT-PCR method to detect bone The effects of different concentrations of metabolic factor.3. on osteoblast differentiation: Wistar rats were induced to differentiate into osteoblasts by P3 generation BMSCs, adding different concentrations (25m M, 50 m M, 100 m M and 200 m M) with oleic acid and palmitic acid in the osteogenic induction medium. The effect of two fatty acids on osteoblast differentiation was observed. After three generations, the surface molecular markers CD29 and CD44 were positive.P3 generation BMSCs differentiated into adipocytes with 21 d under the action of fat induced medium, and the oil red O staining was positive. 21 d differentiated into osteoblasts under the action of osteogenic induction medium, and the whole bone marrow fluid of alizarin red staining positive.Wistar rats was used in osteoclast culture medium. The lower 28 d was differentiated into osteoclast, and the.2. adipocyte resistant to tartaric acid acid phosphatase stained positive adipocytes continued to induce differentiation. The number of twenty-first D adipocytes was significantly higher than fortieth D (P0.001), but the volume was significantly lower than fortieth D adipocytes (P0.05). As the induction time increased, the expression of TG, TNF- A and IL-6 increased in the adipocytes. The expression of the induced 40 d was significantly higher than that of twenty-first D (P0.01). In the course of non direct contact and direct contact co culture at 40 d-50 D, the number of calcium nodules in the co culture group of adipocytes and osteoblasts induced to 21 d increased significantly (P0.001), and the induction to 40 d of adipocytes and osteoblasts was in common. The number of calcium nodules in the culture group was significantly lower than that in the positive control group (P0.05).4. induced to 21 d adipocyte co culture group, the expression of OPG expression in the co culture of 40 d adipocytes was significantly higher (P0.001), and the expression of RANKL was significantly reduced (P0.001) in the osteogenic inducement medium with different concentrations of oleic acid (25m M, 50m) 200m M), the majority of BMSCs differentiated into adipocytes, and with the increase of concentration, the number and volume of adipocytes increased, but the osteoblast differentiation was not observed. The different concentrations of palmitic acid were toxic to the cells, and the cell death.200m M oleic acid could be used as an inducer to induce bone marrow mesenchymal stem cells to differentiate into adipocytes. The number of adipocytes induced by the method increased significantly (P0.01) and increased significantly (P0.01). Conclusion 1. the co culture of small fat cells in bone marrow cavity and osteoblast can increase the expression of OPG and promote the formation of.2. bone marrow cells and osteoblasts to co culture the osteoblasts, which can reduce the expression of OPG and inhibit the osteogenesis. Cell formation.

【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R589.2;R580

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