甲状旁腺激素对大鼠骨髓间充质干细胞成脂分化的影响

发布时间：2018-11-16 18:40

【摘要】：目的:1.大鼠骨髓间充质干细胞的分离、培养,观察其形态特征及鉴定表面标志物。2.对大鼠骨髓间充质干细胞成脂分化和成骨分化进行研究。3.观察不同浓度PTH1-34对大鼠骨髓间充质干细胞成脂分化的影响,探讨其可能机制。方法:1.采用贴壁法分离培养大鼠骨髓间充质干细胞(BMSCs),观察其形态、细胞生长曲线,式细胞仪检测表面标志物及细胞周期。2.诱导BMSCs定向分化为脂肪细胞和成骨细胞,并通过组织染色法鉴定分化后的细胞。3.以第三代BMSCs为体外试验模型,不同浓度甲状旁腺激素(PTH1-34)(0、10-10、10-9、10-8mol/L)分别作用于加入成脂诱导液的BMSCs,14天后采用ELISA法测定LPL活性,采用RT-PCR法测定ALP和PPARγ-2 m RNA表达水平,观察不同浓度PTH1-34对BMSCs成脂分化的影响。4.统计学方法采用统计学软件SPSS13.0软件进行分析,计量资料的表示用X±S,采用方差分析和LSD-t检验进行各组间比较,P0.05差异具有统计学意义。结果:1.BMSCs贴壁生长,均一长梭形,生长曲线为“S”型。细胞周期测定大部分处于G1/G0期。P3代BMSCs阳性表达CD44、CD29,阴性表达CD45。2.成脂诱导液诱导BMSCs分化21天后,油红O染色可见胞内脂滴出现,成骨诱导28天后,茜素红染色可见钙结节。3.不同浓度PTH1-34(10-10、10-9、10-8、mol/L)分别作用于BMSCs后,与未加入PTH1-34的对照组比较,LPL、PPARγ-2的表达量下降,ALP活性明显增加,并呈剂量依赖性,差异有统计学意义(P0.05)。结论:1.BMSCs形态为均一长梭形;稳定表达CD44、CD29,不表达CD45;BMSCs具有向脂肪细胞和成骨细胞分化的潜能。2.PTH1-34抑制BMSCs成脂分化,促进其成骨分化,并呈剂量依赖性。3.PTH1-34可能是通过下调PPARγ-2的表达量来抑制BMSCs向脂肪细胞分化。
[Abstract]:Objective: 1. Isolation and culture of rat bone marrow mesenchymal stem cells, observation of their morphological characteristics and identification of surface markers. 2. The adipogenic and osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) was studied. To observe the effects of different concentrations of PTH1-34 on adipogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and to explore its possible mechanism. Methods: 1. Bone marrow mesenchymal stem cells (BMSCs),) were isolated and cultured by adherent method to observe their morphology, cell growth curve, surface markers and cell cycle. 2. 2. BMSCs was induced to differentiate into adipocytes and osteoblasts. The third generation of BMSCs was used as the model in vitro. Different concentrations of parathyroid hormone (PTH1-34) (10 ~ (-10) 10 ~ (-9) (10 ~ (-8) mol 路L ~ (-1) were used to determine the activity of LPL by ELISA method after BMSCs,14 was added into the lipids. The expression levels of ALP and PPAR 纬 -2 m RNA were measured by RT-PCR assay, and the effects of different concentrations of PTH1-34 on the adipogenic differentiation of BMSCs were observed. 4. The statistical method was analyzed by SPSS13.0 software, X 卤S was used to express the measurement data, and the analysis of variance and LSD-t test were used to compare each group. The difference was statistically significant (P0.05). Results: 1.BMSCs adherent to the wall, uniform long spindle shape, the growth curve is "S" type. Most of the cell cycle measurements were in the G1/G0 phase. P3 generation BMSCs positive expression CD44,CD29, negative expression of CD45.2. After 21 days of BMSCs differentiation induced by lipopolysaccharide, lipid droplets could be seen in oil red O staining, calcium nodules could be seen in alizarin red staining after 28 days of osteogenesis induction. Compared with the control group without PTH1-34, the expression of LPL,PPAR 纬-2 decreased and the activity of ALP increased in a dose-dependent manner after different concentrations of PTH1-34 (10-10 ~ (-9) -10 ~ (-8) mol / L were treated with BMSCs. The difference was statistically significant (P0.05). Conclusion: the morphology of 1.BMSCs is uniform and fusiform, and stable expression of CD44,CD29, does not express CD45;. BMSCs has the potential to differentiate into adipocytes and osteoblasts. 2.PTH1-34 inhibits the adipogenic differentiation of BMSCs and promotes its osteogenic differentiation. In a dose-dependent manner, 3.PTH1-34 may inhibit the differentiation of BMSCs into adipocytes by down-regulating the expression of PPAR 纬 -2.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580

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