【摘要】：目的:1.大鼠骨髓間充質(zhì)干細胞的分離、培養(yǎng),觀察其形態(tài)特征及鑒定表面標志物。2.對大鼠骨髓間充質(zhì)干細胞成脂分化和成骨分化進行研究。3.觀察不同濃度PTH1-34對大鼠骨髓間充質(zhì)干細胞成脂分化的影響,探討其可能機制。方法:1.采用貼壁法分離培養(yǎng)大鼠骨髓間充質(zhì)干細胞(BMSCs),觀察其形態(tài)、細胞生長曲線,式細胞儀檢測表面標志物及細胞周期。2.誘導BMSCs定向分化為脂肪細胞和成骨細胞,并通過組織染色法鑒定分化后的細胞。3.以第三代BMSCs為體外試驗模型,不同濃度甲狀旁腺激素(PTH1-34)(0、10-10、10-9、10-8mol/L)分別作用于加入成脂誘導液的BMSCs,14天后采用ELISA法測定LPL活性,采用RT-PCR法測定ALP和PPARγ-2 m RNA表達水平,觀察不同濃度PTH1-34對BMSCs成脂分化的影響。4.統(tǒng)計學方法采用統(tǒng)計學軟件SPSS13.0軟件進行分析,計量資料的表示用X±S,采用方差分析和LSD-t檢驗進行各組間比較,P0.05差異具有統(tǒng)計學意義。結(jié)果:1.BMSCs貼壁生長,均一長梭形,生長曲線為“S”型。細胞周期測定大部分處于G1/G0期。P3代BMSCs陽性表達CD44、CD29,陰性表達CD45。2.成脂誘導液誘導BMSCs分化21天后,油紅O染色可見胞內(nèi)脂滴出現(xiàn),成骨誘導28天后,茜素紅染色可見鈣結(jié)節(jié)。3.不同濃度PTH1-34(10-10、10-9、10-8、mol/L)分別作用于BMSCs后,與未加入PTH1-34的對照組比較,LPL、PPARγ-2的表達量下降,ALP活性明顯增加,并呈劑量依賴性,差異有統(tǒng)計學意義(P0.05)。結(jié)論:1.BMSCs形態(tài)為均一長梭形;穩(wěn)定表達CD44、CD29,不表達CD45;BMSCs具有向脂肪細胞和成骨細胞分化的潛能。2.PTH1-34抑制BMSCs成脂分化,促進其成骨分化,并呈劑量依賴性。3.PTH1-34可能是通過下調(diào)PPARγ-2的表達量來抑制BMSCs向脂肪細胞分化。
[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 緯 -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 鹵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 緯-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 緯 -2.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R580

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1 唐雯菁;杜艷萍;洪維;李慧林;程群;;甲狀旁腺素對骨髓間充質(zhì)干細胞增殖、分化和旁分泌功能的影響[J];中華骨質(zhì)疏松和骨礦鹽疾病雜志;2014年01期

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