富血小板血浆诱导骨髓基质细胞碱性磷酸酶表达的研究

发布时间：2018-04-04 09:04

本文选题：富血小板血浆　切入点：骨髓基质细胞　出处：《青岛大学》2005年硕士论文

【摘要】：目的:体外观察不同浓度富血小板血浆(PRP)对自体骨髓基质细胞碱性磷酸酶活性和mRNA表达的影响。对PRP诱导骨髓基质细胞向成骨细胞分化的生物学效应进行初步评价。方法:抽取狗静脉血,采用改进Appel方法制备PPP和不同血小板浓度的PRP。取狗骨髓,于含15%胎牛血清的D-MEM-F12培养液中培养,经传代培养筛选,获得足够数量稳定的骨髓基质细胞(BMSCs)。实验分5组,分别加入诱导物(A组PPP+BMSCs,B组PRP+BMSCs,C组4倍PRP+BMSCs,D组rhBMP+BMSCs,E组为空白对照组),分别在第3、6、9、12天用酶动力学法检测碱性磷酸酶活性,用RT-PCR法在第6天检测碱性磷酸酶mRNA的表达,Von kossa染色鉴定成骨细胞。结果:(1)原代培养的骨髓基质细胞24h开始贴壁,12d后细胞融汇成单层,细胞多呈成纤维梭形细胞形态,经诱导液传代培养后,细胞形态渐变为方形、多角形。(2)酶动力学法测定各实验组细胞第3、6、9、12天检测碱性磷酸酶活性,经过配对t检验,各组第6、9、12天的ALP活性水平均较第3天明显升高,差异具有统计学意义(P0.05)。(3)用RT-PCR法检测第6天碱性磷酸酶mRNA的表达,结果显示PRP低浓度组ALPmRNA表达明显高于对照组。(4)Von kossa染色鉴定成骨细胞:细胞密集区出现大面积黑染区域,即钙结节。结论:一定浓度的PRP能诱导骨髓基质细胞碱性磷酸酶mRNA的表达增加,并提高细胞碱性磷酸酶的活性,PRP能有效的促使BMSCs向前期成骨细胞分化。骨组织学观察证实PRP能诱导BMSCs向成骨细胞分化。
[Abstract]:Aim: to investigate the effects of different concentrations of platelet-rich plasma on alkaline phosphatase (ALP) activity and mRNA expression in autologous bone marrow stromal cells (BMSCs).The biological effects of PRP induced bone marrow stromal cell differentiation into osteoblasts were preliminarily evaluated.Methods: PPP and different platelet concentrations were prepared by modified Appel method.The bone marrow of dog was cultured in D-MEM-F12 medium containing 15% fetal bovine serum, and a sufficient number of stable bone marrow stromal cells (BMSCs) were obtained by subculture and screening.The experiment was divided into five groups. The activity of alkaline phosphatase was detected by enzyme kinetic method on the 12th day of the 3rd day after the addition of inducer A, PPP BMSCs, B, PRP BMSCs, C, 4 times PRP BMSCs, D, rhBMP, BMSCs, and E, as blank control group.The expression of alkaline phosphatase (mRNA) in osteoblasts was detected by RT-PCR method and Von kossa staining was used to identify osteoblasts.Results the primary cultured bone marrow stromal cells were fused into a monolayer after 12 days of adherent adhesion. The cells were mostly fibroblast-like cells. After the passage of the induction medium, the cell morphology gradually changed to square.The activity of alkaline phosphatase (ALP) was measured by polygonal kinetic assay on the 12th day of the 3rd day, and the activity of ALP was significantly higher than that of the 3rd day after paired t test.The expression of alkaline phosphatase mRNA was detected by RT-PCR on the 6th day. The results showed that the expression of ALPmRNA in the low PRP group was significantly higher than that in the control group. Von kossa staining was used to identify the osteoblasts.Calcium nodules.Conclusion: PRP can induce the expression of alkaline phosphatase mRNA in bone marrow stromal cells and increase the activity of alkaline phosphatase in bone marrow stromal cells. It can effectively induce the differentiation of BMSCs into osteoblasts.Bone histological observation showed that PRP could induce BMSCs to differentiate into osteoblasts.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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