研究BMP-2对骨髓间充质干细胞促进造血干细胞增殖的影响

发布时间：2018-01-02 00:10

  本文关键词：研究BMP-2对骨髓间充质干细胞促进造血干细胞增殖的影响　出处：《四川医科大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 造血干细胞 间充质干细胞 BMP-2 微龛 增殖

【摘要】：目的:生存在骨髓微环境即微龛(niche)中的造血干细胞(HSCs)为所有成熟血细胞的原始造血细胞,其具有高度自我更新能力与多向分化潜能。微龛极其复杂,由基质细胞及其前体间充质干细胞(MSCs)、细胞因子、细胞外基质等组成,它们对于HSC生理功能的调控起着重要的作用。niche主要分为“成骨微龛”和“血管微龛”,前者主要维持HSC静息状态,后者可以调节HSC增殖、分化、迁移等。骨形态发生蛋白(BMP)-2是转化生长因子(TGF-β)超家族中的一员,是一个多功能蛋白,作为重要的成骨因子可刺激MSC分化为成骨细胞,构成“成骨niche”的主体结构,此外它还能促进血管的形成,关于它在成骨niche中的研究较多,而其在血管niche方面的作用研究较少。BMP-2在造血调控中也起重要作用,关于其在胚胎期造血中的研究较多,而其在成人造血中的作用研究得较少。目前关于MSC促进HSC增殖的研究较多,而关于BMP-2对MSC促进HSC增殖的影响的研究较少。本研究通过transwell非接触共培养CD34+细胞与MSC,并用BMP-2干预,在第三天时检测HSC的增殖指标,可以了解MSC和BMP-2对CD34+细胞增殖的影响以及BMP-2信号通路对MSC促进HSC增殖的影响。这能为骨髓CD34+细胞体外扩增的深入研究提供依据,同时为我们下一步探讨相关机制的研究奠定基础,且这有利于造血干细胞移植(HSCT)的发展及恶性血液病的治疗。方法:①结合MSCs贴壁生长的特性采用全骨髓法体外扩增成人BMMSCs至P3,并用流式细胞术(FCM)鉴定MSCs的表面标志及纯度;②通过密度梯度离心法分离骨髓单个核细胞(MNCs),再用mini MACS磁珠分选仪从MNCs中分选成人骨髓CD34+细胞,并用FCM鉴定其纯度;③利用Transwell小室(微网孔径0.4um)非接触共培养骨髓CD34+细胞与P3代的BMMSCs,并用BMP-2干预,具体分组为:单纯HSC组、HSC+BMP2组、HSC+MSC组、HSC+MSC+BMP2组。培养至72小时收集样本;④用以下方法检测HSC在不同组的增殖情况:计数板计数每组HSC的四个复孔的数目;TRIZOL法提取各组HSC的RNA后测定RNA浓度并计算各组HSC的RNA的含量;实时荧光定量PCR法检测各组HSC的增殖基因Ki67的m RNA水平的表达,PCR的Ct值利用2-△△Ct分析法进行相对定量(RQ)分析;⑤统计学分析方法:所有统计学数据均用SPSS17.0软件进行统计学分析,两两比较用单因素方差分析,均以“均数±标准差(X±SD)”表示,当P0.05时差异被认为具有统计学意义。结果:①全骨髓法培养的BMMSCs成梭形,贴壁生长,排列成旋涡状;流式细胞术检测显示传至第3代的BMMSCs高表达CD105,而CD45、CD34阴性表达,MSCs的纯度为52.4%。②mini MACS磁珠分选出了较高纯度的CD34+细胞,纯度为81.9%。③各组HSC的计数(×104个):共培养组(3.03±0.50)、HSC+BMP2组(1.70±0.51)均高于HSC单纯培养组(1.2±0.43);共培养+BMP2组(5.29±0.20)高于共培养组(3.03±0.50);P值均0.05。④各组HSC的RNA量(RNA浓度的单位为ng/ul,总RNA还需×50ul):HSC+BMP2组(138±3.464)及共培养组(163±3.559)高于HSC单纯培养组(113±2.944);共培养+BMP2组(241±3.651)高于共培养组(163±3.559);P值均0.05。⑤荧光定量PCR法检测HSC的Ki67的m RNA相对表达量,即RQ值:共培养组(3.27±0.08)及HSC+BMP2组(2.44±0.26)高于HSC单纯培养组(1.00±0.13);共培养+BMP2组(5.08±0.20)高于共培养组(3.27±0.08);P值均0.05。结论:①利用全骨髓法成功培养出MSC;②应用免疫磁珠分选法成功分选出HSC;③BMP-2可以促进HSC增殖;④MSC可以促进HSC增殖;⑤MSC与BMP-2具有协同效应,即BMP-2信号通路可以促进MSC对HSC的增殖作用。
[Abstract]:Objective: to survive in the bone marrow microenvironment micro niche (niche) in hematopoietic stem cells (HSCs) as primitive hematopoietic cells all mature blood cells, which have high self-renewal capacity and multilineage differentiation potential. The micro niche is extremely complex, and its precursors by stromal cell derived mesenchymal stem cells (MSCs), cytokine extracellular matrix, etc., for they regulate physiological functions of HSC plays an important role for.Niche is mainly divided into "bone micro niche" and "micro vascular niche", the former mainly maintain HSC resting state, which can regulate HSC proliferation, differentiation, migration, bone morphogenetic protein (BMP) -2 transformation growth factor (TGF-) superfamily, is a multifunctional protein, as an important factor of bone can stimulate the differentiation of MSC into osteoblasts, constitute the main structure of bone niche, it can also promote the formation of blood vessels, about it in the bone in niche More research, and study the role of niche in vascular less.BMP-2 in hematopoietic regulation play an important role in embryonic hematopoiesis on its research and its role in adult hematopoiesis was less studied. More research on MSC currently promote the proliferation of HSC, and about BMP-2 of MSC effect the proliferation of HSC is less. This study by Transwell co culture CD34+ cells and MSC, and treated with BMP-2 at third days to detect the proliferation of HSC index, can understand the impact of MSC and BMP-2 on CD34+ cell proliferation and BMP-2 signaling pathway on MSC promote proliferation of HSC. This can provide the basis for further research for the amplification of bone marrow CD34+ cells in vitro, and to explore the related mechanisms of the study laid the foundation for our next step, and this is conducive to hematopoietic stem cell transplantation (HSCT) treatment and development of malignant hematological diseases. Methods: 1. Amplification of adult BMMSCs to P3 binding characteristics of MSCs cultured in vitro by the whole bone marrow method, and flow cytometry (FCM) identification of surface markers MSCs and purity; the separation of bone marrow mononuclear cells by density gradient centrifugation (MNCs), and mini MACS beads sorter sorting from MNCs adult bone marrow CD34+ cells the purity, and identified by FCM; using Transwell chamber (micro aperture 0.4um) bone marrow CD34+ cells and P3 generation BMMSCs non-contact co culture, and treated with BMP-2 specific groups: simple HSC group, HSC+BMP2 group, HSC+MSC group, HSC+MSC+ BMP2 group. Training 72 hours to collect samples with the following; detection of HSC in the proliferation of different groups: four holes the number count of each HSC group; group HSC RNA TRIZOL extraction method for determination of RNA concentration and HSC content were calculated by RNA; the proliferation was detected by HSC based real-time fluorescence quantitative PCR method 鍥燢i67鐨刴 RNA姘村钩鐨勮〃杈，

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