高糖环境下NCoR siRNA对骨髓间充质干细胞成骨分化调控的实验研究

发布时间：2018-04-26 06:45

  本文选题：骨髓间充质干细胞 + NCoR　； 参考：《山东大学》2017年硕士论文

【摘要】：研究背景:骨髓间充质干细胞(BMSCs)具有分化成不同细胞组织的能力,其中包括成骨细胞,脂肪细胞,软骨细胞以及肌细胞。体外培养骨髓间充质干细胞的相关研究中发现,在合适的环境和条件下,骨髓间充质干细胞能够在体外进行34—42次的分裂增殖,增殖过程中始终保持梭形并呈漩涡状排列。不仅如此,骨髓间充质干细胞在冷冻保存进行复苏后,仍然具备干细胞的特性。BMSCs不仅存在于骨髓中,还游离于骨组织外。当其在适当信号刺激时,可游离出骨组织,到达效应靶器官组织中,这种生物学行为叫"归巢"。在适当的生物和物理因素与足够的特异性信号传导的存在,BMSCs能够分化成所需的特定细胞组织。由于这些性质,BMSC已经成为肌肉骨骼组织再生治疗研究的主要细胞来源。BMSCs分化成骨细胞受细胞外信号和转录水平的级联放大调控,这需要大量基因表达之间的精细时空调节。研究表明,原发性成骨分化诱导主要由Runt相关转录因子2(Runx2)关键转录因子调控。Runx2激活和调节成骨作为许多信号通路的靶基因,包括但不限于转化生长因子-β(TGF-b1),骨形态发生蛋白(BMP),Winglesstype1(Wnt),Hedgehog(HH)和(Nel)样蛋白1型(NELL-1)。诱导后,成骨细胞分化下游的前成骨细胞分化成成骨细胞,成骨细胞成熟和基质矿化的过程受其他转录因子调节,包括Osterix,BSP,OCN和OPN。这个过程也受到许多其他物理和生物因素的影响,其中大多数已经在体外证明,并且仍然在仔细审查。影响BMSCs的增殖和成骨分化的物理因素,如高葡萄糖,已经很好地建立并通过几个体外研究证明。临床证据表明,与正常人相比,糖尿病患者的骨折不愈合和延迟愈合更高,此外糖尿病患者早期骨质疏松的发生率高。最近的体外研究已经证明高葡萄糖微环境降低细胞BMP的表达,细胞BMP是成骨分化的终末期期间的重要调节蛋白,因此抑制该过程。进一步了解高葡萄糖对骨代谢的负面影响并设计减轻这种合并症的方法的研究正在进行。核受体辅助抑制因子(nuclear receptor corepressor,NCoR)属于核受体辅助抑制因子,通过与许多其它转录因子(包括NF-Eb,AKT和PPARα)相互作用而发挥各种抑制功能。近年来发现NCoR可以与HDAC结合成复合物,通过影响蛋白的转录,在机体代谢、炎症以及肿瘤发生中扮演着重要角色。JinZ报道HDAC9与视黄酸和甲状腺激素受体(SMRT)/NCoR共抑制因子的沉默介体协同抑制PPARγ活性,并将HDAC9鉴定为骨重建和骨骼稳态的重要和生理学相关的调节剂。最近,YiQin证明NCoR在标准成骨培养基中通过P13K/AKT细胞信号通路调节大鼠间充质干细胞的成骨分化。因此,在高葡萄糖微环境下,NCoR基因敲除对BMSCs的成骨分化的作用是什么?研究目的:观察NCoRsiRNA对BMSCs在高葡萄糖微环境下增殖和成骨分化的影响。研究方法:1、根据NCoRsiRNA靶位点的选择原则,从GenBank数据库中择优选取三条siRNA序列,通过构建干扰及过表达载体,筛选出最佳序列。2、检测NCoR对BMSCs在高葡萄糖微环境下增殖的影响。在96孔板上按1×106/孔接种大鼠BMSCs,设置空白对照组、阴性对照组(无目标siRNA和NCoRsiRNA组,每组设3复孔。从转染后第1天开始,达到65%的汇合后,将细胞样品在含有5.5,16.5,25和35mmol/L葡萄糖浓度的成骨培养基中培养。培养9天后,使用甲基噻唑基四唑(MTT)测定细胞增殖,并在490nm测定吸光度。3、观察NCoR对大鼠BMSCs诱导成骨分化的影响。在含有25mmol/L(高葡萄糖)和5.5mmol/L葡萄糖(对照)的成骨培养基中分别培养用NCoRsiRNA或无目标siRNA转染的细胞21天,然后通过测量细胞ALP活性,钙沉积RT-PCR检测相关成骨基因;Run2,Osterix,OCN,OPN和BSP的表达,来测定成骨分化。结果:1、成功构建了 NCoRsiRNA及无目标siRNA质粒载体,并筛选了最佳干扰序列,载体由广州Cyagen生物科技公司合成。2、NCoRsiRNA可使BMSCs的增殖降低。用NCoRsiRNA或无目标siRNA转染的BMSCs在含有四个不同葡萄糖浓度(5.5,16.5,25和35mmol/L)的成骨培养基中培养。与对照组相比,第1d三组细胞增殖差异不大。从第3d开始,在所用葡萄糖浓度,NCoRsiRNA组细胞数量及增殖明显低于对照组。提示NCoR可促进BMSCs增殖。3、NCoRsiRNA可在高葡萄糖下促进BMSCs的成骨分化。与对照组相比,通过ALP-ELISA试剂盒和钙诊断试剂盒测定的,NCoRsiRNA组细胞的ALP活性以及矿化(钙沉淀)在25mmol/L(高葡萄糖)和5.5mmol/L葡萄糖下都明显增加。此外,实时RT-PCR对成骨分化相关的五个基因Run2,Osterix,OCN,OPN和BSP的检测显示,与对照组相比,NCoRsiRNA组Run2,Osterix,OCN,OPN和BSP表达都显著增加(P0.01)。提示NCoR负性调控大鼠BMSCs的成骨分化。结论:我们利用RNA干扰技术成功设计并构建了 NCoR基因特异性的小干扰siRNA,以及设计并构建了无目标siRNA。经证实,基因沉默的效果良好。我们成功将NCoRsiRNA和无目标siRNA转染至大鼠骨髓间充质干细胞内,并证实转染效率较高,然后在四个不同葡萄糖浓度下(5.5,16.5,25和35mmol/L)的成骨培养基中培养。然后检测BMSCs的增殖发现,NCoRsiRNA组BMSCs的增殖明显下降,提示NCoR可促进BMSCs的增殖。沉默NCoR时,在正常(5.5mmol/L)和高糖(25mmol/L)浓度下,大鼠BMSCs诱导成骨分化,成骨细胞分化标志基因Run2,Osterix,OCN,OPN和BSP的表达明显增强。提示NCoR负性调控大鼠BMSCs的成骨分化。结合糖浓度分析,NCoR siRNA可中和高糖对大鼠BMSCs成骨分化的抑制作用。综合既往研究,NCoR siRNA能改善机体胰岛素敏感性,NCoR可作为潜在靶点,在糖尿病病人中改善机体胰岛素敏感性,降糖的同时,保护糖尿病患者骨质疏松。
[Abstract]:Background: bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate into different cellular tissues, including osteoblasts, adipocytes, chondrocytes, and myocytes. In the study of bone marrow mesenchymal stem cells in vitro, bone marrow mesenchymal stem cells can carry out 34 - 42 in the suitable environment and conditions. In addition, after the resuscitation of cryopreservation, bone marrow mesenchymal stem cells still possess the characteristics of stem cells, not only in the bone marrow, but also free from the bone tissue. When the stem cells are stimulated by proper signal,.BMSCs can free the bone tissue and reach the effect target. In the official organization, this biological behavior is called "homing". In the presence of adequate biological and physical factors and sufficient specific signal transduction, BMSCs can differentiate into specific cellular tissues required. Because of these properties, BMSC has become a major cell source for the study of musculoskeletal tissue regeneration,.BMSCs differentiation into osteoblasts from cells. Cascade amplification regulation of signal and transcriptional levels requires fine space-time regulation between a large number of gene expressions. Studies have shown that primary osteogenic differentiation is induced by Runt related transcription factor 2 (Runx2) key transcription factors to regulate.Runx2 activation and regulation of bone formation as a target gene for many signaling pathways, including but not limited to transformation growth factors. Sub beta (TGF-b1), bone morphogenetic protein (BMP), Winglesstype1 (Wnt), Hedgehog (HH) and (Nel) like protein 1 (NELL-1). After induction, osteoblasts are differentiated into osteoblasts downstream of osteoblast differentiation, and the process of osteoblast maturation and matrix mineralization is regulated by other transcription factors, including Osterix, BSP, OCN, and OPN.. The effects of many other physical and biological factors, most of which have been proven in vitro, are still under scrutiny. Physical factors affecting the proliferation and osteogenesis of BMSCs, such as Hyperglucose, have been well established and proved by several in vitro studies. Clinical evidence suggests that the fracture nonunion of diabetic patients is compared with normal people. And delayed healing is higher, in addition to the high incidence of early osteoporosis in diabetic patients. Recent in vitro studies have shown that hyperglycemic microenvironment reduces the expression of cell BMP, and cell BMP is an important regulatory protein during the end stage of osteogenic differentiation, thus inhibiting the process. The study of methods designed to mitigate this complication is ongoing. Nuclear receptor corepressor (NCoR) is a nuclear receptor auxiliary inhibitor, which plays a variety of inhibitory functions by interacting with many other transcription factors (including NF-Eb, AKT and PPAR alpha). In recent years, it is found that NCoR can be combined with HDAC. Compounds, by influencing the transcription of protein, play an important role in body metabolism, inflammation, and oncology,.JinZ reports that HDAC9 is synergistic to inhibit PPAR gamma activity with retinoic acid and thyroid hormone receptor (SMRT) /NCoR co suppressor, and to identify HDAC9 as an important and physiological regulation of bone remodeling and bone homeostasis Recently, YiQin has shown that NCoR regulates the osteogenic differentiation of rat mesenchymal stem cells by P13K/AKT cell signaling pathway in the standard osteogenic medium. So, what is the effect of NCoR knockout on the osteogenic differentiation of BMSCs in the Hyperglucose microenvironment? The purpose of this study was to observe the proliferation and formation of BMSCs in the Hyperglucose microenvironment of BMSCs. The effect of bone differentiation. 1. According to the selection principle of NCoRsiRNA target site, three siRNA sequences were selected from the GenBank database, and the optimal sequence.2 was screened by constructing the interference and overexpression vector. The effect of NCoR on the proliferation of BMSCs in the hyperglycemic microenvironment was detected. The rat BMSCs was inoculated on the 96 orifice plate by 1 x 106/ holes. In the blank control group, the negative control group (no target siRNA and NCoRsiRNA group, each set 3 compound holes. After first days after transfection, the cell samples were cultured in the osteogenic medium containing 5.5,16.5,25 and 35mmol/L glucose concentration. After 9 days, the cell proliferation was measured using methyl thiazolidazole four azoles (MTT), and 490nm was measured. " The effect of NCoR on the osteogenic differentiation induced by BMSCs in rats was observed. The cells transfected with NCoRsiRNA or non target siRNA were cultured in the osteogenic medium containing 25mmol/L (high glucose) and 5.5mmol/L glucose (control) for 21 days, and then by measuring the cell ALP activity, calcium deposition RT-PCR was used to detect the related osteogenic genes; Run2, Osterix, OCN, and OCN. The expression of PN and BSP to determine osteogenic differentiation. Results: 1, NCoRsiRNA and non target siRNA plasmid vectors were successfully constructed, and the optimal interference sequence was screened. The carrier was synthesized from.2 in Guangzhou Cyagen biotechnology company, NCoRsiRNA could reduce the proliferation of BMSCs. BMSCs in NCoRsiRNA or non target siRNA was contained in four different glucose concentrations ( 5.5,16.5,25 and 35mmol/L were cultured in the osteogenic medium. Compared with the control group, there was little difference in cell proliferation in the group 1D Three. From 3D, the number and proliferation of cells in the NCoRsiRNA group were significantly lower than that in the control group. It suggests that NCoR can promote BMSCs proliferation.3, NCoRsiRNA can promote the osteogenesis of BMSCs under high glucose. Compared with the ALP-ELISA kit and the calcium diagnostic kit, the ALP activity of the NCoRsiRNA group and the mineralization (calcium precipitation) were significantly increased under 25mmol/L (high glucose) and 5.5mmol/L glucose. In addition, the real-time RT-PCR for the five genes related to osteogenesis differentiation, Run2, Osterix, OCN, OPN and BSP, showed that the control group was with the control group. The expression of Run2, Osterix, OCN, OPN and BSP in the NCoRsiRNA group increased significantly (P0.01). It suggests that NCoR negatively regulates the osteogenic differentiation of BMSCs in rats. Conclusion: we successfully designed and constructed the small interference siRNA of NCoR gene by RNA interference technique, and the design and construction of a non target gene have proved that the effect of gene silencing is good. NCoRsiRNA and non target siRNA were successfully transfected into rat bone marrow mesenchymal stem cells and confirmed that the transfection efficiency was high and then cultured in the osteogenic medium of four different glucose concentrations (5.5,16.5,25 and 35mmol/L). Then the proliferation of BMSCs was detected, and the proliferation of BMSCs in the NCoRsiRNA group decreased significantly, suggesting that NCoR promoted the increase of BMSCs. At the concentration of normal (5.5mmol/L) and high glucose (25mmol/L), rat BMSCs induced osteogenic differentiation, and the expression of osteoblast differentiation marker gene Run2, Osterix, OCN, OPN and BSP increased obviously at the concentration of normal (5.5mmol/L) and high glucose (25mmol/L), suggesting that NCoR negatively regulated the osteogenic differentiation of BMSCs rats. In a comprehensive study, NCoR siRNA can improve the body's insulin sensitivity. NCoR can be a potential target for improving the body's insulin sensitivity and reducing glucose in diabetic patients, while protecting the osteoporosis in diabetic patients.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1;R580

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