lncRNA对SPA作用下人骨髓间充质干细胞成骨分化能力的作用及机制研究

发布时间：2017-12-27 04:04

本文关键词：lncRNA对SPA作用下人骨髓间充质干细胞成骨分化能力的作用及机制研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的:骨髓炎是一种急性或慢性的骨组织感染,以化脓性炎症、异常骨重建、难控性骨吸收为特征,常伴有骨缺损、骨不连等骨科疾病的出现,导致肢体功能障碍、截肢,甚至危及生命,严重威胁患者身心健康,其治疗一直是骨科医师面临的难题之一。骨髓炎并骨不连的发生发展机制并不清楚。金黄色葡萄球菌(Staphylococcus aureus,SA)是最常见的引发骨髓炎的微生物,葡球菌A蛋白(StaphylococcalproteinA,SPA)是SA的主要毒力成分,研究发现在骨髓炎发生发展过程中,SPA能直接刺激病灶区成骨细胞的凋亡,导致病灶区的骨破坏和骨丢失,说明SPA在骨髓炎的发生发展过程起着关键的作用。研究已证实:在骨再生的修复过程中,骨折部位需要聚集足够数量的骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs),这些干细胞向成骨细胞的分化能力直接影响到了骨组织的新骨生成和骨愈合能力。以往研究主要集中于SA的毒力因子引起成骨细胞的凋亡,而对SA的菌体成分的研究,如SPA对成骨细胞的祖细胞-BMSCs的分化能力影响尚未见报道。本课题组前期首次通过SPA对BMSCs成骨分能力的影响做了相关研究,发现SPA确实能显著抑制BMSCs的成骨分化能力,提示毒力因子SPA可模拟体内的炎症环境,同时对SPA如何影响BMSCs成骨分化能力的机制则不是很清楚。因此,深入研究在SPA作用下hBMSCs成骨分化能力下降的原因,对进一步理解骨髓炎情况下骨生成不足的原因具有重要意义。长链非编码RNA(long non-coding RNA,lncRNA)是一类在基因组转录中不参与编码蛋白、长度超过200个核苷酸的转录产物,涉及一系列的发育过程和疾病进展。研究表明一些lncRNAs如AK141205、ANCR、H19和MEG3在调节干细胞的成骨分化过程中起到重要的作用,可促进成骨细胞分化。以上研究均表明lncRNA对干细胞的成骨分化具有正向调节作用。而也有研究表明部分lncRNA对干细胞的成骨分化存在负向调节作用。研究发现过表达AK035085能够抑制C3H10T1/2细胞的成骨分化。说明lncRNA对干细胞的成骨分化能力有着明显的调控作用,但是在骨髓炎状态下lncRNA对hBMSCs的成骨分化能力的作用则未见相关报道。因此,我们采用不同浓度的SPA处理hBMSCs,通过检测炎症因子的表达以及成骨活性变化情况,构建模拟骨髓炎细胞模型;然后,通过基因芯片分析SPA处理hBMSCs成骨分化的过程中lncRNA差异的变化,根据生物信息学分析的结果,确定lncRNA的靶基因;进一步分析lncRNA通过调控其靶基因在SPA处理的hBMSCs成骨诱导中的作用,明确lncRNA在骨髓炎状态下调控hBMSCs成骨分化的机制。本研究是首次从lncRNA水平去阐述骨髓炎情况下hBMSCs成骨能力不足的原因,为骨髓炎并缺损的研究提供新的理论基础及研究方向,为下一步的研究提供坚实的基础。研究方法:1..SPA作用下体外骨髓炎模型的构建为了模拟骨髓炎细胞模型,不同浓度的SPA(0μg/ml、0.1 μg/ml,0.5 μg/ml,1 μg/ml,10μg/mL,100μg/mL)处理hBMSCs细胞。处理72h后,24孔培养板细胞上清被收集进行ELISA检测IL-la、IL-6和TNF-a。添加不同浓度的SPA的成骨分化培养基培养3周,然后收集细胞进行茜素红染色检测钙结节沉积和碱性磷酸酶检测;2.分析及验证SPA处理下hBMSCs成骨分化过程中IncRNA的变化hBMSCs在含有(SPA组)或者不含有(对照组)1μg/ml SPA的成骨分化培养基中培养2周后,收集细胞进行lncRNA基因芯片分析。然后分析即表达差异显著又和成骨分化密切相关的lncRNA,将以上差异基因进行qRT-PCR验证,筛选出目的基因。3.差异lncRNA对SPA骨毮炎模型下BMMSC成骨分化功能的影响合成 3 条 NONHSAT009968 干扰序列(siRNA-1,siRNA-2 和 siRNA-3)。培养hBMSCs细胞,根据操作说明书,使用Lipofectamine 2000试剂将大约200nmsiRNA转染到hBMSCs。干扰效率采用qRT-PCR检测。采用siRNA-2序列进行慢病毒包装。根据标准操作进行慢病毒生产、浓度和滴定测定、感染条件确认以及稳定细胞株筛选。慢病毒表达NONHSAT009968-shRNA或空的慢病毒,感染后3天,收集骨髓间充质干细胞检测NONHSAT009968确认是否成功沉默。慢病毒表达NONHSAT009968-shRNA或空的慢病毒感染的hBMSCs细胞培养14天和21天。采用茜素红染色检测钙结节沉积。收集细胞进行western blot检测Runx2、OCN、OPN和COL1A1的表达,ELISA检测IL-Ia、IL-6、TNF-a和碱性磷酸酶检测。分析干扰NONHSAT009968对SPA处理下的hBMSCs的成骨分化能力的影响。4差异IncRNA对SPA骨髓炎模型下BMMSC成骨分化功能影响的作用机制慢病毒表达NONHSAT009968-shRNA或空的慢病毒感染的hBMSCs细胞培养14天和21天。收集细胞采用qRT-PCR和western blot检测Wnt3a、β-catenin和GSK-3[β的表达,分析干扰NONHSAT009968对Wnt3a、β-catenin和GSK-3β的表达的影响。研究结果:1.SPA骨髓炎模型构建成功SPA可以显著增加hBMSCs细胞成骨分化过程中炎症因子IL-1a、IL-6和TNF-a的分泌(P0.05),而且炎症因子的分泌对SPA呈浓度依赖性(0、0.1、0.5、1、10和100 μg/mL)。另外,茜素红染色显示不同浓度的SPA能够明显抑制钙结节沉积,而且抑制程度和SPA的浓度有密切联系。然后ALP检测结果表明SPA处理能够抑制ALP的活性(P0.05),并且抑制能力呈浓度依赖性。以上结果表明在1 μg/ml SPA能够明显增加炎症因子IL-1a、IL-6和TNF-a的分泌,抑制钙结节沉积和ALP的表达。说明SPA可以促进炎症因子的表达及抑制hBMSCs成骨能力的下降,这一结果与体内骨髓炎状态表现一致。因此,在体外采用1μg/ml SPA处理hBMSCs能够模拟体内骨髓炎状态,体外可构建SPA骨髓炎模型。2.IncRNA基因芯片结果分析(1)SPA处理的骨髓间充质干细胞和对照组相比,存在2033个异常表达的lncRNAs。其中 641 个 lncRNAs 下调,1392 个 lncRNAs 上调(倍数变化2.0,P0.05)。从mRNA表达分析数据中发现SPA处理的骨髓间充质干细胞和对照组相比,存在449个异常表达的mRNA,其中318个mRNA显著下调,131个mRNA显著上调(倍数变化2.0,P0.05)。(2)基于KEGG途径分析,表达上调的mRNA包含20个不同的信号通路,最丰富的通路是唾液分泌,谷氨酸能突触,嗅觉传导和胰岛素分泌。基于KEGG途径分析,表达下调的mRNA包含20个不同的信号通路,最丰富的通路是造血,视黄醇的新陈代谢,药物代谢,细胞粘附分子和细胞色素代谢外源性物质。为了理解差异表达基因的功能,所有的差异表达基因映射到GO数据库中并比较其背景。基于对生物过程进行GO分析,上调的转录本主要富集在纤溶酶原激活和水转运,而下调的转录本主要富集在对糖皮质激素刺激的细胞反应和细胞糖脂化。基于对分子功能进行GO分析,上调的转录本主要富集在神经递质转运体活动和钙调素结合,而下调的转录本主要富集在对配体-依赖的核受体转录共激活和葡萄糖醛酸基转移酶活性。(3)通过cis-lOOk分析,五个潜在的和成骨分化相关且表达差异显著的lncRNA被鉴定出来,包括 NONHSAT125464、ENST00000504555、NONHSAT098635、NONHSAT054627和NONHSAT009968。lncRNA的潜在靶向调控目标分别包括骨形态形成蛋白1、SMAD 1、SMAD 1、胶原蛋白I型αl和Wnt3a,这些蛋白和成骨细胞分化密切相关。然后将差异的1ncRNA采用qRT-PCR验证,qRT-PCR结果表明SPA处理中和对照组中 NONHSAT125464、ENST00000504555、NONHSAT098635、NONHSAT054627和NONHSAT009968的表达显著上升(P0.05),和基因芯片的结果一致。其中NONHSAT009968的表达结果提高的最为明显,后续实验采用NONHSAT009968以及其靶基因Wnt3a进行研究。3.沉默]NONHSAT009968能够逆转SPA对hBMSCs细胞成骨分化的影响在siRNA的检测中siRNA-2和siRNA-3转染后能够显著降低NONHSAT009968的表达(P0.05),其中siRNA-2转染后NONHSAT009968的表达量最低,干扰效率最好,可以用于后续NONHSAT009968的干扰序列。当感染NONHSAT009968-shRNA后采用 qRT-PCR 检测 NONHSAT009968 的表达,结果表明 NONHSAT009968-shRNA干扰能够显著降低 NONHSAT009968 的表达(P0.05)。NONHSAT009968-shRNA 和NC在1 μg/ml SPA中培养14天和21天。茜素红染色结果表明:无论在培养后14天还是21天,NONHSAT009968沉默组和NC组相比,都能够增加钙结节沉积(P0.05)。碱性磷酸酶活性检测结果表明无论在培养后14天还是21天,NONHSAT009968沉默和NC组相比,都能够增加碱性磷酸酶活性(P0.05)。另外,沉默NONHSAT009968能够增加成骨分化相关的Runx2、OCN、OPN和COL1A1蛋白的表达(P0.05)。这些结果表明沉默NONHSAT009968能逆转SPA抑制的hBMSCs成骨分化。4.沉默 NONHSAT009968 促进 Wnt3a 以及 β-catenin 和 GSK-3β 的表达SPA处理能够显著抑制Wnt3a、β-catenin和GSK-3β的表达(P0.05),且该抑制结果还呈时间依赖性。该结果还表明干扰NONHSAT009968的表达能够增强Wnt3a、β-catenin和GSK-3β的表达(P0.05)。该结果表明干扰NONHSAT009968促进成骨分化的机制可能和Wnt3a以及β-catenin和GSK-3β的表达相关。研究结论:(1)本研究1 μg/ml的SPA处理能够促进IL-la、IL-6和TNF-a的表达,降低碱性磷酸酶的表达和钙结节沉积,抑制Wnt3a、β-catenin和GSK-3β的表达,抑制hBMSCs的成骨分化,成功模拟骨髓炎状态。(2)在SPA处理hBMSCs的成骨分化的过程中,通过基因芯片分析发现存在5个和成骨分化相关的 lncRNA,有 NONHSAT125464、ENST00000504555、NONHSAT098635、NONHSAT054627 和 NONHSAT009968。其中 lncRNA NONHSAT009968的表达上升最为明显,而且lncRNA NONHSAT009968潜在的cis-regulated mRNA 目标为 Wnt3a。(3)进一步研究发现通过干扰lncRNA NONHSAT009968的表达能够显著提高Wnt3a、β-catenin和GSK-3β的表达,促进钙结节沉积,提高ALP的表达,促进成骨相关蛋白Runx2、OCN、OPN和COL1A1的表达,改善SPA抑制的hBMSCs的成骨分化。(4)本研究首次从lncRNA角度阐明骨髓炎状态下成骨能力下降的原因及其作用机制,为骨髓炎状态下成骨能力下降的原因提出了新的解释及研究方向。
[Abstract]:Background and objective: osteomyelitis is a kind of acute or chronic bone infection with purulent inflammation, abnormal bone remodeling, refractory bone absorption, often accompanied by bone defect and nonunion of diseases in Department of orthopedics, leading to limb dysfunction, amputation, and even endanger the life, a serious threat to the physical and mental health of patients and its treatment has been one of the problems in Medical Department of orthopedics. The pathogenesis of osteomyelitis with bone nonunion is not clear. Staphylococcus aureus (Staphylococcus aureus SA) is the most common cause of microorganisms, staphylococcus protein A (StaphylococcalproteinA, SPA) is a main toxic component of SA, the study found that in the process of the occurrence and development of osteomyelitis, SPA lesions can directly stimulate the apoptosis of osteoblasts, resulting in bone destruction and bone lesions. Lost, indicating that SPA plays a key role in the occurrence and development of osteomyelitis. Research has confirmed that in the repairing process of bone regeneration in the fracture site need to gather a sufficient number of bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells, BMSCs), these stem cells differentiation into osteogenic cells directly affects the formation of new bone and bone tissue healing ability. Previous studies have focused on the virulence factors of SA, which induce the apoptosis of osteoblasts. However, the effect of SPA on the differentiation of osteoblasts from progenitor cells, such as -BMSCs, has not been reported in SA. The research group for the first time through the early effects of SPA on osteogenic ability of BMSCs to do related research, found that SPA can significantly inhibit BMSCs osteogenic differentiation, suggesting that virulence factor SPA can mimic the in vivo inflammatory environment, at the same time the effect of SPA on osteogenic differentiation of the BMSCs mechanism is not very clear. Therefore, in-depth study of the causes of the decline of hBMSCs osteogenic differentiation under the action of SPA is of great significance for further understanding the cause of osteogenesis. Long chain non coding RNA (long non-coding RNA, lncRNA) is a class of transcription products that do not participate in encoding protein and over 200 nucleotides in genome transcription, and involves a series of development processes and disease progression. Studies have shown that some lncRNAs, such as AK141205, ANCR, H19 and MEG3, play an important role in regulating the osteogenic differentiation of stem cells, and promote osteoblast differentiation. All of these studies suggest that lncRNA has a positive regulatory effect on the osteogenic differentiation of stem cells. However, some studies have shown that partial lncRNA has a negative regulatory effect on the osteogenic differentiation of stem cells. It was found that the expression of AK035085 could inhibit the osteogenic differentiation of C3H10T1/2 cells. It indicates that lncRNA plays a significant role in regulating the osteogenic differentiation ability of stem cells. However, in the condition of osteomyelitis, the effect of lncRNA on the osteogenic differentiation of hBMSCs has not been reported. Therefore, we use the SPA treatment of different concentrations of hBMSCs, by detecting the expression of inflammatory factors and changes of bone activity, construction simulation of osteomyelitis cell model; then, changes of lncRNA SPA hBMSCs during osteogenic differentiation by gene chip analysis of the difference, according to the results of a bioinformatics analysis, to identify the target gene of lncRNA lncRNA; further analysis through the regulation of its target gene into bone in the induction of SPA treatment of hBMSCs, lncRNA in the clear state of osteomyelitis regulation of hBMSCs differentiation mechanism. This study is the first time to expound the causes of insufficient hBMSCs osteogenesis ability from lncRNA level, and provide a new theoretical basis and research direction for the study of osteomyelitis and defect, and provide a solid foundation for further research. Research method: 1..SPA was used to construct the model of extracorporeal osteomyelitis. In order to simulate the osteomyelitis cell model, hBMSCs cells were treated with different concentrations of SPA (0 g/ml, 0.1 g/ml, 0.5 g/ml, 1 g/ml, 10 g/mL, 100 g/mL). After the treatment of 72h, the 24 hole culture plate cell supernatant was collected for ELISA detection of IL-la, IL-6 and TNF-a. The effects of different concentrations of SPA osteogenic differentiation medium for 3 weeks, then the cells were collected for detection of alizarin red staining calcium nodules and alkaline phosphatase deposition; 2. analysis and verification of SPA under the treatment of hBMSCs hBMSCs IncRNA changes in the differentiation of bone containing (SPA group) or without (control group) after 2 weeks 1 g/ml SPA osteogenic differentiation medium, cells were collected for analysis of lncRNA gene chip. Then the analysis is to express the lncRNA which is closely related to the differentiation of osteogenesis, and the above differentiating genes are verified by qRT-PCR and the target genes are screened. 3. different effects of lncRNA on osteogenic differentiation of BMMSC SPA bone Sha inflammation model of the synthesis of 3 NONHSAT009968 interference sequence (siRNA-1, siRNA-2 and siRNA-3). HBMSCs cells were cultured and Lipofectamine 2000 reagents were used to transfect about 200nmsiRNA to hBMSCs according to the operation instructions. The interference efficiency was detected by qRT-PCR. The siRNA-2 sequence is used to pack the lentivirus. Lentivirus production, concentration and titration, infection condition confirmation and stable cell line screening were carried out according to the standard operation. The lentivirus expressed NONHSAT009968-shRNA or empty lentivirus, and 3 days after infection, bone marrow mesenchymal stem cells were collected to determine whether NONHSAT009968 was successfully silent. The lentivirus expressed NONHSAT009968-shRNA or empty lentivirus infection of hBMSCs cells for 14 days and 21 days. Alizarin red staining was used to detect the deposition of calcium nodules. The cells were collected for Western blot to detect the expression of Runx2, OCN, OPN and COL1A1, and ELISA was used to detect IL-Ia, IL-6, TNF-a and alkaline phosphatase. The effects of interfering NONHSAT009968 on the osteogenic differentiation of hBMSCs treated with SPA were analyzed. 4, the mechanism of the effect of differential IncRNA on the osteogenic differentiation function of BMMSC in SPA osteomyelitis model. Lentivirus expressing NONHSAT009968-shRNA or empty lentivirus infected hBMSCs cells were cultured for 14 days and 21 days. Collecting cells using qRT-PCR and Western blot to detect Wnt3a, beta -catenin and GSK-3
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R681.2

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