雷奈酸锶在高糖诱导的成骨细胞损伤中的保护作用

目的:探究雷奈酸锶(SR)在高糖诱导的成骨细胞损伤中的保护作用。方法:体外培养MC3T3-E1成骨细胞,用MTT法筛选出能够诱导成骨细胞活性显著降低的高糖浓度,以及能够促进成骨细胞活性的最优SR浓度,随后观察SR对高糖诱导的成骨细胞的作用效果。采用MTT法检测细胞增殖情况,流式细胞仪检测凋亡水平,PCR和ELISA分别检测骨形成相关基因表达和蛋白分泌水平。结果:0.50 mmol/L的SR能够显著增强高糖诱导的MC3T3-E1成骨细胞的增殖能力和细胞活性,降低细胞凋亡水平,改善细胞形态,并显著促进Runx2、OCN、COL-1、BMP-2、IGF-1等的表达。结论:SR能够通过增加成骨细胞活性、抑制成骨细胞凋亡、促进骨形成相关因子的表达和分泌,发挥对高糖诱导的成骨细胞损伤的保护作用,为SR发挥糖尿病骨质疏松的治疗作用提供了实验依据。     

淫羊藿苷对体外牙周膜细胞增殖、成骨分化以及成骨和破骨因子表达的影响

目的:明确淫羊藿苷对于体外分离培养的人牙周膜细胞的增殖、成骨分化以及成骨和破骨因子表达的影响。方法:原代分离培养人牙周膜细胞,实验组细胞加入0.01 mg/L淫羊藿苷溶液。采用MTT法检测牙周膜细胞的增殖速率,采用碱性磷酸酶活性检测试剂盒评估牙周膜细胞的成骨分化能力,分别通过real-time PCR和Western印迹检测成骨相关的OCN、Runx2、BMP2以及破骨相关的RANKL的基因和蛋白表达。结果:MTT结果揭示,淫羊藿苷显著提高了人牙周膜细胞的增殖速率(P0.05),也显著促进了碱性磷酸酶活性的提升(P0.05),揭示其对于牙周膜细胞的成骨分化具有促进作用;real-time PCR和Western印迹结果均表明,淫羊藿苷能够上调OCN、Runx2和BMP2的基因和蛋白表达(P0.05),并下调RANKL的基因和蛋白表达(P0.05)。结论:淫羊藿苷能够促进体外牙周膜细胞的增殖、成骨分化潜能和成骨功能活性。本研究有望为牙周炎治疗以及基于组织工程技术的牙周组织的损伤修复提供新思路。     

非典型腺苷酸激酶AK6/hCINAP的结构和功能

腺苷酸激酶广泛存在于各种生物中,在维持细胞中核苷酸的正常含量以及能量代谢活动中发挥重要作用。腺苷酸激酶6 (AK6,又称人coilin相互作用核ATP酶蛋白hCINAP)是一种非典型的腺苷酸激酶,既具有腺苷酸激酶的活性,又具有ATP酶的活性。本课题组对AK6/hCINAP的结构、酶学特征和生物学功能开展了长期的研究,证明其在调控基因转录、核糖体质量、早期胚胎发育、细胞衰老、细胞代谢、细胞增殖和凋亡、DNA损伤反应、炎症反应、肿瘤发展等诸多生物学过程中均发挥关键作用。本文对AK6/hCINAP的结构特征、生物学功能及上游调控因子进行了总结,阐明该酶生物学作用和分子机制具有重要的生物学意义,有助于开发AK6/hCINAP选择性抑制剂并应用于临床治疗。     

人股骨抽提物的骨诱导活性分析

骨诱导因子在骨的发生及损伤修复过程中起重要作用,因而它们在治疗骨缺损、骨质疏松等病症方面有广阔的应用前景。本实验测定了人股骨内活性因子的骨诱导活性。结果表明,人胎骨抽提物能在小鼠体内诱导未分化的间充质细胞向骨细胞转化,对培养人骨细胞的ＤＮＡ合成与碱性磷酸酶活性均有刺激作用。抽提物经初步纯化后,活性有所增强。     

Sirtl在免疫调节中的研究进展

沉默信息调节因子1(Sirt1),属于Ⅲ类组蛋白去乙酰化酶,是依赖于NAD+的去乙酰化酶,具有高度保守的催化结构域,通过对多种底物进行去乙酰化作用在机体内参与一系列生物学活动.Sirt1去乙酰化调节多种转录因子活性,介导包括细胞凋亡、自噬、代谢、蛋白质内稳态、炎症等多种功能.近年研究发现,Sirt1在免疫调节中也具有重要作用,Sirtl可调节巨噬细胞功能,参与T细胞增殖、分化,在维持T细胞耐受中也具有一定作用,Sirt1还可能通过CD38介导的相关途径调节B细胞功能.大量文献报道多种疾病的发生与Sirt1活性水平有关,Sirt1活性受损可增加炎症和自身免疫性疾病的发生.本文通过综述Sirtl对NF-κB、AP-1信号通路的调节机制,来明确Sirt1在天然免疫和获得性免疫应答中的作用,为临床治疗自身免疫性疾病提供理论依据和可能的治疗靶点.     

碱性成纤维细胞生长因子的研究现状

ｂＦＧＦ为多种细胞的促分裂剂,是神经血管的主要营养因子,对机体损伤有广泛的修复作用。是一具有发展潜力的治疗制剂。本文就ｂＦＧＦ的生物学特性,临床治疗及制瘤性等方面作一介绍。     

细胞致死性膨胀毒素损伤宿主防御机制研究进展

细胞致死性膨胀毒素(cytolethal distending toxin, CDT)属于AB₂毒素,由多种革兰氏阴性菌产生。CDT是第一种被描述的细菌基因毒素,编码3种多肽：CDTA、CDTB和CDTC。CdtB是活性部分,有损伤多种细胞类型的能力。CDT具有一种新的分子作用模式,会干扰真核细胞周期的进展,从而导致G2/M停滞和细胞凋亡,该作用机制针对细胞,而且现阶段对于CDT的研究更多也是细胞层面,但是CDT作为毒力因子最终作用是损伤宿主造成疾病。但目前对CDT与宿主相互作用的分子机制了解尚不清晰。本文对细胞致死性膨胀毒素作为毒力因子从损伤上皮屏障、适应性免疫以及促进炎症反应三方面来综合阐述其对宿主防御机制途径的损伤,以期了解其致病机制以及为其临床治疗提供理论依据和新思路。     

白细胞介素18的生物学作用

白细胞介素 18(IL - 18)是新近发现的细胞因子 ,具有诱导 IFN-γ的产生 ,促进 T细胞的增殖 ,促进 Th1细胞的发育和增殖 ,增强 NK细胞及 Th1细胞的细胞毒性 ,增强粒细胞 -巨噬细胞集落刺激因子 (GM- CSF)的产生 ,抗肿瘤效应 ,抑制 Ig E的生成以及破骨细胞的形成等多种生物学活性和作用     

碱性成纤维生长因子（bFGF）对软骨生物学的影响

bFGF的生物学作用极其广泛,特别在促进创伤愈合与组织修复、组织再生起着十分重要的作用,它作为重要的有丝分裂促进因子,可传递发育的信号促进软骨细胞分裂,同时也是软骨细胞形态发生和分化的诱导因子,参与软骨的生长发育和组织损伤修复过程,特别是在软骨细胞的增殖分化起到重要作用,在解决软骨工程中面临的问题以及治疗骨关节炎等研究中具有的参考意义。本文对bFGF对软骨的分化、增殖、凋亡等不同生物学阶段的影响作用做一个综述。     

表皮形态发生素对肝细胞癌SK-HEP-1细胞生物学活性的影响

目的：明确表皮形态发生素（EPM）对盱细胞癌SK-HEP-1细胞生物学行为的影响。方法：构建高表达EPM的SK-HEP-1细胞,real-timePCR和Westem印迹检测EPM在肿瘤细胞内的表达,CCK8分析和克隆形成实验检测细胞的增殖能力,Matrigel-transwell实验检测细胞的浸润能力。结果：EPM在肿瘤细胞内的高表达不影响细胞的增殖能力,怛明显增强肿瘤细胞的浸润能力。结论：肝癌肿瘤微环境有可能通过EPM影响肿瘤细胞的生物学活性,对其作用机制的进一步明确,将有助于阐明肝癌发生发展的病理机制,发现新的恶性肿瘤诊断和治疗手段。     

尼氟酸对晚期内皮祖细胞生物学特性的影响

目的:探讨ClCa通道抑制剂--尼氟酸(Niflumic,NFA)对大鼠骨髓来源的晚期内皮祖细胞(endothelial progenitor cells,EPCs)生物学特性的影响。方法:密度梯度离心法分离大鼠骨髓单核细胞,应用EGM-2完全培养液进行体外培养,以第三代或第四代的晚期EPCs作为靶细胞,应用RT-PCR检测晚期EPCs上是否存在Cl Ca通道标志基因TMEM16A和Cl Ca4的表达。采用CCK-8法、Ed U标记法、划痕实验、Boyden小室实验及Matrigel法分别检测10μmol/L NFA对细胞增殖、迁移及体外血管形成能力的影响;应用荧光定量PCR及流式细胞术检测内皮分化标志v WF和CD31基因及蛋白的表达。结果:晚期EPCs表达ClCa通道标志基因TMEM16A和ClCa4;NFA抑制晚期EPCs的迁移功能(P0.05);但对EPCs的增殖、分化及成血管能力有促进作用。NFA上调了晚期EPCs的CD31和v WF基因和蛋白表达。结论:NFA能促进EPCs的增殖、分化及成血管能力,抑制EPCs的迁移能力。NFA对EPCs生物学特性的这类影响将为心血管疾病治疗药物选择方面提供一定的参考依据。     

Mesenchymal stem cells induce dermal fibroblast responses to injury

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.     

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

ABSTRACT: BACKGROUND: Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC), satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2) is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS). RESULTS: Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL) region of the lumbar spinal cord (LSC) in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM) resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. CONCLUSION: We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.     

Cellular responses and expression profiling of human bone marrow stromal cells stimulated with enamel matrix proteins in vitro

Objectives:  The aim of this study was to investigate biological effects and gene expression profiles of enamel matrix proteins (EMPs), on human bone marrow stromal cells (HBMSCs), for preliminary understanding of mechanisms involved in promoting periodontal regeneration by EMPs.
Materials and methods:  EMPs were extracted using the acetic acid method, and HBMSCs from human bone marrow aspirates were cultured. Attachment levels, level of cells morphologically attenuated, cell proliferation, alkaline phosphatase (ALP) activity and staining of HBMSCs were measured in the absence and in the presence of EMPs. Microarray analysis was performed to detect gene profiles of HBMSCs by treatment with 200 μg/ml EMPs, for 5 days. Four differential genes were selected for validation of the microarray data using real-time PCR.
Results:  EMPs promoted proliferation and ALP activity of HBMSCs in a time- and dose-dependent manner, and at a concentration of 200 μg/ml significantly enhanced proliferation and ALP expression. However, there were no significant changes between EMP-treated groups and the control group in cell attachment and cell process attenuation levels. Twenty-seven genes were differentially expressed by HBMSCs in the presence of EMPs. Expressions of 18 genes were upregulated and expressions of nine genes were found to be downregulated. There was good consistency between data obtained from the validation group and microarray results.
Conclusions:  EMPs promoted cell proliferation and differentiation and gene expression profiles of HBMSCs were affected. This may help elucidation of mechanisms involved in promoting regeneration of periodontal tissues by EMPs.     

骨唾液酸蛋白基因沉默抑制乳腺癌细胞与骨基质粘附

旨在探索骨唾液酸蛋白 (Bone sialoprotein,BSP) 基因沉默对亲骨转移乳腺癌细胞 (MDA-MB-231BO) 与骨基质粘附能力的影响,为以BSP为靶点的乳腺癌骨转移预防和靶向治疗提供实验依据。体外检测BSP基因沉默对乳腺癌细胞与小鼠骨基质粘附能力的影响,MTS法检测细胞增殖能力;扫描电镜观察骨片表面肿瘤细胞粘附情况和骨吸收状况;ELISA法检测骨基质细胞粘附培养上清中TGF-β1和RANKL表达分泌量差异;左心室注射法构建裸鼠骨转移模型,检测不同细胞株在裸鼠体内转移能力。结果提示BSP     

FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Silencing stromal interaction molecule 1 by RNA interference inhibits the proliferation and migration of endothelial progenitor cells

Knockdown of stromal interaction molecule 1 (STIM1) significantly suppresses neointima hyperplasia after vascular injury. Endothelial progenitor cells (EPCs) are the major source of cells that respond to endothelium repair and contribute to re-endothelialization by reducing neointima formation after vascular injury. We hypothesized that the effect of STIM1 on neointima hyperplasia inhibition is mediated through its effect on the biological properties of EPCs. In this study, we investigated the effects of STIM1 on the proliferation and migration of EPCs and examined the effect of STIM1 knockdown using cultured rat bone marrow-derived EPCs. STIM1 was expressed in EPCs, and knockdown of STIM1 by adenoviral delivery of small interfering RNA (siRNA) significantly suppressed the proliferation and migration of EPCs. Furthermore, STIM1 knockdown decreased store-operated channel entry 48 h after transfection. Replenishment with recombinant human STIM1 reversed the effects of STIM1 knockdown. Our data suggest that the store-operated transient receptor potential canonical 1 channel is involved in regulating the biological properties of EPCs through STIM1. STIM1 is a potent regulator of cell proliferation and migration in rat EPCs and may play an important role in the biological properties of EPCs.     

DDR2基因缺失对小鼠骨髓间充质干细胞生物学特性的影响

目的:骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)是骨再生工程中重要的种子细胞,它对骨组织缺损的修复有着良好的效果。但是BMSCs向成骨细胞分化并修复骨组织缺损是是由细胞外因子共同作用产生的结果。DDR2(Discoidin Domain Receptor 2)作为I型胶原的特异性受体在成骨细胞的分化中发挥重要的调节作用。而对于其在BMSCs向成骨细胞的分化过程中的所起到的作用还鲜有研究,对其作用机理尚不明确。因此我们希望通过分离、培养并鉴定比较DDR2基因缺失小鼠与野生型小鼠来源的骨髓间充质干细胞了解其生物学特性,为后续的实验奠定理论基础。方法:采用改良型的全骨髓贴壁细胞分离方法分离培养两种小鼠来源的骨髓间充质干细胞,采用流式细胞技术鉴定其表面标记物的表达,并利用诱导培养液诱导骨髓间充质干细胞向成骨细胞和成脂肪细胞分化。结果:分离培养的两种骨髓间充质干细胞形态一致,增殖能力和自我更新能力强,流式细胞术检测其表面标记物CD29,Sca-1均表达阳性,CD105,CD45表达为阴性,分离得到的两种细胞均有向成骨细胞和成脂肪细胞分化的能力,但可以明显观察到DDR2基因缺失小鼠的骨髓间充质干细胞的成骨分化能力减弱。结论:本实验通过对于DDR2基因缺失小鼠BMSCs分离、培养和鉴定,初步探索DDR2基因缺失在在成骨过程中的作用结果,为进一步研究提高BMSCs的成骨分化能力奠定理论基础。经实验证明,DDR2基因缺失小鼠来源的骨髓间充质干细胞虽然仍具备干细胞的生物学特性,但其向成骨细胞的分化能力明显减弱,说明DDR2基因缺失对其骨髓间充质干细胞的成骨分化等有着重要的影响。     

Live attenuated Salmonella carrying platelet factor 4 cDNAs as radioprotectors

To determine whether live attenuated Salmonella carrying platelet factor 4 cDNAs can protect mice from radiation damage, the attenuated Salmonella SL3261 was used as oral vector for targeted gene delivery. The recovery of mice receiving sublethal total-body irradiation (TBI) was investigated after the oral administration of attenuated Salmonella carrying cDNA for platelet factor 4 (PF4) or truncated PF4. This oral gene therapy protected mice from radiation damage after TBI. The number of bone marrow cells and high proliferative potential colony-forming cells (HPP-CFCs) increased significantly at day 7. Similarly, the administration of PF4 or PF4(17-70) protein also improved the survival of mice after TBI. Both PF4 gene therapy and protein administration accelerated hematopoietic recovery in vivo in mice after irradiation. In vitro, PF4 also promoted survival and proliferation of 5-fluorouracil-resistant hematopoietic stem/progenitor cells after irradiation. These data demonstrate a novel biological function of PF4 as a protector against radiation injury and suggest that attenuated Salmonella could be used in vivo as a PF4 DNA delivery vector in the management of radiation injury.