人肺泡骨膜间充质干细胞的分离及维生素 D 对骨膜衍生细胞成骨活性的影响
Summary
本文提出了一种研究维生素 c (维生素 c) 和125二羟基维生素 D [125-(OH)2d3] 诱导的骨膜衍生细胞 (PDCs) mRNA 表达标志物的协议。此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。
Abstract
间充质干细胞 (MSCs) 存在于各种组织中, 可分化为多种细胞类型, 包括成骨细胞。在骨髓间充质干细胞的牙科来源中, 骨膜是一种易于接触的组织, 被确定为包含层间的 mscs。然而, 这个来源还没有得到广泛的研究。
维生素 D3和 125-(OH)2D3已被证明可以刺激体外诱导干细胞分化为成骨细胞。此外, 维生素 C 有助于胶原蛋白的形成和骨骼细胞的生长。然而, 还没有研究维生素 D3和维生素 C 对 MSCs 的影响。
本文提出了一种从人肺泡骨膜中分离 MSCs 的方法, 并对 125-(OH)2D3可能对这些细胞施加骨诱导影响的假说进行了研究。我们还研究了骨髓间充质干细胞在人肺泡骨膜中的存在, 并评估了干细胞黏附和增殖。要评估维生素 C (作为控制) 和各种浓度的 125-(OH)2D3 (10−10) 的能力, 10−9, 10−8, 10−7 M) 改变关键 mRNA 生物标志物采用实时聚合酶链反应 (rt-pcr) 测定了碱性磷酸酶、骨涎蛋白 (BSP)、核结合因子 alpha-1 (CBFA1)、collagen-1 和骨钙素 (OCN) 的分离 MSCs mRNA 表达。
Introduction
虽然近年来已开发了许多相关技术, 但骨重建仍然受到多种限制因素的限制, 估计必要重建的程度往往是不可能的。除了获得良好的长期成功率外, 还需要硬组织增强来实现审美和功能目标。这种手术常用的方法包括自体和同种异体骨移植、xenografting 和异体骨移植。在各种类型的骨移植中, 自体骨移植被认为是最有效的。然而, 供体部位的发病率, 受损的血管, 和有限的组织可用性,1一直是主要的缺点, 自生骨移植。另外, 同种异体骨移植和异基因移植也与疾病传播有关。目前, 人工骨移植广泛用于解决这些问题。然而, 由于缺乏成骨潜能, 临床结局也有很大差异。纤维素等材料与体积波动、感染和强度缺乏有关。
用组织工程学进行骨隆增引起了相当大的兴趣。在这种技术中, 骨髓间充质干细胞 (MSCs) 最初用于促进成骨细胞分化, 然后移植到骨质丢失部位以实现骨修复。本程序目前已应用于细胞治疗。与其他方法相比, 提取有限数量的组织来实现骨重建更简单、更少侵入。
MSCs 作为一种用于牙科再生的细胞疗法的工具的潜在作用, 是各研究小组日益关注的问题之一。研究证实, MSCs 可以区别于以下类型的组织: 骨髓, 脂肪, 滑膜, 周细胞, 小梁骨, 人脐带和牙科组织 2, 3.MSCs 的常见来源包括骨髓、脂肪组织和牙科组织。与脂肪组织和骨髓间的干细胞相比, 牙干细胞的优点是容易获得, 收获后发病率较低。与胚胎干细胞相比, 来自牙科组织的 MSCs 出现 nonimmunogenic, 与复杂的伦理问题没有关联3。
在 2006年, 国际细胞治疗学会建议使用以下标准来识别 MSCs: 首先, mscs 必须能够附着在塑料上。其次, MSCs 必须是阳性的表面抗原 CD105, CD73, CD90 和阴性的标记, 单核, 巨噬细胞, 和 B 淋巴细胞除了造血抗原 CD45 和 CD344。作为最终标准, MSCs 必须能够在体外分化的标准条件下区分成以下三种类型的细胞: 成骨细胞、脂肪细胞和软骨组织4。迄今为止, 已有六种类型的人牙干细胞被分离和表征。第一种是从人牙髓组织中分离出来的, 称为产后牙髓干细胞5。随后, 三种其他类型的牙科干细胞被隔离并被描述为: 脱落的落叶牙齿的干细胞6、牙周韧带7和顶端的乳突8。最近, 牙科卵泡衍生的9, 牙龈组织衍生的10, 口腔芽干细胞 (DBSCs)11, 和根尖囊肿 mscs (hPCy-mscs)12也已确定。
Friedenstein 是第一个定义 MSCs13的。MSCs 具有很高的增殖潜能, 可以在移植前进行区分, 这表明它们是再生过程的理想候选者10。
尽管大多数研究都用骨髓作为干细胞的来源, 但最近14也使用了骨膜衍生细胞 (PDCs)。骨膜比骨髓更容易接近。因此, 在这项技术中, 我们使用肺泡骨膜, 以消除在手术中需要额外的切口和减少手术发病率的病人。骨膜是形成长骨骼外衬的结缔组织, 包括两个不同的层: 由成纤维细胞、胶原蛋白和弹性纤维组成的外层纤维层15, 直接接触的内富细胞形成层骨表面。形成层包含混合细胞种群, 主要是成纤维细胞16, 成骨细胞17, 毛细血管18, 以及一个关键亚群, 确定为 MSCs19,20,21。大多数研究报告说, PDCs 是可比的, 如果不是优越, 骨髓源干细胞的骨骼愈合和再生22,23,24。骨膜容易接近, 具有良好的再生效果。然而, 很少有研究聚焦于骨膜25、26、27。
在骨修复方面, 目前的临床实践涉及在支持性支架内扩增的骨膜祖细胞移植。最近的研究重点是获取有缺陷区域的干细胞, 并使用祖细胞进行组织再生20。牙医还预测牙周骨再生在牙周治疗和牙种植体中的应用前景。对于供体部位, 骨膜可以很容易地收获一般牙科外科医生。这与骨髓基质细胞比较有利, 因为在常规的口腔手术中可以访问骨膜。因此, 本研究的目的是建立一个捕捞 PDCs 的协议, 并评估人骨膜干细胞的形态、附着性、活力和增殖。
维生素 D 代谢物影响体内骨-矿物质动态平衡。一项研究报告说, 24 r、25-(OH)2D3活性维生素 D 对人骨髓间充质干细胞 (hMSCs)28的成骨细胞分化至关重要。骨骼稳态和修复由维生素 D3代谢产物网络调节, 其中 125-(OH)2D3 (骨化三醇) 是最具生物学活性和相关的骨骼健康的调节。维生素 D3对于钙化29至关重要。在一项使用2维昆明白鼠的研究中, 小鼠的胚体显示维生素 C 和维生素 d 补充剂有效促进了 ESC 型成骨细胞30的分化。在它的其他生物活动中, 125-(OH)2D3刺激体外hMSCs 与成骨细胞的分化, 可以根据碱性磷酸酶 (磷酸) 酶活性或 OCN 基因的增加来监测。表达。
很少有研究发现, 与维生素 C 和 125-(OH)2D3的联合治疗在人类 PDCs 中的剂量反应关系, 特别注重骨组织工程。因此, 在本研究中, 我们检查 125-(OH)2D3和维生素 C 的单一或联合治疗的最佳浓度, 以诱导人 PDCs 的成骨分化。本议定书的目的是确定从牙槽骨膜中分离出来的细胞是否包含有 MSC 表型的细胞, 以及这些细胞是否可以在培养中被扩展 (体外) 并分化形成所需的组织。.此外, 我们评估 PDCs 的能力, 以分化为骨, 软骨细胞和脂肪。研究的第二部分评估维生素 C 和 10−10的影响, 10−9, 10−8, 和 10−7 M 125-(OH)2D3 PDCs 的成骨活动。本研究的主要目的是评估维生素 C 和 125 (OH)2D3在 PDCs 的成骨细胞分化过程中的作用, 以及支持成骨细胞的基因, 如碱性磷酸酶、collagen-1、OCN、BSP 和 CBFA1。此外, 本研究根据这些结果确定了人类 PDCs 的最佳骨诱导条件。
Protocol
Representative Results
Discussion
最近发展起来的治疗方式, 即组织工程所涉及的 MSCs, 有许多优点。MSCs 是存在于几种组织类型中的多能细胞, 可分化成多种功能性胚层组织细胞37和其他细胞, 如成骨体。
骨膜充当祖细胞的利基, 作为骨骼的丰富的血管供应, 它包裹着38。在我们的研究中, 34 份调查样本中, 20 成功地取得了 MSC 的菌落, 成功的隔离率为58.8%。本研究根据其增殖和?…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项研究协议已获得长功夫纪念医院 (IRB99-1828B、100-3019C、99-3814B、102-1619C、101-4728B、103-4223C) 临床研究机构审查委员会批准。这项研究得到了长功夫纪念医院 (CMRPG392071、CMRPG3A1141、CMRPG3A1142 和 NMRPG3C0151) 的支持。这篇手稿是由华莱士的学术编辑编辑的。
Materials
| 0.25% trypsin-EDTA | Gibco | 25200-056 | |
| 2-phospho-L-ascorbic acidtrisodium salt | Sigma | 49752 | |
| 35-mm culture dishes | Corning | 430165 | |
| 3-isobutyl-1-methylxanthine | Sigma | I5879 | |
| 6 well plate | Corning | 3516 | |
| Alkaline phosphatase | ABI | Hs01029144_m1 | |
| Alkaline Phosphatase Activity Colorimetric Assay Kit | BioVision | K412-500 | |
| avian myeloblastosis virus reverse transcriptase | Roche | 10109118001 | |
| CD146 | BD | 561013 | |
| CD19 | BD | 560994 | |
| CD34 | BD | 560942 | |
| CD44 | BD | 561858 | |
| CD45 | BD | 561088 | |
| CD73 | BD | 561014 | |
| CD90 | BD | 561974 | |
| Cell banker1 | ZEAOAQ | 11888 | |
| core binding factor alpha-1 | ABI | Hs00231692_m1 | |
| dexamethasone | Sigma | D4902 | |
| DPBS | Gibco | 14190250 | |
| FBS | Gibco | 26140-079 | |
| GAPDH | ABI | Hs99999905_m1 | |
| HLA-DR | BD | 562008 | |
| indomethacin | Sigma | I7378 | |
| insulin | sigma | 91077C | |
| insulin–transferrin–selenium-A | Sigma | I1884 | |
| MicroAmp Fast 96 well reaction plate(0.1ml) | Life | 4346907 | |
| MicroAmp optical adhesive film | Life | 4311971 | |
| Minimum Essential Medium 1X Alpha Modification | HyClone | SH30265.02 | |
| Penicillin/Streptomycin | Gibco | 15140-122 | |
| Permeabilization buffer | eBioscience | 00-8333-56 | |
| Sodium pyruvate | Gibco | 11360070 | |
| STRO-1 | BioLegend | 340103 | |
| SYBER Green PCR Master Mix | AppliedBiosystems | 4309155 | |
| TaqMan Master Mix | Life | 4304437 | |
| transforming growth factor-β | Sigma | T7039 | |
| Trizol reagent (for RNA isolation) | Life | 15596018 | |
| β-glycerophosphate | Sigma | G9422 | |
| collagen-1 | Invitrogen | forward primer 5' CCTCAAGGGCTCCAACGAG-3 | |
| reverse primer 5'-TCAATCACTGTCTTGCCCCA-3' | |||
| OCN | Invitrogen | forward primer 5'-GTGCAGCCTTTGTGTCCAAG-3' | |
| reverse primer 5'-GTCAGCCAACTCGTCACAGT-3' | |||
| BSP | Invitrogen | forward primer 5' AAAGTGAGAACGGGGAACCT-3' | |
| reverse primer 5'-GATGCAAAGCCAGAATGGAT-3' | |||
| Commercial ALP primers | |||
| Commercial CBFA1 primers |
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