模拟早期椎间盘疾病的发炎性、退行性器官培养模型。
Summary
该协议提出了一种新的实验模型的发炎，退化牛器官培养，以模拟早期椎间盘退化。
Abstract
症状性椎间盘 （IVD） 退化 （IDD） 是一个主要的社会经济负担，其特征是炎症和组织退化。由于缺乏因果疗法，迫切需要创新的实验器官培养模型，以研究疾病进展中涉及的机制，寻找治疗目标，减少对动物模型的需求。我们在这里展示一个新的，三维的器官培养模型协议，模仿发炎和催化微环境，这是在DD期间存在。
最初，牛的静脉注射器在组织培养介质中被解剖、清洁和培养。动态生理或病理负荷应用于定制生物反应器，每天2小时。IVDs被分配给一个对照组（高葡萄糖介质，生理负荷，磷酸盐缓冲盐水注射）和病理组（低葡萄糖介质，病理负荷，肿瘤坏死因子-阿尔法注射）四天。对IVD采集的细胞核细胞进行基因表达分析，对条件器官培养介质进行酶相关免疫分析。
我们的数据表明，与对照组相比，在病理组加载后，炎症标记的表达更高，圆盘高度降低。此协议是可靠的模拟IVD炎症和退化，可以进一步扩大其应用范围。
Introduction
背痛 （LBP） 可以影响所有年龄段的个人，是全世界残疾的主要原因1，2，3.与LBP相关的总成本每年超过1000亿美元4，5。症状性椎间盘 （IVD） 退化 （IDD）， 一种以炎症和组织退化为特征的疾病， 是 LBP6，7的主要原因 .具体来说，IDD 的特点是 IVD 的细胞外基质 （ECM） 逐渐分解，由导致加速病理学、神经紊乱并最终残疾的多种因素诱导和触发。此外，IDD与释放发炎细胞因子、改变脊柱生物力学、血管生成和神经内生有关，这增加了疼痛感，共引起慢性LBP（主动不和谐）6，8。迄今为止，治疗方案包括切除术和随后融合相邻的椎骨，植入IVD假肢，或非手术方法，如非类固醇抗炎药物，阿片类药物，和肌肉松弛剂的IDD9患者。目前的标准治疗方案，外科和非手术，只是部分有效，未能解决潜在的生物学问题9，10。早期退行性椎间盘疾病的特点是初始炎症组织反应，特别是肿瘤坏死因子-阿尔法（TNF-阿尔法）表达11的增加。这些早期的椎间盘变化主要发生在细胞水平，而不会破坏光盘架构，以前可以模仿营养缺乏在亲炎症条件下12。因此，精确模拟体内情况，以调查这些退化机制，并找到合适的治疗目标至关重要。此外，在这些分子特性的模拟中，光盘的机械加载环境在IVD的病理和生理变化中起着关键作用。因此，结合这些方法将使我们向前迈出一步，以模拟体内静脉注射器的复杂微环境。目前没有研究考虑动态加载的方面，以及亲炎症和营养设置，以最好的我们所知。
虽然大型动物模型允许调查潜在的相关体内相互作用，他们是昂贵和工作密集型。此外，由于动物模型在研究中的使用长期以来一直是一个有争议的问题，因此减少回答重要研究问题所需的动物数量是人们非常感兴趣的。最后，目前还没有理想的动物模型模仿IDD在IVD研究13，14。因此，有必要建立一个具有成本效益和可靠的替代方法，例如一种器官培养模型，以模拟 IDD 以及相关的炎症和退行性过程。最近，本议定书的应用，建立一个发炎和退行性器官培养模型，以模拟早期椎间盘疾病，使我们能够研究抗炎药物在DD器官培养15的影响。
在这里，我们描述了如何获得牛椎间盘，并通过直接 α在低营养介质条件下在生物反应器中产生催化和增生微环境诱导早期 IDD 状态。 图1 说明了实验模型，并显示了用于模拟退行性和生理负荷条件的生物反应器。
图1：实验设置的插图。A：牛尾巴： B：解剖牛间椎间盘： C：将光盘转移到具有文化媒介的井板： D：在生物反应器中加载模拟： E：静脉注射技术： F： 注射PBS/三潘蓝色染料后IVD显示分布。IDD：椎间盘退化。 请单击此处查看此图的较大版本。
Protocol
实验是使用从当地屠宰场获得的牛尾巴进行的。本研究中使用的生物材料取自食物链，无需瑞士和欧洲法律的道德批准。 1. 牛椎间盘解剖 用自来水彻底冲洗整个尾巴，去除表面上的污垢和头发。注：具有完好的解剖端，每尾最多可使用 9 IVD（共管 1-9）用于实验，具体取决于 IVD 的预期大小。考虑到所需的直径在15-20毫米之间，我们使用12条牛尾，每尾5IVD进行实验。…
Representative Results
低血糖介质的退行性负荷与TNF-α注射相结合，导致蛋白标记物的基因表达显著增加，白细胞介质6（IL-6）和白细胞介质8（IL-8）与NP细胞中的生理对照组相比，经过4天的培养（图2）。相比之下，我们没有观察到NP细胞中蛋白1+（IL-1+）和TNF-α的发炎基因发生显著变化（未显示数据）。此外，退化培养条件并没有改变IL-6和IL-8在AF细胞中的基因表达。 <p class="jove_content" fo:keep-tog…
Discussion
我们在这里提供了一个详细的协议，以模拟退行性和炎症性IVDD。此协议可用于对导致对光盘的破坏性影响的炎症通路进行详细检查。此外，该协议可以帮助确定在疾病进展中涉及的有希望的治疗目标。
我们最近发现，人类重组TNF-α可诱发牛和人类NP细胞21的炎症，这是根据该领域的其他研究证实，TNF-α可用于炎症模拟IVD细胞22，23。…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 AO 基金会和 AO 斯平国际的支持。巴巴克·萨拉维得到了德国脊柱基金会和德国骨关节炎基金会的奖学金支持。格诺特·朗得到了德国弗赖堡大学医学院高级临床科学家贝尔塔-奥滕斯坦方案的支持。
Materials
| 1-Bromo-3-chloropropane(BCP) | Sigma-Aldrich, St. Louis, USA | B9673 | |
| Ascorbate-2-phosphate | Sigma-Aldrich, St. Louis, USA | A8960 | |
| Band saw | Exakt Apparatebau, Norderstedt, Germany | model 30/833 | |
| Betadine | Munndipharma, Frankfurt, Germany | ||
| Bovine IL-8 Do.it-Yourself ELISA | Kingfisher Biotech, St. Paul, USA | DIY1028B-003 | |
| Corning ITS Premix | Corning Inc., New York, USA | 354350 | |
| DMEM high glucose | Gibco by life technologies, Carlsbad, USA | 10741574 | |
| DMEM low glucose | Gibco by life technologies, Carlsbad, USA | 11564446 | |
| Ethanol for molecular biology | Sigma-Aldrich, St. Louis, USA | 09-0851 | |
| Fetal Bovine Serum (FBS) | Gibco by life technologies, Carlsbad, USA | A4766801 | |
| Non-essential amino acid solution | Gibco by life technologies, Carlsbad, USA | 11140050 | |
| Penicillin/Streptomycin(P/S) | gibco by life technologies, Carlsbad, USA | 11548876 | |
| Phosphate Buffer Solution, tablet | Sigma-Aldrich, St. Louis, USA | P4417 | |
| Pronase | Sigma-Aldrich, St. Louis, USA | 10165921001 | |
| Primocin | InvivoGen, Sandiego, USA | ant-pm-05 | |
| Pulsavac Jet Lavage System | Zimmer, IN,USA | ||
| TissueLyser II | Quiagen, Venlo, Netherlands | 85300 | |
| Streptavidinn-HRP | Kingfisher Biotech, St. Paul, USA | AR0068-001 | |
| Superscript VILO | Invitrogen by life Technologies, Carlsbad, USA | 10704274 | |
| cDNA Synthesis Kit | Applied Biosystems by life technologies | 10400745 | |
| TaqMan Universal Master Mix | Applied Biosystems by life technologies | ||
| TNF-alpha, recombinant human protein | R&D systems, Minnesota, USA | 210-TA-005 | |
| TRI Reagent | Molecular Research Center, Cincinnati, USA | TR 118 | |
| Tris-EDTA buffer solution | sigma-Aldrich, St. Louis, USA | 93283 | |
| Gene bIL-6 | Applied Biosystems by life technologies | Custom made probes | Primer fw (5′–3′) TTC CAA AAA TGG AGG AAA AGG A Primer rev (5′–3′) TCC AGA AGA CCA GCA GTG GTT Probe (5′FAM/3′TAMRA) CTT CCA ATC TGG GTT CAA TCA GGC GATT |
| Gene bIL8 | Applied Biosystems by life technologies | Bt03211906_m1 | |
| Gene bTNF-alpha | Applied Biosystems by life technologies | Custom made probes | Primer fw (5′–3′) CCT CTT CTC AAG CCT CAA GTA ACA A Primer rev (5′–3′) GAG CTG CCC CGG AGA GTT Probe (5′FAM/3′TAMRA) ATG TCG GCT ACA ACG TGG GCT ACC G |
| GENE bIL1beta | Applied Biosystems by life technologies | Custom made probes | Primer fw (5′–3′) TTA CTA CAG TGA CGA GAA TGA GCT GTT Primer rev (5′–3′) GGT CCA GGT GTT GGA TGC A Probe (5′FAM/3′TAMRA) CTC TTC ATC TGT TTA GGG TCA TCA GCC TCA A |
| RPLP0 | Applied Biosystems by life technologies | Bt03218086_m1 |
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Citar este artículo
Saravi, B., Lang, G., Grad, S., Alini, M., Richards, R. G., Schmal, H., Südkamp, N., Li, Z. A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.. J. Vis. Exp. (168), e62100, doi:10.3791/62100 (2021).