大鼠椎间盘巢源性干细胞对退化髓核细胞作用的研究

The repair effects of ISN-SCs on senescence nucleus pulposus cells

中文关键词:  椎间盘退变  髓核细胞  衰老  修复

英文关键词:Intervertebral disc degeneration  Nucleus pulposus cell  Senescence  Repair

中文摘要:

  【摘要】 目的：研究大鼠椎间盘巢源性干细胞（stem cells derived from ISN，ISN-SCs）对衰老髓核细胞（nucleus pulposus cells，NPCs）的作用。方法：取10只SD大鼠（雄性，10周龄），处死后分离获取脊柱功能单位，显露髓核及椎间盘干细胞巢（stem cell niches of the intervertebral disc，ISN）区域，解剖显微镜下仔细分离获取髓核和ISN组织，采用Ⅱ型胶原酶消化、细胞滤网过滤后，将原代ISN-SCs和NPCs分别重悬于培养液，接种于普通培养箱培养，细胞达约90%融合后进行传代，第三代（P3）NPCs及第四代（P4）ISN-SCs用于进一步实验。采用三系诱导分化（成骨、成软骨、成脂肪）培养液对P4 ISN-SCs分别进行定向诱导分化培养，采用茜素红、钙钴染色检测其成骨分化能力，阿利新蓝染色检测其成软骨分化能力，油红O染色检测其成脂肪分化能力；采用连续传代法，将P3 NPCs连续传代，制备NPCs衰老模型，衰老相关β-半乳糖苷酶（senescence associated-β-galactosidase，SA-β-Gal）染色法检测衰老模型的有效性。研究将细胞培养体系进一步分为正常对照组、衰老组、共培养组，正常对照组采用P3NPCs接种，衰老组采用衰老NPCs接种，共培养组采用P4 ISN-SCs与衰老NPCs以1∶1混匀后接种，各组样本细胞总数及培养环境相同。培养1周后，采用甲苯胺蓝染色检测各组蛋白聚糖表达水平，采用免疫组化检测各组Ⅱ型胶原蛋白表达水平，采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction，qPCR）检测各组ACAN和COL2a1的mRNA表达水平。结果：在特定诱导环境下，ISN-SCs可以定向成骨、成软骨、成脂肪分化。SA-β-Gal染色显示连续传代后NPCs衰老率随着传代次数而逐渐增加，P3 NPCs未出现明显的衰老[衰老率（3.0±0.5）%]，P5 NPCs衰老率为（18.3±0.7）%，P10 NPCs达到严重衰老标准[衰老率为（86.0±4.6）%]。胞外基质蛋白检测结果显示：与正常对照组相比，衰老组的蛋白聚糖甲苯胺蓝染色明显减弱，Ⅱ型胶原免疫组化染色强度定量也显著减弱（P<0.05）；而接触共培养后，与衰老组相比，共培养组中的蛋白聚糖甲苯胺蓝染色明显增强，Ⅱ型胶原免疫组化染色强度定量显著增强（P<0.05）并恢复至正常水平。qPCR结果显示：与正常对照组相比，衰老组的ACAN和COL2a1的mRNA表达水平均显著性降低（分别为1.00±0.05 vs 0.43±0.03，P<0.05；1.00±0.03 vs 0.40±0.02，P<0.05）；接触共培养后，与衰老组相比，共培养组中ACAN和COL2a1的mRNA表达水平均显著性增加（分别为0.43±0.03 vs 0.99±0.05，P<0.05；0.40±0.02 vs 1.07±0.04，P<0.05），且与正常组相比无显著性差异。结论：ISN-SCs具有较好的三系分化能力，通过接触共培养能够提高胞外基质蛋白和基因的表达水平，对衰老NPCs产生良好的修复作用，为后续ISN-SCs修复椎间盘的体内研究打下理论基础。

英文摘要:

  【Abstract】 Objectives: To investigate the repair effects of ISN-SCs(stem cells derived from stem cell niches of intervertebral disc) on senescence NPCs(nucleus pulposus cells). Methods: 10 Sprague-Dawley rats(male, 10-week-old) were used for this study. The functional spinal units were isolated after the animals being sacrificed. Then, the tissues of ISN (stem cell niches of the intervertebral disc) and nucleus pulposus were isolated anatomically under dissecting microscope. After being digested and filtrated, the harvested primary ISN-SCs and NPCs were resuspended, seeded and cultured under normal condition. The cells were passaged, and P4 ISN-SCs and P3 NPCs were used for further experiments. P4 ISN-SCs were cultured in specific differentiation induction medium (osteogenesis, chondrogenesis and adipogenesis) for 1 induction period before being tested. Alizarin red staining and calcium cobalt staining were used for detecting osteogenesis, alcian blue staining was used for detecting chondrogenesis, and Oil red O staining was used for detecting adipogenesis. The senescence model of NPCs was established by continuous passage, and the effectiveness of model was detected by the staining of senescence associated-β-galactosidase(SA-β-Gal). The cell culture systems were divided into 3 groups: normal control group, senescence group, and co-culture group. Treatments were set as follows P3 NPCs were seeded in the normal control group, senescence NPCs were seeded in the senescence group, and in the co-culture group, P4 ISN-SCs and senescence NPCs were mixed at the blending ratio 1:1 and seeded, total cell number and the culture environment of each specimen were the same. After being cultured for 7 days, further detections were conducted in each group, including toluidine blue staining for aggrecan, immumohistochemical staining for type Ⅱ collagen, and quantitative real-time polymerase chain reaction(qPCR) for the mRNA expression level of ACAN and COL2a1, which were compared among groups. Results: Osteogenic, chondrogenic and adipogenic differentiation of ISN-SCs were observed under the certain induction environment respectively. SA-β-Gal staining indicated that NPCs gradually became senescence as passage increased, with no obvious senescence cells in passage 3(3.0±0.5)%, less than 20% senescence cells in passage 5(18.3±0.7)%, and more than 80% senescence cells in passage 10 (86.0±4.6)%. When compared to the normal control group, the senescence group expressed less aggrecan and type Ⅱ collagen (quantification, P<0.05). While after co-culturing, the co-culture group expressed more aggrecan and type Ⅱ collagen (quantification, P<0.05) when compared to the senescence group, which was comparable to the normal control group. QPCR results showed that the mRNA expression levels of ACAN and COL2a1 in senescence group significantly decreased when compared to the normal control group (1.00±0.05 vs 0.43±0.03, P<0.05 and 1.00±0.03 vs 0.40±0.02, P<0.05, respectively), and that the mRNA expression levels of ACAN and COL2a1 in co-culture group were significantly increased when compared to the senescence group (0.43±0.03 vs 0.99±0.05, P<0.05 and 0.40±0.02 vs 1.07±0.04, P<0.05, respectively), which was comparable to the normal control group. Conclusions: ISN-SCs possess excellent multilineage differentiation capacities. The senescence NPCs can be remarkably repaired in co-culture model, through elevating the expression levels of extra cellular matrix protein and mRNA. ISN-SCs can be utilized for further research on the repair of intervertebral disc degeneration.