来自由软骨下骨成骨细胞直接转化为功能性软骨细胞及其关节软骨再生潜力的研究

Articular cartilage damage is a highly prevalent and impairing condition in the osteoarthritic joint, progressively worsening in association with continuous cartilage loss but a limited intrinsic capacity of self-healing. Current treatment options for full-thickness cartilage lesions are particularly insufficient. Whereas implantation of autologous chondrocytes from donor cartilage relies on the very restricted retrieval efficiency, commercially available therapies using exogenous stem cells have yet to show more complete morphological formation and functional recovery of native-like cartilage tissue. These limitations have motivated us to search within the joint for an alternative reliable self-cellular material and a therapeutic strategy that could offer a novel rationale for future approach to the regenerative healing of full-thickness cartilage defects in situ. Indeed, the only other functional cells in the lesion are the osteoblasts from the subchondral bone plate localized near the deep zone of the cartilage. We hypothesized that direct reprogramming of osteoblasts of the bare bone exposed may convert them into chondrocyte-like cells and evoke regenerative potential toward forming neo-cartilaginous tissue that would heal the defect. In the present study, we are aiming at establishing a procedure to directly convert subchondral bone osteoblasts (SBO) into functional chondrocytes by transducing a set of defined transcription factors and implanting the cells over the damaged area to allow formation of neo-cartilaginous tissue. We thus have first isolated the mouse knee cartilage and subchondral bone of the hindlimb of mice and performed real-time PCR and DNA microarray to detect changes of differentially expressed transcription factor genes between samples of the two different tissue origins. We have also screened candidate transcription factors for conversion into chondrocyte-like cells from the cell line created from the primary culture of SBO. Efficacy of the direct conversion will be next verified on freshly isolated SBO cells. We have also established a novel mouse model for full-thickness articular cartilage lesion by means of the micro-cutting technique we recently developed. Both cell morphology and gene expression profiles as well as functionality will be analyzed on the induced chondrogenic cell line after transduction of SBO with defined transcription factors. Further, SBO transduced with defined transcription factors will be implanted into the articular cartilage defects created in mice and any hyaline cartilage formation will be determined. We believe that, by direct conversion of nearby osteoblasts into reparative chondrogenic cells and functional cartilage, our approach may eventually lead to the development of a new effective in-situ regenerative treatment regimen for full-thickness cartilage defect in subjects who suffer from osteoarthritis and other joint diseases.

关节软骨损伤发病率日益增加，但有限的软骨自愈能力致使损伤不断加重。除全关节转换术外，目前尚缺乏修复软骨全层缺损恢复关节功能的有效治疗方法。鉴于自体软骨细胞和外源干细胞移植的局限性，我们积极寻找更为适合的种子细胞。基于软骨全层缺损暴露缺损底部软骨下骨的现象，以缺损面上唯一细胞类型软骨下骨成骨细胞（SBO）作为软骨再生的细胞来源可能是修复治疗全层缺损的新希望。为此，本项目拟研究利用细胞重编程技术诱导SBO向功能性软骨细胞直接转化的治疗策略。前期工作中，我们进行了转录组差异表达分析，并通过SBO细胞系筛选出重编程所需的候选转录因子组合。本项目将开展诱导因子组合优化，提高重编程效率，在小鼠软骨全层缺损位点接种重编程启动后的SBO验证体内形成新生软骨组织潜力等一系列研究。我们希望在NSFC资助下，通过该项研究将为基于毗邻细胞的原位组织修复技术的临床转化奠定基础，为重度软骨缺损患者带来崭新机遇。

关节软骨分解和软骨下骨板暴露常见于大多数骨关节炎疾病，而找到关键细胞类型满足晚期软骨损伤组织再生的迫切需要仍悬而未决。除关节软骨细胞外，唯一主要靠近全层软骨病变的功能细胞是软骨深区下方的软骨下成骨细胞。而两种细胞都来自共同祖细胞，这为采用软骨下成骨细胞作为ACC的新来源提供了细胞与分子基础。为此，我们建立一个重编系统，筛选可诱导转分化的转录因子（TF）。cDNA微阵列表明，超过20个基因具有高度软骨特异性和随着年龄的增长而显着下调的特性。经初步筛选后，我们根据同时诱导II型胶原蛋白和聚蛋白多糖的表达，确定了一些具有重编效应的TF，并最终优化了含有Sox9和Sox5的两个3-TF组合，分别联合Plagl1或c-Myc后表现出重编倾向，即深层成熟软骨细胞和表层前体细胞表型，且据具有形成软骨微球能力。该研究结果对开发用于重建全层软骨缺损的新型原位再生策略具有重要意义。作为淋巴细胞重要组成部分的T细胞，在炎症过程中具有关键作用，可使骨关节炎中的ACC退变。T细胞经分化形成大量可进入外周组织的效应细胞和少数局限于循环且无效应功能的中枢记忆细胞。因此，限制效应细胞的产生或将它们分化为记忆细胞，有可能消弱炎症反应。我们发现具有免疫调节和抑炎作用的天然药物产品小檗碱显着抑制了效应细胞增殖，而同时显着促进了记忆分化；这主要是通过激活的AMPK和Stat5进行协调。因此，这项研究将有助于对天然产物的免疫调节作用及其在治疗自身免疫性疾病和可能改善组织再生方面潜在机制的理解。基于纳米技术的药物输送系统正在成为改善药物吸收/生物利用度的有效方法。我们设计了一种用于组织靶向小分子药物递送系统的微纳米封装混合递送系统。该系统通过将载药的聚（乳酸-乙醇酸）纳米颗粒（PLP）封装到微环境敏感的药物预包埋聚丙烯酸甲酯（PMA）基质中，形成由游离药物和含药PLP组成的复合微粒。一旦进入适当的环境，游离小分子可几乎立即完全释放发挥作用，而PLP所含药物随后可进行持续释放，提供长期药效。混合递送系统代表了将治疗（如重编程因子或抗炎分子）递送至靶组织或靶细胞的新型药物传输手段。选择恰当的软骨生成细胞类型的重要性在于可能产生奇效。此外，结合纳米制剂、微胶囊、纳米胶束或活载体等先进的递送技术，装载生物效应更好的化合物或特别是天然产物，可能有助于那些总是伴有炎性的缺损软骨组织再生的靶向治疗。

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