基于Integrin信号通路的生物化学和生物力学双模式刺激抑制软骨细胞去分化

The in-vitro expanded chondrocytes are more likely to dedifferentiate. The cells lose their typical phenotype and reduce the production of extracellular matrix (ECM). After implantation, the newly formed cartilage is prone to degeneration, which can affect the functional outcomes.We once used siRNA to inhibit the expression of MMP for preventing dedifferentiation.We also found that multiple stimulations through different ways might be more efficient to inhibit dedifferentiaion.Both biochemical and biomechanical stimulations could be transmitted through Integrin singaling pathway.In this study, Ras and Sox9 were selected as target genes according to reports and our primary data. The lentiviral vector-mediated transgenic Sox9 and shRNA suppressing Ras were constructed.Then it was used to transfect the expanded chondrocytes.The transfected cells were loaded into alginate-collagen-hyaluroan gel particle and transferred to bioreactor to receive biomechanical stimulation. The biomechancial and biochemical stimulations in this system were enhanced each other to reduce the dedifferentiation.The interaction between these two stimulation ways was also assesed.Thereafter,the expanded chondrocytes were loaded onto scaffolds to repair cartilage defects in rabbit model. The degeneration of newly formed cartilage and dedifferentiation of chondrocytes were investigated. This study will optimize the techniques of cartilage tissue engineering and improve the clinical therapeutic effects.

体外扩增的软骨细胞易发生去分化，导致表型丧失，特异性细胞外基质分泌减少，植入体内后再生软骨易发生退变，影响疗效。申请人既往使用小干扰RNA抑制MMP表达对抗软骨细胞去分化。研究中发现多途径对抗去分化可能更为有效。有关Integrin信号通路的研究显示该途径可传导生物化学和生物力学两种信号。本研究针对该信号通路的不同节点构建了Ras shRNA和Sox9基因双表达慢病毒载体，用其转染软骨细胞，将转染后的细胞接种于海藻酸钠-II型胶原-透明质酸凝胶球上并放在生物反应器内进行力学刺激。分别从生物化学和生物力学两种途径对Integrin信号通路的不同靶点进行干预，对抗去分化；同时研究生物化学和生物力学信号通路间的相互作用。将双模式刺激后的软骨细胞同支架复合、植入体内修复软骨缺损，观察对抗软骨细胞去分化和抑制再生软骨退变作用。本研究有助于优化软骨组织工程技术，提高临床疗效。

体外扩增的软骨细胞易发生去分化，导致表型丧失，特异性细胞外基质分泌减少，植入体 内后再生软骨易发生退变，影响疗效。申请人既往使用小干扰RNA抑制MMP表达对抗软骨细 胞去分化。研究中发现多途径对抗去分化可能更为有效。有关Integrin信号通路的研究显 该途径可传导生物化学和生物力学两种信号。本研究针对该信号通路的不同节点构建了 Ras shRNA和Sox9基因双表达慢病毒载体，用其转染软骨细胞，将转染后的细胞接种于海 藻酸钠-II型胶原-透明质酸凝胶球上并放在生物反应器内进行力学刺激。分别从生物化学和生物力学两种途径对Integrin信号通路的不同靶点进行干预，对抗去分化；同时研究生物化学和生物力学信号通路间的 相互作用。将双模式刺激后的软骨细胞同支架复合、植入 体内修复软骨缺损，观察对抗软骨细胞去分化和抑制再生软骨退变作用。结果表明：四种慢病毒均可成功转染软骨细胞，目的基因在转染的软骨细胞内正常表达，但转染效率还有待进一步提高；Sox 9过表达+Hras shRNA双基因病毒可成功转染软骨细胞，对目的基因的表达分别起到增强和抑制作用，但效果不及两种单基因慢病毒分别干预；含透明质酸钠的海藻酸钠水凝胶可以更好地维持软骨细胞表型，抑制软骨细胞去分化；培养液内含ITS可以有效促进软骨细胞的增殖和活力，但海藻酸钠水凝胶球和/或培养液中添加ITS对抑制软骨细胞去分化无明显作用。本研究有助于优化软骨组织工程技术，提高临床疗效。