AAV-p65shRNA和AAV-BMP4联合应用抑制早期骨性关节炎软骨细胞退变的实验研究

Inflammation and repair of chondrocytes are the two main mechanisms underlying the cartilage degeneration of early phase of osteoarthritis. They play important role in triggering and inhibiting the process, respectively. Both inhibiting the inflammation and improving the repair of chondrocytes will slow down the process of cartilage degeneration. However, the relation between the inflammation and repair of OA chondrocytes is not clear as well as its' effect on cartilage degeneration. Our previous studies showed that BMP4 gene therapy can promote the repair of OA chondrocytes and deter the OA process. In the present study, we hypothesize that combined inhibiting the NF-KB inflammation signal by AAV-p65shRNA and promoting repair by AAV-BMP4 will yield the synergistic inhibiting effect of OA chondrocytes degeneration; to maximize the synergistic inhibiting effect needs certain ratio of AAV-p65shRNA and AAV-BMP4 transduction; based on the in vitro result, combined injection of certain ratio of AAV-p65shRNA and AAV-BMP4 particles into the sheep knee joint will inhibit the degeneration process of OA chondrocytes. In the present study, we will use AAV-p65shRNA and AAV-BMP4 to transduce human OA chondrocytes in vitro, and use 3D cell pellet culture to induce the transduced chondrocytes and study their chondrogenic differentiation. In vivo, we will inject both AAV-p65shRNA and AAV-BMP4 particles at certain ratio into the sheep OA knee and examine the cartilage degeneration at different time points. We believe that the combined application of certain ratio of AAV-p65shRNA and AAV-BMP4 will have an inhibiting synergistic effect on OA cartilage chondrocytes degeneration, which may enlighten a new therapy for the early stage of OA.

软骨细胞的炎症、修复是骨性关节炎早期病程中软骨细胞退变的两个重要机制，分别起着诱发、减缓该病程的作用。抑制炎症和促进修复均可减缓软骨细胞的退变，但目前尚不清楚炎症和修复两者的内在联系对其退变的影响。我们前期研究发现，BMP4基因治疗可促进软骨细胞修复而抑制其退变。本次研究中，我们提出三个假说：1）体外抑制NF-KB炎症信号并联合应用BMP4对软骨细胞的退变可产生协同抑制作用；2）体外协同抑制作用发挥最大化的条件是两者具有合理比例的作用强度；3）基于体外试验结果，关节腔内注射合理比例的两种病毒颗粒可减缓羊骨性关节炎的软骨细胞退变。我们拟采用体外转染人骨性关节炎软骨细胞AAV-p65shRNA和AAV-BMP4的方法， 经软骨细胞3D培养和羊关节腔内病毒颗粒注射试验，分别从体外和体内实验观察软骨细胞的退变，从而进一步揭示软骨细胞炎症和修复机制对软骨退变影响，探索治疗早期骨性关节炎的新方法。

膝关节的骨性关节炎（Osteoarthritis, OA）是一种常见的非对称性、无全身性征象的疾病，是骨科临床的常见病和多发病，多表现为膝关节的疼痛、畸形、不同程度的功能障碍，严重影响患者的生活质量和劳动能力。.目的：本研究希望能通过基因治疗技术对骨性关节炎早期病程中软骨细胞的炎症、修复过程进行调节，通过AAV-BMP4转染炎性软骨细胞促进其修复，而AAV-p65shRNA转染该细胞抑制NF-kb介导的炎性传导通路抑制软骨细胞的炎症。联合两者的作用，来揭示其治疗软骨细胞退变的机制，从而探索出治疗早期骨性关节炎的新方法.方法：采用体外培养人骨性关节炎的软骨细胞，分别转染腺病毒相关病毒AAV-BMP4和AAV-p65shRNA，经体外软骨细胞3D 培养技术和骨性关节炎大鼠体内关节腔内联合两种病毒颗粒注射试验，在不同时间点观察软骨细胞的退变和分化。.结果：体外培养的炎性软骨细胞，分别转染AAV-BMP4和AAV-P65shRNA后其软骨细胞标志物蛋白多糖，II型胶原表达增强，而两种病毒按1:1数目比例混合的细胞表现出的软骨分化能力最强。当比较AAV-BMP4和AAV-p65shRNA按1:1数目比例混合体外3D培养条件下，该组细胞成软骨能力要优于AAV-BMP4和AAV-p65shRNA按50:1, 5:1, 1:5, 1:50的比例配伍，具有统计学差异。当比较AAV-BMP4和AAV-p65shRNA的1:1组与5:1组的病毒，按不同指数级浓度注射入大鼠膝关节骨性关节炎模型的关节腔内的时候，结果显示AAV-BMP4和AAV-p65shRNA的1:1组的治疗效果最好，要优于5:1组，结果具有显著性差异。.结论：当应用AAV-BMP4和AAV-p65shRNA转染炎性软骨细胞具有诱导其逆分化的能力。当两者按1:1数目的比例联合应用时，体内和体外实验结果均表明其对软骨细胞的逆分化诱导作用具有协同作用，而且在1:1的情况下，该协同作用能最大化的发挥。