Gene-mediated restoration of cartilage matrix by combination insulin-like growth factor-I/interleukin-1 receptor antagonist therapy
Gene-mediated restoration of cartilage matrix by combination insulin-like growth factor-I/interleukin-1 receptor antagonist therapy
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DOI：
10.1038/sj.gt.3302396
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发表时间：
2005-01-01
期刊：
影响因子：
5.1
通讯作者：
Ghivizzani, S
中科院分区：
文献类型：
--
作者：
Nixon, AJ;Haupt, JL;Ghivizzani, S
将生长因子基因增强的软骨基质合成与白细胞介素-1 受体拮抗蛋白 (IL-1Ra) 相结合，消除软骨基质降解，可能会减少并可能逆转滑膜炎和骨关节炎中的软骨损失。在体外共培养系统中研究了滑膜与两种可能作用于软骨稳态的基因共转导的可行性。单层培养的滑膜细胞与 E1 缺失的腺病毒载体共转导，一个包含受巨细胞病毒 (CMV) 启动子控制的 IGF-I 编码序列（200 感染复数 (moi)），第二个包含受 CMV 启动子控制的 IL-1Ra 序列（100 moi）。滑膜单层培养物的腺病毒-IGF-I (AdIGF-I)转导和AdIGF-I/AdIL-1Ra共转导导致IGF-I mRNA和配体表达增加，并且类似地AdIL-1Ra和AdIGF-I/AdIL-1Ra转导的培养物表达高水平的IL-1Ra。 Northern 分析证实了单个 mRNA 转录物的大小适合 IGF-I 和 IL-1Ra 转基因表达。使用滑膜细胞单层和软骨外植体共培养实验来检查转导的滑膜细胞表达的IGF-I和IL-1Ra蛋白对正常和IL-1耗尽的软骨的影响。转导的单层培养物在转导后48小时产生峰值培养基IGF-I含量114+/-20.2ng/ml和IL-1Ra水平241.8+/-10.5ng/ml。这些IGF-I浓度足以使在AdIGF-I和AdIGF-I/AdIL-1Ra转导的滑膜细胞条件化的培养基中培养的正常软骨的蛋白聚糖(PG)含量显着增加。 Interleukin-1暴露的软骨明显缺乏PG，并且这种分解代谢状态在AdIGF-I转导的培养物中部分逆转，并且通过AdIGF-I/AdIL-1Ra转导的滑膜共培养物完全逆转。这些数据表明，培养的滑膜细胞很容易被含有恢复关节健康活性的转基因的两种重组腺病毒载体共转导。 AdIL-1Ra 和 AdIGF-I 转基因大量表达，分泌产物在第 2 天达到治疗浓度。基质生物合成的增加使软骨 PG 含量恢复到正常水平。这些数据表明，滑膜共转导对于减轻与滑膜炎、损伤或早期关节炎相关的软骨软化可能具有重要价值。
Combination of growth factor gene-enhanced cartilage matrix synthesis with interleukin-1 receptor antagonist protein (IL-1Ra) abrogation of cartilage matrix degradation may reduce and possibly reverse cartilage loss in synovitis and osteoarthritis. The feasibility of cotransduction of synovial membrane with two such genes that may act on cartilage homeostasis was investigated in an in vitro coculture system. Cultured synoviocytes in monolayer were cotransduced with E1-deleted adenoviral vectors, one containing IGF-I coding sequence under cytomegalovirus (CMV) promoter control ( 200 multiplicities of infection (moi)), and the second containing IL-1Ra sequence under CMV promoter control ( 100 moi). Adenovirus-IGF-I (AdIGF-I) transduction and AdIGF-I/AdIL-1Ra cotransduction of synovial monolayer cultures resulted in increased IGF-I mRNA and ligand expression, and similarly AdIL-1Ra and AdIGF-I/AdIL-1Ra-transduced cultures expressed high levels of IL-1Ra. Northern analysis confirmed a single mRNA transcript of the appropriate size for both IGF-I and IL-1Ra transgene expression. Synovial cell monolayer and cartilage explant coculture experiments were used to examine the effects of IGF-I and IL-1Ra protein expressed by transduced synoviocytes on normal and IL-1-depleted cartilage. Transduced monolayer cultures produced peak medium IGF-I content of 114 +/- 20.2 ng/ml and IL-1Ra levels of 241.8 +/- 10.5 ng/ml at 48 h after transduction. These IGF-I concentrations were sufficient to produce significantly increased proteoglycan ( PG) content of normal cartilage cultured in medium conditioned by AdIGF-I and AdIGF-I/ AdIL-1Ra-transduced synoviocytes. Interleukin-1-exposed cartilage was markedly depleted of PG, and this catabolic state was partially reversed in AdIGF-I-transduced cultures and fully reversed by AdIGF-I/ AdIL-1Ra-transduced synovial cocultures. These data indicate that cultured synoviocytes are readily cotransduced by two recombinant adenoviral vectors containing transgenes active in restoring joint health. The AdIL-1Ra and AdIGF-I transgenes were abundantly expressed and the secreted products achieved therapeutic concentrations by day 2. The resulting increase in matrix biosynthesis returned cartilage PG content to normal levels. These data suggest that there may be significant value in cotransduction of synovial membrane to attenuate cartilage malacia associated with synovitis, injury, or early arthritis.