Kakutani Kenichiro, Nishida Kotaro, Uno Koki, Takada Toru, Shimomura Takatoshi, Maeno Koichiro, Kurosaka Masahiro, Doita Minoru
Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan.
J Orthop Res. 2006 Jun;24(6):1271-8. doi: 10.1002/jor.20171.
To investigate the efficacies and the longevity of RNA interference in nucleus pulposus cells from rat and human, two reporter luciferase plasmids (Firefly and Renilla) were used. These plasmids were cotransfected with siRNA targeting Firefly luciferase to the nucleus pulposus cells extracted from Sprague Dawley rats and scoliosis patients. The inhibitory effects were evaluated by dual luciferase assay for 3 weeks. Proliferation activity of fibroblast-like cells extracted from the subcutaneous tissue of Sprague Dawley rats and the nucleus pulposus cells were measured by proliferation assay (WST-8 assay) every 2 days after plating. The expression of Firefly luciferase was drastically inhibited both in rats (94.7%) and in humans (93.7%). The inhibitory effects were maintained for 2 weeks and had disappeared completely by 3 weeks. The proliferation activity of nucleus pulposus cells was significantly lower than fibroblast-like cells. We have shown, for the first time, siRNA-mediated gene silencing in rat and human disc cells for a relatively sustained period, probably due to the stability of the nucleus pulposus cells in terms of cell proliferation. The demonstration of this study may allow further exploration of the use of siRNA for scientific research and the treatment of disc degenerative diseases.
为了研究RNA干扰在大鼠和人类髓核细胞中的效果及持续时间，使用了两种报告荧光素酶质粒（萤火虫荧光素酶和海肾荧光素酶）。这些质粒与靶向萤火虫荧光素酶的小干扰RNA（siRNA）共转染到从Sprague Dawley大鼠和脊柱侧弯患者提取的髓核细胞中。通过双荧光素酶测定法评估3周的抑制效果。接种后每2天通过增殖测定法（WST-8测定法）测量从Sprague Dawley大鼠皮下组织提取的成纤维样细胞和髓核细胞的增殖活性。萤火虫荧光素酶的表达在大鼠（94.7%）和人类（93.7%）中均受到显著抑制。抑制效果维持了2周，到3周时完全消失。髓核细胞的增殖活性明显低于成纤维样细胞。我们首次证明了siRNA介导的基因沉默在大鼠和人类椎间盘细胞中能持续相对较长时间，这可能是由于髓核细胞在细胞增殖方面的稳定性。本研究的结果可能有助于进一步探索siRNA在科研和椎间盘退行性疾病治疗中的应用。
