小干扰RNA沉默TNF-α表达并抑制骨溶解的实验研究_《中国修复重建外科杂志》

作者：

余博 , 郝绍文 , 孙首选 , 郭浩辉 , 杨小春 , 马晓军 , 金群华

宁夏医科大学总医院骨三科（银川，750004）;

关键词：

关节置换骨溶解基因治疗 TNF-α 重组腺病毒小干扰RNA 小鼠

DOI：

10.7507/1002-1892.20130216

视频：

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摘要 全文 图表 视频 参考文献 施引文献 补充材料

目的通过构建沉默TNF-α的重组腺病毒并观察其对小鼠假体骨溶解模型的影响，探讨基因治疗人工关节假体周围骨溶解的可能性。方法设计沉默TNF-α的小干扰RNA（small interfering RNA，siRNA）编码序列的引物，扩增后利用RAPAD腺病毒包装系统将该序列载入腺病毒，并构建出E1、E3区双缺失的重组腺病毒Ad5-TNF-α-siRNA-CMVeGFP。取8～10周龄SPF级BABL/C雌性小鼠64只，体重20～25 g，随机分为4组（n=16）。空白对照组（A组）制备假体模型，颗粒对照组（B组）、空病毒组（C组）、治疗组（D组）制备假体松动骨溶解模型；造模第2周开始向关节腔内分别注入以下溶液（两次各注射40 μL）：A组注入PBS溶液，24 h后再次注入；B、C、D组注入钛颗粒悬浊液，24 h后分别注入PBS溶液、腺病毒Ad5 E1-CMVeGFP溶液（1 × 109 PFU/mL）、重组腺病毒Ad5-TNF-α-siRNA-CMVeGFP溶液（1 × 109 PFU/mL）；每隔2周重复1次至第10周。术后观察小鼠一般情况，术后12周取材进行组织学观察及Western blot检测TNF-α表达。结果目的基因载入腺病毒载体后经双切酶鉴定及DNA测序确定获得阳性克隆，重组腺病毒 Ad5-TNF-α-siRNA-CMVeGFP成功转染HEK293细胞。术后各组小鼠均存活至实验完成。组织学观察示，A组炎性细胞及破骨细胞少，骨质形成良好；B、C组见大量炎性细胞浸润，破骨细胞多，骨质破坏明显；D组少量炎性细胞浸润，破骨细胞较B、C组少，未出现明显骨质破坏。各组界膜厚度及破骨细胞计数均为A组 lt; D组 lt; B组 lt; C组，组间比较差异均有统计学意义（P lt; 0.05）。Western blot 检测显示各组均有TNF-α表达， A、B、C、D组TNF-α表达量分别为0.235 ± 0.022、0.561 ± 0.031、0.731 ± 0.037、0.329 ± 0.025，组间比较差异均有统计学意义（P lt; 0.05）。结论成功构建沉默TNF-α的重组腺病毒，并可有效沉默小鼠假体周围组织中TNF-α的表达，从而抑制骨溶解。

引用本文： 余博,郝绍文,孙首选,郭浩辉,杨小春,马晓军,金群华. 小干扰RNA沉默TNF-α表达并抑制骨溶解的实验研究. 中国修复重建外科杂志, 2013, 27(8): 994-999. doi: 10.7507/1002-1892.20130216 复制

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2.	Boyce BF, Li P, Yao Z, et al. TNF-alpha and pathologic bone resorption. Keio J Med, 2005, 54(3): 127-131.
3.	Yang SY, Yu H, Gong W, et al. Murine model of prosthesis failure for the long-term study of aseptic loosening. J Orthop Res, 2007, 25(5): 603-611.
4.	Athanasou NA. Peri-implant pathology—relation to implant failure and tumor formation. J Long Term Eff Med Implants, 2007, 17(3): 193-206.
5.	Petruska J, Durcanský D, Makarevic A, et al. Morphological-functional characteristic of periarticular tissue after total hip arthroplasty: histological, cytochemical and electron microscopy aspects. Acta Chir Orthop Traumatol Cech, 2008, 75(5): 375-381.
6.	Romas E, Gillespie MT, Martin TJ. Involvement of receptor activator of NFkappaB ligand and tumor necrosis factor-alpha in bonedestruction in rheumatoid arthritis. Bone, 2002, 30(2): 340-346.
7.	Holding CA, Findlay DM, Stamenkov R, et al. The correlation of RANK, RANKL and TNF-alpha expression with bone loss volume and polyethylene wear debris around hip implants. Biomaterials, 2006, 27(30): 5212-5219.
8.	Sabokbar A, Kudo O, Athanasou NA. Two distinct cellular mechanisms of osteoclast formation and bone resorption in periptosthetic osteolysis. J Orthop Res, 2003, 21(1): 73-80.
9.	陈志荣, 张亮, 吴兴临, 等. 依那西普对磨屑诱导骨溶解影响的实验研究. 中国矫形外科杂志, 2008, 16(4): 285-287.
10.	王真, 高希武, 孙克宁, 等. 辛伐他汀防治人工关节无菌性松动的实验研究. 中国修复重建外科杂志, 2010, 24(5): 544-547.
11.	Herzog RW, Cao O, Srivastava A. Two decades of clinical gene therapy—success is finally mounting. Discov Med, 2010, 9(45): 105-111.
12.	Khare R, Chen CY, Weaver EA, et al. Advances and future challenges in adenoviral vector pharmacology and targeting. Curr Gene Ther, 2011, 11(4): 241-258.
13.	Johnson M, Huyn S, Burton J, et al. Differential biodistribution of adenoviral vector in vivo as monitored by bioluminescence imaging and quantitative polymerase chain reaction. Hum Gene Ther, 2006, 17(12): 1262-1269.
14.	Katayama K, Furuki R, Yokoyama H, et al. Enhanced in vivo gene transfer into the placenta using RGD fiber-mutant adenovirus vector. Biomaterials, 2011, 32(17): 4185-4193.
15.	Bakhtiyari S, Haghani K, Basati G, et al. SiRNA therapeutics in the treatment of diseases. Ther Deliv, 2013, 4(1): 45-57.
16.	Zheng X, Mao Q, Wang D, et al. A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector. J Biotechnol, 2011, 155(2): 135-139.
17.	Goodship AE, Blunn GW, Green J, et al. Prevention of strain-related osteopenia in aseptic loosening of hip prostheses using perioperative bisphosphonate. J Orthop Res, 2008, 26(5): 693-703.
18.	Wise LM, Waldman SD, Kasra M, et al. Effect of zoledronate on bone quality in the treatment of aseptic loosening of hip arthroplasty in the dog. Calcif Tissue Int, 2005, 77(6): 367-375.
19.	Ren W, Yang SY, Wooley PH. A novel murine model of orthopaedic wear-debris associated osteolysis. Scand J Rheumatol, 2004, 33(5): 349-357．.
20.	Zhang C, Tang T, Ren W, et al. Influence of mouse genetic background on wear particle-induced in vivo inflammatory osteolysis. Inflame Res, 2008, 57(5): 211-215．.
21.	McCaffrey AP, Fawcett P, Nakai H, et al. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Mol Ther, 2008, 16(5): 931-941.

1. Mäkelä KT, Eskelinen A, Pulkkinen P, et al. Total hip arthroplasty for primary osteoarthritis in patients fifty-five years of age or older. An analysis of the Finnish arthroplasty registry. J Bone Joint Surg (Am), 2008, 90(10): 2160-2170.
2. Boyce BF, Li P, Yao Z, et al. TNF-alpha and pathologic bone resorption. Keio J Med, 2005, 54(3): 127-131.
3. Yang SY, Yu H, Gong W, et al. Murine model of prosthesis failure for the long-term study of aseptic loosening. J Orthop Res, 2007, 25(5): 603-611.
4. Athanasou NA. Peri-implant pathology—relation to implant failure and tumor formation. J Long Term Eff Med Implants, 2007, 17(3): 193-206.
5. Petruska J, Durcanský D, Makarevic A, et al. Morphological-functional characteristic of periarticular tissue after total hip arthroplasty: histological, cytochemical and electron microscopy aspects. Acta Chir Orthop Traumatol Cech, 2008, 75(5): 375-381.
6. Romas E, Gillespie MT, Martin TJ. Involvement of receptor activator of NFkappaB ligand and tumor necrosis factor-alpha in bonedestruction in rheumatoid arthritis. Bone, 2002, 30(2): 340-346.
7. Holding CA, Findlay DM, Stamenkov R, et al. The correlation of RANK, RANKL and TNF-alpha expression with bone loss volume and polyethylene wear debris around hip implants. Biomaterials, 2006, 27(30): 5212-5219.
8. Sabokbar A, Kudo O, Athanasou NA. Two distinct cellular mechanisms of osteoclast formation and bone resorption in periptosthetic osteolysis. J Orthop Res, 2003, 21(1): 73-80.
9. 陈志荣, 张亮, 吴兴临, 等. 依那西普对磨屑诱导骨溶解影响的实验研究. 中国矫形外科杂志, 2008, 16(4): 285-287.
10. 王真, 高希武, 孙克宁, 等. 辛伐他汀防治人工关节无菌性松动的实验研究. 中国修复重建外科杂志, 2010, 24(5): 544-547.
11. Herzog RW, Cao O, Srivastava A. Two decades of clinical gene therapy—success is finally mounting. Discov Med, 2010, 9(45): 105-111.
12. Khare R, Chen CY, Weaver EA, et al. Advances and future challenges in adenoviral vector pharmacology and targeting. Curr Gene Ther, 2011, 11(4): 241-258.
13. Johnson M, Huyn S, Burton J, et al. Differential biodistribution of adenoviral vector in vivo as monitored by bioluminescence imaging and quantitative polymerase chain reaction. Hum Gene Ther, 2006, 17(12): 1262-1269.
14. Katayama K, Furuki R, Yokoyama H, et al. Enhanced in vivo gene transfer into the placenta using RGD fiber-mutant adenovirus vector. Biomaterials, 2011, 32(17): 4185-4193.
15. Bakhtiyari S, Haghani K, Basati G, et al. SiRNA therapeutics in the treatment of diseases. Ther Deliv, 2013, 4(1): 45-57.
16. Zheng X, Mao Q, Wang D, et al. A novel system for rapid screening of effective siRNA target sites by one step transfection with a single vector. J Biotechnol, 2011, 155(2): 135-139.
17. Goodship AE, Blunn GW, Green J, et al. Prevention of strain-related osteopenia in aseptic loosening of hip prostheses using perioperative bisphosphonate. J Orthop Res, 2008, 26(5): 693-703.
18. Wise LM, Waldman SD, Kasra M, et al. Effect of zoledronate on bone quality in the treatment of aseptic loosening of hip arthroplasty in the dog. Calcif Tissue Int, 2005, 77(6): 367-375.
19. Ren W, Yang SY, Wooley PH. A novel murine model of orthopaedic wear-debris associated osteolysis. Scand J Rheumatol, 2004, 33(5): 349-357．.
20. Zhang C, Tang T, Ren W, et al. Influence of mouse genetic background on wear particle-induced in vivo inflammatory osteolysis. Inflame Res, 2008, 57(5): 211-215．.
21. McCaffrey AP, Fawcett P, Nakai H, et al. The host response to adenovirus, helper-dependent adenovirus, and adeno-associated virus in mouse liver. Mol Ther, 2008, 16(5): 931-941.

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小干扰RNA沉默TNF-α表达并抑制骨溶解的实验研究

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