小干擾RNA沉默TNF-α表達并抑制骨溶解的實驗研究_《中國修復重建外科雜志》

作者：

余博 , 郝紹文 , 孫首選 , 郭浩輝 , 楊小春 , 馬曉軍 , 金群華

寧夏醫科大學總醫院骨三科（銀川，750004）;

關鍵詞：

關節置換骨溶解基因治療 TNF-α 重組腺病毒小干擾RNA 小鼠

DOI：

10.7507/1002-1892.20130216

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目的通過構建沉默TNF-α的重組腺病毒并觀察其對小鼠假體骨溶解模型的影響，探討基因治療人工關節假體周圍骨溶解的可能性。方法設計沉默TNF-α的小干擾RNA（small interfering RNA，siRNA）編碼序列的引物，擴增后利用RAPAD腺病毒包裝系統將該序列載入腺病毒，并構建出E1、E3區雙缺失的重組腺病毒Ad5-TNF-α-siRNA-CMVeGFP。取8～10周齡SPF級BABL/C雌性小鼠64只，體重20～25 g，隨機分為4組（n=16）。空白對照組（A組）制備假體模型，顆粒對照組（B組）、空病毒組（C組）、治療組（D組）制備假體松動骨溶解模型；造模第2周開始向關節腔內分別注入以下溶液（兩次各注射40 μL）：A組注入PBS溶液，24 h后再次注入；B、C、D組注入鈦顆粒懸濁液，24 h后分別注入PBS溶液、腺病毒Ad5 E1-CMVeGFP溶液（1 × 109 PFU/mL）、重組腺病毒Ad5-TNF-α-siRNA-CMVeGFP溶液（1 × 109 PFU/mL）；每隔2周重復1次至第10周。術后觀察小鼠一般情況，術后12周取材進行組織學觀察及Western blot檢測TNF-α表達。結果目的基因載入腺病毒載體后經雙切酶鑒定及DNA測序確定獲得陽性克隆，重組腺病毒 Ad5-TNF-α-siRNA-CMVeGFP成功轉染HEK293細胞。術后各組小鼠均存活至實驗完成。組織學觀察示，A組炎性細胞及破骨細胞少，骨質形成良好；B、C組見大量炎性細胞浸潤，破骨細胞多，骨質破壞明顯；D組少量炎性細胞浸潤，破骨細胞較B、C組少，未出現明顯骨質破壞。各組界膜厚度及破骨細胞計數均為A組 lt; D組 lt; B組 lt; C組，組間比較差異均有統計學意義（P lt; 0.05）。Western blot 檢測顯示各組均有TNF-α表達， A、B、C、D組TNF-α表達量分別為0.235 ± 0.022、0.561 ± 0.031、0.731 ± 0.037、0.329 ± 0.025，組間比較差異均有統計學意義（P lt; 0.05）。結論成功構建沉默TNF-α的重組腺病毒，并可有效沉默小鼠假體周圍組織中TNF-α的表達，從而抑制骨溶解。

引用本文： 余博,郝紹文,孫首選,郭浩輝,楊小春,馬曉軍,金群華. 小干擾RNA沉默TNF-α表達并抑制骨溶解的實驗研究. 中國修復重建外科雜志, 2013, 27(8): 994-999. doi: 10.7507/1002-1892.20130216 復制

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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.
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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.	陳志榮, 張亮, 吳興臨, 等. 依那西普對磨屑誘導骨溶解影響的實驗研究. 中國矯形外科雜志, 2008, 16(4): 285-287.
10.	王真, 高希武, 孫克寧, 等. 辛伐他汀防治人工關節無菌性松動的實驗研究. 中國修復重建外科雜志, 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. 陳志榮, 張亮, 吳興臨, 等. 依那西普對磨屑誘導骨溶解影響的實驗研究. 中國矯形外科雜志, 2008, 16(4): 285-287.
10. 王真, 高希武, 孫克寧, 等. 辛伐他汀防治人工關節無菌性松動的實驗研究. 中國修復重建外科雜志, 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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小干擾RNA沉默TNF-α表達并抑制骨溶解的實驗研究

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