重組人BMP-2調節VEGF表達的濃度和時間依賴性研究_《中國修復重建外科雜志》

作者：

曹鑫 ¹ , 陳慧錦 ² , 崔正禮 ¹ , 金格勒 ² , 楊毅 ¹ , 王亞楠 ¹ , 肖偉 ¹

1勝利油田中心醫院骨科中心（山東東營，257000）;;
2新疆醫科大學第一附屬醫院VIP外二科;

關鍵詞：

重組人BMP-2 VEGF 脂肪來源干細胞生物學規律

DOI：

10.7507/1002-1892.20130200

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目的通過基因水平和蛋白水平研究不同誘導濃度以及不同誘導時間重組人BMP-2（recombinant human BMP-2，rhBMP-2）調節人脂肪來源干細胞（adipose-derived stem cells，ADSCs）表達VEGF的生物學特點。方法從成人脂肪組織中分離培養ADSCs。取第3代細胞，分別用終濃度為0、50、100、200 ng/mL rhBMP-2誘導，24 h后收集樣本；另采用濃度為100 ng/mL rhBMP-2誘導（rhBMP-2誘導組），3、6、12、18、24、36、48 h后收集樣本，并設未誘導的相應時間點細胞作為對照組；采用RT-PCR和ELISA檢測VEGF mRNA和蛋白表達。結果RT-PCR及ELISA檢測示，不同濃度rhBMP-2誘導下VEGF mRNA相對表達量和蛋白表達均呈濃度依賴性，其中100 ng/mL組表達最高。50、100、200 ng/mL組VEGF mRNA相對表達量均高于0 ng/mL組（P lt; 0.05）；100 ng/mL組VEGF mRNA相對表達量最高，與其余兩組比較差異有統計學意義（P lt; 0.05）。100 ng/mL組VEGF蛋白表達量顯著高于其余各組，差異均有統計學意義（P lt; 0.05）。100 ng/mL rhBMP-2誘導ADSCs后VEGF mRNA及蛋白表達呈時間依賴性。rhBMP-2誘導組誘導3、6 h時VEGF mRNA相對表達量及蛋白表達均低于對照組（P lt; 0.05）；12 h時VEGF表達較低，兩組比較差異無統計學意義（P gt; 0.05）；18、24 h達高峰，rhBMP-2誘導組顯著高于對照組（P lt; 0.05）；36、48 h 兩組VEGF表達均明顯減少，比較差異無統計學意義（P gt; 0.05）。結論rhBMP-2調節人ADSCs表達VEGF具有濃度依賴性和時間依賴性。rhBMP-2最適誘導濃度為100 ng/mL，誘導至18～24 h是利用rhBMP-2促進組織工程骨血管化的關鍵時間段。

引用本文： 曹鑫,陳慧錦,崔正禮,金格勒,楊毅,王亞楠,肖偉. 重組人BMP-2調節VEGF表達的濃度和時間依賴性研究. 中國修復重建外科雜志, 2013, 27(8): 904-909. doi: 10.7507/1002-1892.20130200 復制

1.	Novosel EC, Kleinhans C, Kluger PJ, et al. Vascularization is the key challenge in tissue engineering. Advanced Drug Delivery Reviews, 2011, 63(4-5): 300-311.
2.	徐紅珍, 蘇儉生. 組織工程骨的血管化研究進展. 中華臨床醫師雜志(電子版), 2010, 4(4): 456-458.
3.	Schubert T, Xhema D, Vériter S, et al. The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells. Biomaterials, 2011, 32(34): 8880-8891.
4.	Dae Kyun Park, Yi Hwa Ji, Eun-Sang Dhong, et al. Transplantation of rat adipose-derived stem cells (ASCs) to improve the survival of autologous aspirated fat grafts. Tissue Engineering and Regenerative Medicine, 2012, 9(2): 69-74.
5.	楊毅, 金格勒, 唐周舟, 等. 復合骨在兔腰椎融合過程中相關基因表達調控的影響. 中華實驗外科雜志, 2010, 27(2): 245-247.
6.	Trubiani O, Fulle S, Traini T, et al. Functional assay, expression of growth factors and proteins modulating bone-arrangement in human osteoblasts seeded on an anorganic bovine bone biomaterial. Eur Cells Mater, 2010, 20: 72-83.
7.	Kim SE, Song SH, Yun YP, et al. The effect of immobilization of heparin and bone morphogenic protein-2 (BMP-2) to titanium surfaces on inflammation and osteoblast function. Biomaterials, 2011, 32(2): 366-378.
8.	Hwang CJ, Vaccaro AR, Lawrence JP, et al. Immunogenicity of bone morphogenetic proteins. J Neurosurg Spine, 2009, 10(5): 443-451.
9.	Tumialán LM, Rodts GE. Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion. Spine J, 2007, 7(4): 509-510.
10.	Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 2011, 6(3): 300-311.
11.	Seebach C, Henrich D, Kähling C, et al. Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance earlyvascularization and bone healing in a critical-sized bone defect in rats. Tissue Eng Part A, 2010, 16(6): 1961-1970.
12.	Zou DH, Zhang ZY, He J, et al. Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1a mediated BMSCs. Biomaterials, 2012, 33(7): 2097-2018.
13.	Song I, Kim BS, Kim CS, et al. Effects of BMP-2 and vitamin D3 on the osteogenic differentiation of adipose stem cells. Biochem Biophys Res Commun, 2011, 408(1): 126-131.
14.	Yan MN, Dai KR, Tang TT, et al. Reconstruction of peri-implant bone defects using impacted bone allograft and BMP-2 gene-modified bone marrow stromal cells. J Biomed Mater Res Part A, 2010, 93(1): 304-313.
15.	傅德皓, 楊進華, 劉國輝, 等. 骨形態發生蛋白-2促進鼠胚成骨細胞血管內皮生長因子表達的機制研究. 中華創傷骨科雜志, 2007, 9(10): 964-966.
16.	金格勒, 任龍龍, 楊小豐, 等. 重組人骨形態發生蛋白-2/異體骨復合骨用于兔腰椎融合的正電子發射計算機體層攝影-CT研究. 中華創傷骨科雜志, 2010, 12(11): 1065-1068.
17.	Mladenov KV, Kunkel P, Stuecker R. The use of recombinant human BMP-2 as a salvage procedure in the pediatric spine: a report on 3 cases. Eur Spine J, 2010, 19 Suppl: S135-139.
18.	Carlisle E, Fischgrund JS. Bone morphogenetic proteins for spinal fusion. Spine J, 2005, 5(12): 240S-249S.
19.	Brower RS, Vickroy NM. A case of psoas ossification from the use of BMP-2 for posterolateral fusion at L4-L5. Spine (Phila Pa 1976), 2008, 33(18): E653-655.
20.	Perri B, Cooper M, Lauryssen C, et al. Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion: a case study. Spine J, 2007, 7(2): 235-239.
21.	Mroz TE, Wang JC, Hashimoto R, et al. Complications related to osteobiologics use in spine surgery: a systematic review. Spine (Phila Pa 1976), 2010, 35(9 Suppl): S86-104.

1. Novosel EC, Kleinhans C, Kluger PJ, et al. Vascularization is the key challenge in tissue engineering. Advanced Drug Delivery Reviews, 2011, 63(4-5): 300-311.
2. 徐紅珍, 蘇儉生. 組織工程骨的血管化研究進展. 中華臨床醫師雜志(電子版), 2010, 4(4): 456-458.
3. Schubert T, Xhema D, Vériter S, et al. The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells. Biomaterials, 2011, 32(34): 8880-8891.
4. Dae Kyun Park, Yi Hwa Ji, Eun-Sang Dhong, et al. Transplantation of rat adipose-derived stem cells (ASCs) to improve the survival of autologous aspirated fat grafts. Tissue Engineering and Regenerative Medicine, 2012, 9(2): 69-74.
5. 楊毅, 金格勒, 唐周舟, 等. 復合骨在兔腰椎融合過程中相關基因表達調控的影響. 中華實驗外科雜志, 2010, 27(2): 245-247.
6. Trubiani O, Fulle S, Traini T, et al. Functional assay, expression of growth factors and proteins modulating bone-arrangement in human osteoblasts seeded on an anorganic bovine bone biomaterial. Eur Cells Mater, 2010, 20: 72-83.
7. Kim SE, Song SH, Yun YP, et al. The effect of immobilization of heparin and bone morphogenic protein-2 (BMP-2) to titanium surfaces on inflammation and osteoblast function. Biomaterials, 2011, 32(2): 366-378.
8. Hwang CJ, Vaccaro AR, Lawrence JP, et al. Immunogenicity of bone morphogenetic proteins. J Neurosurg Spine, 2009, 10(5): 443-451.
9. Tumialán LM, Rodts GE. Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion. Spine J, 2007, 7(4): 509-510.
10. Novosel EC, Kleinhans C, Kluger PJ. Vascularization is the key challenge in tissue engineering. Adv Drug Deliv Rev, 2011, 6(3): 300-311.
11. Seebach C, Henrich D, Kähling C, et al. Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance earlyvascularization and bone healing in a critical-sized bone defect in rats. Tissue Eng Part A, 2010, 16(6): 1961-1970.
12. Zou DH, Zhang ZY, He J, et al. Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1a mediated BMSCs. Biomaterials, 2012, 33(7): 2097-2018.
13. Song I, Kim BS, Kim CS, et al. Effects of BMP-2 and vitamin D3 on the osteogenic differentiation of adipose stem cells. Biochem Biophys Res Commun, 2011, 408(1): 126-131.
14. Yan MN, Dai KR, Tang TT, et al. Reconstruction of peri-implant bone defects using impacted bone allograft and BMP-2 gene-modified bone marrow stromal cells. J Biomed Mater Res Part A, 2010, 93(1): 304-313.
15. 傅德皓, 楊進華, 劉國輝, 等. 骨形態發生蛋白-2促進鼠胚成骨細胞血管內皮生長因子表達的機制研究. 中華創傷骨科雜志, 2007, 9(10): 964-966.
16. 金格勒, 任龍龍, 楊小豐, 等. 重組人骨形態發生蛋白-2/異體骨復合骨用于兔腰椎融合的正電子發射計算機體層攝影-CT研究. 中華創傷骨科雜志, 2010, 12(11): 1065-1068.
17. Mladenov KV, Kunkel P, Stuecker R. The use of recombinant human BMP-2 as a salvage procedure in the pediatric spine: a report on 3 cases. Eur Spine J, 2010, 19 Suppl: S135-139.
18. Carlisle E, Fischgrund JS. Bone morphogenetic proteins for spinal fusion. Spine J, 2005, 5(12): 240S-249S.
19. Brower RS, Vickroy NM. A case of psoas ossification from the use of BMP-2 for posterolateral fusion at L4-L5. Spine (Phila Pa 1976), 2008, 33(18): E653-655.
20. Perri B, Cooper M, Lauryssen C, et al. Adverse swelling associated with use of rh-BMP-2 in anterior cervical discectomy and fusion: a case study. Spine J, 2007, 7(2): 235-239.
21. Mroz TE, Wang JC, Hashimoto R, et al. Complications related to osteobiologics use in spine surgery: a systematic review. Spine (Phila Pa 1976), 2010, 35(9 Suppl): S86-104.

《中國修復重建外科雜志》

重組人BMP-2調節VEGF表達的濃度和時間依賴性研究

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《中國修復重建外科雜志》

重組人BMP-2調節VEGF表達的濃度和時間依賴性研究

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