插頭/翼狀螺旋轉錄因子C2基因慢病毒載體構建及其在兔BMSCs中的表達_《中國修復重建外科雜志》

作者：

尤武林 , 王坤正 , 段大鵬 , 王春生 , 樊立宏 , 劉瑞宇

西安交通大學醫學院第二附屬醫院骨一科（西安，710004）;

關鍵詞：

BMSCs 插頭/翼狀螺旋轉錄因子C2基因慢病毒基因治療兔

DOI：

10.7507/1002-1892.20130120

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目的構建插頭/翼狀螺旋轉錄因子C2（homo sapiens forkhead box C2，Foxc2）基因慢病毒載體并轉染兔BMSCs，檢測其在BMSCs中的表達，為進一步應用攜帶Foxc2基因的BMSCs移植治療股骨頭缺血性壞死奠定實驗基礎。方法通過RT-PCR法獲得人Foxc2基因片段，將該片段克隆至包含綠色熒光蛋白（green fluorescent protein，GFP）的慢病毒載體LV-GFP中，重組獲得Foxc2慢病毒質粒，將其與pGC-LV載體、pHelper1.0載體、pHelper2.0載體共轉染293T細胞，獲得Foxc2基因慢病毒載體；檢測病毒滴度。分離、培養兔BMSCs，以Foxc2基因慢病毒載體轉染第3 代BMSCs，通過熒光表達法判定最佳感染復數（multiplicity of infection，MOI），并以最佳MOI進行轉染，倒置熒光顯微鏡觀察、Western blot法檢測轉染1、3、7 d BMSCs中Foxc2的表達；對轉染后BMSCs行成骨誘導，茜素紅染色法觀察礦化物結節形成情況。結果成功構建Foxc2基因慢病毒載體，經酶切及測序鑒定完全正確，能轉染293T細胞并表達GFP，病毒滴度為2 × 108 TU/mL。Foxc2基因慢病毒轉染BMSCs的最佳MOI為200，轉染BMSCs 3 d后經免疫熒光檢測84.5% ± 4.8%的BMSCs可表達Foxc2。Western blot 結果顯示Foxc2在BMSCs中高表達，且在7 d內表達水平逐漸升高。成骨誘導培養2周后，茜素紅染色示細胞質中有大量紅色的鈣化基質沉積。結論成功構建并包裝獲得較高滴度的Foxc2基因慢病毒載體，慢病毒可高效并穩定轉染BMSCs，Foxc2表達增加，為基因治療股骨頭缺血性壞死奠定了基礎。

引用本文： 尤武林,王坤正,段大鵬,王春生,樊立宏,劉瑞宇. 插頭/翼狀螺旋轉錄因子C2基因慢病毒載體構建及其在兔BMSCs中的表達. 中國修復重建外科雜志, 2013, 27(5): 535-540. doi: 10.7507/1002-1892.20130120 復制

1.	Mont MA, Hangerford DS. Non-traumatic avasclar necrosis of femarol head. J Bone Joint Surg (Am), 1995, 77(1): 459-474.
2.	Jones JP Jr. Concepts of etiology and early pathogensis of osteonecrotisis. Instr Course Lect, 1994, 43: 499-512.
3.	McMahon JM, Conroy S, Lyons M, et al. Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors. Stem Cells Dev, 2006, 15(1): 87-96.
4.	Naldini L, Blomer U, Gage FH, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A, 1996, 93(21): 11382-11388.
5.	Cederberg A, Grønning LM, Ahrén B, et al. FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance. Cell, 2001, 106(5): 563-573.
6.	Kim SH, Cho K, Choi HS, et al. The forkhead transcription factor Foxc2 stimulates osteoblast differentiation. Biochem Biophys Res Commun, 2009, 386(3): 532-536.
7.	龐永剛, 陳文弦, 崔鵬程. 人骨髓間質干細胞的優化培養. 中國修復重建外科雜志, 2004, 18(3): 220-224.
8.	Tsakano-Murakami R, Tokunaga N, Kondo N, et al. Glucocorticoid inhibits bone regeneration after osteonecrosis of the femoral head in aged female rats. Tohoku J Exp Med, 2009, 217(1): 51-58.
9.	Kuribayashi M, Fujioka M, Takahashi KA, et al. Combination analysis of three polymorphism for predicting the risk for steroid-induced osteonecrosis of the femoral head. J Orthop Sci, 2008, 13(4): 297-303.
10.	Séguin C, Kassis J, Busque L, et al. Non-traumatic necrosis of bone (osteonecrosis) is associated with endothelial cell activation but not thrombophilia. Rheumatology (Oxford), 2008, 47(8): 1151-1155.
11.	Deten A, Volz HC, Clamors S, et al. Hematopoietic stem cells do not repair the infracted mouse heart. Cardiovasc Res, 2005, 65(1): 52-63.
12.	Priller J. Grenzgänger: adult bone marrow cells populate the brain. Histoehem Cell Biol, 2003, 120(2): 85-91.
13.	Davis KE, Moldes M, Farmer SR. The forkhead transcription factor Foxc2 inhibits white adipocyte differentiation. J Biol Chem, 2004, 279(41): 42453-42461.
14.	Pandya S, Klimatcheva E, Planelles V. Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opin Biol Ther, 2001, 1(1): 17-40.
15.	Relph K, Harrington K, Pandha H. Recent developments and current status of gene therapy using viral vectors in the United Kingdom. BMJ, 2004, 329 (7470): 839-842.
16.	Hargrove PW, Kepes S, Hanawa H, et al. Globin Lentiviral vector insertion can perturb the expression of endogenous genes in betathalassemic hematopoietic cells. Mol Ther, 2008, 16(3): 525-533.
17.	Migliaccio AR, Quarto R, Piacibello W. Cell therapy: filling the gap between basic science and clinical trials October 15-17, 2001, Rome, Italy. Stem Cells, 2003, 21(3): 348-356.
18.	Lou S, Gu P, Chen F, et al. The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Spragu-Dawley rats. Brain Res, 2003, 968(1): 114-121.
19.	Paul R, Haydon RC, Cheng HW, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine (Phila Pa 1976), 2003, 28(8): 755-763.
20.	Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol, 2000, 28(8): 875-884.

1. Mont MA, Hangerford DS. Non-traumatic avasclar necrosis of femarol head. J Bone Joint Surg (Am), 1995, 77(1): 459-474.
2. Jones JP Jr. Concepts of etiology and early pathogensis of osteonecrotisis. Instr Course Lect, 1994, 43: 499-512.
3. McMahon JM, Conroy S, Lyons M, et al. Gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors. Stem Cells Dev, 2006, 15(1): 87-96.
4. Naldini L, Blomer U, Gage FH, et al. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci U S A, 1996, 93(21): 11382-11388.
5. Cederberg A, Grønning LM, Ahrén B, et al. FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance. Cell, 2001, 106(5): 563-573.
6. Kim SH, Cho K, Choi HS, et al. The forkhead transcription factor Foxc2 stimulates osteoblast differentiation. Biochem Biophys Res Commun, 2009, 386(3): 532-536.
7. 龐永剛, 陳文弦, 崔鵬程. 人骨髓間質干細胞的優化培養. 中國修復重建外科雜志, 2004, 18(3): 220-224.
8. Tsakano-Murakami R, Tokunaga N, Kondo N, et al. Glucocorticoid inhibits bone regeneration after osteonecrosis of the femoral head in aged female rats. Tohoku J Exp Med, 2009, 217(1): 51-58.
9. Kuribayashi M, Fujioka M, Takahashi KA, et al. Combination analysis of three polymorphism for predicting the risk for steroid-induced osteonecrosis of the femoral head. J Orthop Sci, 2008, 13(4): 297-303.
10. Séguin C, Kassis J, Busque L, et al. Non-traumatic necrosis of bone (osteonecrosis) is associated with endothelial cell activation but not thrombophilia. Rheumatology (Oxford), 2008, 47(8): 1151-1155.
11. Deten A, Volz HC, Clamors S, et al. Hematopoietic stem cells do not repair the infracted mouse heart. Cardiovasc Res, 2005, 65(1): 52-63.
12. Priller J. Grenzgänger: adult bone marrow cells populate the brain. Histoehem Cell Biol, 2003, 120(2): 85-91.
13. Davis KE, Moldes M, Farmer SR. The forkhead transcription factor Foxc2 inhibits white adipocyte differentiation. J Biol Chem, 2004, 279(41): 42453-42461.
14. Pandya S, Klimatcheva E, Planelles V. Lentivirus and foamy virus vectors: novel gene therapy tools. Expert Opin Biol Ther, 2001, 1(1): 17-40.
15. Relph K, Harrington K, Pandha H. Recent developments and current status of gene therapy using viral vectors in the United Kingdom. BMJ, 2004, 329 (7470): 839-842.
16. Hargrove PW, Kepes S, Hanawa H, et al. Globin Lentiviral vector insertion can perturb the expression of endogenous genes in betathalassemic hematopoietic cells. Mol Ther, 2008, 16(3): 525-533.
17. Migliaccio AR, Quarto R, Piacibello W. Cell therapy: filling the gap between basic science and clinical trials October 15-17, 2001, Rome, Italy. Stem Cells, 2003, 21(3): 348-356.
18. Lou S, Gu P, Chen F, et al. The effect of bone marrow stromal cells on neuronal differentiation of mesencephalic neural stem cells in Spragu-Dawley rats. Brain Res, 2003, 968(1): 114-121.
19. Paul R, Haydon RC, Cheng HW, et al. Potential use of Sox9 gene therapy for intervertebral degenerative disc disease. Spine (Phila Pa 1976), 2003, 28(8): 755-763.
20. Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol, 2000, 28(8): 875-884.

《中國修復重建外科雜志》

插頭/翼狀螺旋轉錄因子C2基因慢病毒載體構建及其在兔BMSCs中的表達

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《中國修復重建外科雜志》

插頭/翼狀螺旋轉錄因子C2基因慢病毒載體構建及其在兔BMSCs中的表達

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