EXPERIMENTAL STUDY ON TRANSFECTION OF PLEIOTROPHIN GENE INTO ADIPOSE-DERIVED STEM CELLS OF MICE_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CHEN Changjin ¹ , YUAN Hui ¹ , GU Haiying ¹ , YANG Xuemei ¹ , ZHAO Ziyi ²

1Central Laboratory of Chengdu University of Traditional Chinese Medicine, Chengdu Sichuan, 610075, P.R.China;;
2Alan Garen Laboratory, West China Second University Hospital, Sichuan University. Corresponding author: CHEN Changjin, E-mail: cjczhzha2002@yahoo.com;

Corresponding author：

Keywords：

Vascular tissue engineering; Adipose-derived stem cells; Transfection Pleiotrophin; Gene therapy; Mouse

DOI：

10.7507/1002-1892.20130199

Video：

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Objective To study the transfection and expression of pleiotrophin (Ptn) gene in mice adipose-derived stem cells (ADSCs) so as to provide a new approach for the treatment of ischemic injury. Methods ADSCs from clean inbred C57BL/6W mice (weighing, 15-20 g) were isolated and cultured in vitro. The cell surface markers (CD29 and CD44) of ADSCs were identified by flow cytometry. The ADSCs were transfected with plasmid pIRES2-LEGFPN1 (containing Ptn gene coding sequence) as experimental group (group A) and with plasmid pLEGFP-N1 (containing GFP gene coding sequence) as control group (group B). After ADSCs were transfected by different plasmids respectively, the cells containing Ptn gene were selected by G418 (the best selected concentration was 200 μg/mL), and the immunophenotype of the cells was identified by flow cytometry after transfection. Meanwhile, real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells. Results The mice ADSCs were isolated and cultured successfully in vitro. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%, respectively; the double positive rate of CD44 and CD29 was 93.6%. The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%, respectively after transfection of Ptn gene; the double positive rate of CD44 and CD29 was 93.4%. The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B (P lt; 0.05). Conclusion The ADSCs can be stablely transfected by Ptn gene, the transfected ADSCs can express PTN protein highly, which is a new idea for tissue engineering of vascular reconstruction.

Citation： CHEN Changjin,YUAN Hui,GU Haiying,YANG Xuemei,ZHAO Ziyi. EXPERIMENTAL STUDY ON TRANSFECTION OF PLEIOTROPHIN GENE INTO ADIPOSE-DERIVED STEM CELLS OF MICE. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(8): 897-903. doi: 10.7507/1002-1892.20130199 Copy

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2.	Wakayama T, Rodriguez I, Perry ACF, et al. Mice cloned from embryonic stem cells. Proc Natl Acad Sci USA, 1999, 96(26): 14984-14989.
3.	Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res, 2005, 65(8): 3035-3039.
4.	McGrath J, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science, 1983, 220(4603): 1300-1302.
5.	Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by dened factors. Cell, 2006, 126(4): 663-676.
6.	孙超, 李定国. 基因修饰干细胞移植治疗研究现状. 国外医学: 内科学分册, 2006, 33(6): 264-267.
7.	Milner PG, Li YS, Hoffman RM, et al. A novel 17 kD heparin-binding growth factor (HBGF-8) in bovine uterus: purification and N-terminal amino acid sequence. Biochem Biophys Res Commun, 1989, 165(3): 1096-1103.
8.	Choudhuri R, Zhang HT, Donnini S, et al. An angiogenic role for the neurokines midkine and pleiotrophin in tumorigenesis. Cancer Res, 1997, 57(9): 1814-1819.
9.	马文丽, 李凌. 分子生物学实验手册. 北京: 人民军医出版社, 2011: 139-141.
10.	梁爽, 袁桂峰, 钟毓娟, 等. 小鼠脂肪干细胞的免疫原性及移植安全性 . 中国组织工程研究, 2012, 16(1): 47-50.
11.	Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cell from liposuction aspirates. Cytotherapy, 2004, 6(1): 7-14.
12.	Cook SD, Baffes GC, Wolfe MW, et al. Recombinant human bone morphogenetic protein-7induces healing in a canine long-bone segmental devect model. Clin Rothop, 1994, (301): 302-312.
13.	祝联, 陈付国, 刘伟, 等. 骨髓基质干细胞转染腺病毒BMP7后的成骨改变. 中华整形外科杂志, 2006, 22(1): 59-62.
14.	Wagner J, Akerud P, Csdtro DS, et al. Induction of a midbrain dopaminergic phenotype in Nurr1-ovrexpressing neural stem cells by type 1 astrocytes. Nat Biotechnol, 1999, 17(7): 653-659.
15.	Nakajima-Nagata N, Sakurai T, Mitaka T, et al. In vitro induction of adult hepatic progenitor cells into insulin-producing cells. BiochemBiophys Res Commun, 2004, 318(3): 625-630.
16.	Grierson I, Heathcote L, Hiscott P, et al. Hepatocyte growth factor/scatter facto I the eye. Prog Retin Eye Res, 2000, 19(6): 779-802.
17.	Ferraa N, Dvais-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev, 1997, 18(1): 4-25.
18.	陈菊祥,唐榕, 应康, 等. 肿瘤转移相关基因的表达谱研究. 第二军医大学学报, 2000, 21(9): 806-811, F003.
19.	Duenne AA, Werner JA. Functional anatomy of lymphatic vessels under the aspect of tumor invasion. Recent Results Cancer Res, 2002, 157: 82-89.
20.	Sharifi BG, Zeng Z, Wang L, et al. Pleiotrophin induces transdifferentiation of monocytes into functional endothelial cells. Arterioscler Thromb Vasc Biol, 2006, 26(6): 1273-1280.
21.	Zeng Z, Yuan X, Liu G, et al. Manipulation of proliferation and differentiation of human bone marrow-derived neural stem cells in vitro and in vivo. J Neurosci Res, 2007, 85(2): 310-320.
22.	Chen H, Campbell RA, Chang Y, et al. Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis. Blood, 2009, 113(9): 1992-2002.
23.	吴海霞, 张维铭, 李晓眠. 基因转染技术的发展与现状. 国际生物医学工程杂志, 2007, 30(6): 376-379.
24.	Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA, 1987, 84(21): 7413-7417.
25.	Wang D, Jing NH, Lin QS. Stearylamine liposome as a new efficient reagent for DNA transfection of eukaryotic cells. Biochem Biophys Res Commun, 1996, 226(2): 450-455.

1. Kleinsmith LJ, Pierce GB Jr. Multipotentiality of single embryonal carcinaoma cells. Caner Res, 1964, 24: 1544-1551.
2. Wakayama T, Rodriguez I, Perry ACF, et al. Mice cloned from embryonic stem cells. Proc Natl Acad Sci USA, 1999, 96(26): 14984-14989.
3. Rubio D, Garcia-Castro J, Martin MC, et al. Spontaneous human adult stem cell transformation. Cancer Res, 2005, 65(8): 3035-3039.
4. McGrath J, Solter D. Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science, 1983, 220(4603): 1300-1302.
5. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by dened factors. Cell, 2006, 126(4): 663-676.
6. 孙超, 李定国. 基因修饰干细胞移植治疗研究现状. 国外医学: 内科学分册, 2006, 33(6): 264-267.
7. Milner PG, Li YS, Hoffman RM, et al. A novel 17 kD heparin-binding growth factor (HBGF-8) in bovine uterus: purification and N-terminal amino acid sequence. Biochem Biophys Res Commun, 1989, 165(3): 1096-1103.
8. Choudhuri R, Zhang HT, Donnini S, et al. An angiogenic role for the neurokines midkine and pleiotrophin in tumorigenesis. Cancer Res, 1997, 57(9): 1814-1819.
9. 马文丽, 李凌. 分子生物学实验手册. 北京: 人民军医出版社, 2011: 139-141.
10. 梁爽, 袁桂峰, 钟毓娟, 等. 小鼠脂肪干细胞的免疫原性及移植安全性 . 中国组织工程研究, 2012, 16(1): 47-50.
11. Aust L, Devlin B, Foster SJ, et al. Yield of human adipose-derived adult stem cell from liposuction aspirates. Cytotherapy, 2004, 6(1): 7-14.
12. Cook SD, Baffes GC, Wolfe MW, et al. Recombinant human bone morphogenetic protein-7induces healing in a canine long-bone segmental devect model. Clin Rothop, 1994, (301): 302-312.
13. 祝联, 陈付国, 刘伟, 等. 骨髓基质干细胞转染腺病毒BMP7后的成骨改变. 中华整形外科杂志, 2006, 22(1): 59-62.
14. Wagner J, Akerud P, Csdtro DS, et al. Induction of a midbrain dopaminergic phenotype in Nurr1-ovrexpressing neural stem cells by type 1 astrocytes. Nat Biotechnol, 1999, 17(7): 653-659.
15. Nakajima-Nagata N, Sakurai T, Mitaka T, et al. In vitro induction of adult hepatic progenitor cells into insulin-producing cells. BiochemBiophys Res Commun, 2004, 318(3): 625-630.
16. Grierson I, Heathcote L, Hiscott P, et al. Hepatocyte growth factor/scatter facto I the eye. Prog Retin Eye Res, 2000, 19(6): 779-802.
17. Ferraa N, Dvais-Smyth T. The biology of vascular endothelial growth factor. Endocr Rev, 1997, 18(1): 4-25.
18. 陈菊祥,唐榕, 应康, 等. 肿瘤转移相关基因的表达谱研究. 第二军医大学学报, 2000, 21(9): 806-811, F003.
19. Duenne AA, Werner JA. Functional anatomy of lymphatic vessels under the aspect of tumor invasion. Recent Results Cancer Res, 2002, 157: 82-89.
20. Sharifi BG, Zeng Z, Wang L, et al. Pleiotrophin induces transdifferentiation of monocytes into functional endothelial cells. Arterioscler Thromb Vasc Biol, 2006, 26(6): 1273-1280.
21. Zeng Z, Yuan X, Liu G, et al. Manipulation of proliferation and differentiation of human bone marrow-derived neural stem cells in vitro and in vivo. J Neurosci Res, 2007, 85(2): 310-320.
22. Chen H, Campbell RA, Chang Y, et al. Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis. Blood, 2009, 113(9): 1992-2002.
23. 吴海霞, 张维铭, 李晓眠. 基因转染技术的发展与现状. 国际生物医学工程杂志, 2007, 30(6): 376-379.
24. Felgner PL, Gadek TR, Holm M, et al. Lipofection: a highly efficient lipid-mediated DNA transfection procedure. Proc Natl Acad Sci USA, 1987, 84(21): 7413-7417.
25. Wang D, Jing NH, Lin QS. Stearylamine liposome as a new efficient reagent for DNA transfection of eukaryotic cells. Biochem Biophys Res Commun, 1996, 226(2): 450-455.

Chinese Journal of Reparative and Reconstructive Surgery

EXPERIMENTAL STUDY ON TRANSFECTION OF PLEIOTROPHIN GENE INTO ADIPOSE-DERIVED STEM CELLS OF MICE

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