多效生長因子基因轉染小鼠脂肪來源干細胞的實驗研究_《中國修復重建外科雜志》

作者：

陳昌金 ¹ , 袁惠 ¹ , 辜海英 ¹ , 楊雪梅 ¹ , 趙梓亦 ²

1 成都中醫藥大學附屬醫院中心實驗室（成都，610075）;;
2 四川大學華西第二醫院Alan Garen實驗室;

關鍵詞：

血管組織工程脂肪來源干細胞轉染多效生長因子基因基因治療小鼠

DOI：

10.7507/1002-1892.20130199

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目的探討多效生長因子基因（pleiotrophin，Ptn）轉染小鼠脂肪來源干細胞（adipose-derived stem cells，ADSCs）作為組織工程血管的重建種子細胞的可行性，并檢測其基因表達，為缺血性病損治療提供新手段。方法取3只清潔級近交系C57BL/6W雌性小鼠（體重15～20 g）腹股溝皮下脂肪，采用酶消化法體外分離、培養ADSCs，流式細胞儀行細胞表面標志物CD29、CD44鑒定。實驗分為轉染pIRES2-LEGFPN1組（含Ptn基因編碼序列，A組）和轉染pLEGFP-N1負對照組（含GFP基因編碼序列，B組）。ADSCs轉染后，A組以最佳篩選濃度200 μg/mL 的G418對轉染后細胞進行篩選，流式細胞儀鑒定轉染后細胞免疫表型。實時熒光定量PCR、Western blot檢測A、B組細胞Ptn基因和PTN蛋白表達。結果實驗成功分離、培養小鼠ADSCs；細胞表面標志物CD29、CD44陽性率分別達99.5%、95.8%，雙陽性率達93.6%。A組轉染Ptn后細胞表面標志物CD29、CD44陽性率分別達99.1%、95.6%，雙陽性率達93.4%。實時熒光定量PCR、Western blot檢測示，A組Ptn基因和PTN蛋白相對表達水平均顯著高于B組（P lt; 0.05）。結論Ptn基因能轉染小鼠ADSCs并高表達PTN蛋白，為組織工程血管構建種子細胞選擇提供了新思路。

引用本文： 陳昌金,袁惠,辜海英,楊雪梅,趙梓亦. 多效生長因子基因轉染小鼠脂肪來源干細胞的實驗研究. 中國修復重建外科雜志, 2013, 27(8): 897-903. doi: 10.7507/1002-1892.20130199 復制

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.	孫超, 李定國. 基因修飾干細胞移植治療研究現狀. 國外醫學: 內科學分冊, 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.	馬文麗, 李凌. 分子生物學實驗手冊. 北京: 人民軍醫出版社, 2011: 139-141.
10.	梁爽, 袁桂峰, 鐘毓娟, 等. 小鼠脂肪干細胞的免疫原性及移植安全性 . 中國組織工程研究, 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.	祝聯, 陳付國, 劉偉, 等. 骨髓基質干細胞轉染腺病毒BMP7后的成骨改變. 中華整形外科雜志, 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.	陳菊祥,唐榕, 應康, 等. 腫瘤轉移相關基因的表達譜研究. 第二軍醫大學學報, 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.	吳海霞, 張維銘, 李曉眠. 基因轉染技術的發展與現狀. 國際生物醫學工程雜志, 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. 孫超, 李定國. 基因修飾干細胞移植治療研究現狀. 國外醫學: 內科學分冊, 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. 馬文麗, 李凌. 分子生物學實驗手冊. 北京: 人民軍醫出版社, 2011: 139-141.
10. 梁爽, 袁桂峰, 鐘毓娟, 等. 小鼠脂肪干細胞的免疫原性及移植安全性 . 中國組織工程研究, 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. 祝聯, 陳付國, 劉偉, 等. 骨髓基質干細胞轉染腺病毒BMP7后的成骨改變. 中華整形外科雜志, 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. 陳菊祥,唐榕, 應康, 等. 腫瘤轉移相關基因的表達譜研究. 第二軍醫大學學報, 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. 吳海霞, 張維銘, 李曉眠. 基因轉染技術的發展與現狀. 國際生物醫學工程雜志, 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.

《中國修復重建外科雜志》

多效生長因子基因轉染小鼠脂肪來源干細胞的實驗研究

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《中國修復重建外科雜志》

多效生長因子基因轉染小鼠脂肪來源干細胞的實驗研究

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