SCF 對體外轉染p EGFP2C1/ Akt 的骨髓間充質干細胞中c-kit 、Akt 及VEGF 表達的影響_《中國普外基礎與臨床雜志》

作者：

1.中國醫科大學附屬第一醫院干診科( 沈陽110001) ;;
2.大慶油田總醫院內分泌科(大慶163000) ;;
3.中國醫科大學附屬第一醫院循環科(沈陽110001) ;;
4.中國醫科大學附屬第一醫院內分泌科(沈陽110001);

關鍵詞：

骨髓間充質干細胞,Akt 基因,干細胞因子,血管內皮生長因子

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目的　克隆構建綠色熒光蛋白( GFP) / Akt 表達載體,觀察其在鼠骨髓間充質干細胞(MSCs) 中的表
達和定位,并探討干細胞因子( SCF) 通過PI3-Akt 途徑對轉染p EGFP-C1/ Akt 的MSCs 中c-kit 、Akt 和VEGF
mRNA及蛋白表達的影響。方法　采用酶切法將Akt 重組于GFP 表達載體中,經酶切序列鑒定后,將該重組質粒
GFP/ Akt 及GFP 空質粒通過脂質體轉染骨髓MSCs ; 熒光顯微鏡觀察其轉染及在細胞中的分布; 分別采用
Western blot 和反轉錄多聚酶鏈反應(RT-PCR) 方法檢測p EGFP-C1 和p EGFP-C1/ Akt 轉染MSCs 后對c-kit 、Akt
及VEGF mRNA 和蛋白表達的影響, p EGFP-C1/ Akt 轉染MSCs 后不同濃度SCF 對c-kit 、Akt 及VEGF
mRNA 和蛋白表達的影響。結果　GFP/ Akt 基因表達載體經酶切鑒定和測序分析后確認構建成功,并在MSCs
中表達。熒光顯微鏡下見p EGFP-C1 轉染后,綠色熒光均勻分布于整個細胞中; p EGFP-C1/ Akt 轉染后,綠色熒光
分布于細胞質中。與p EGFP-C1 組相比較,p EGFP-C1/ Akt 轉染組Akt 及VEGF 的mRNA 和蛋白表達均明顯增
強( P lt; 0. 05) , c-kit mRNA 和蛋白表達則無明顯變化( P gt; 0. 05) 。加入SCF 后其能增強實驗組( SCF + p EGFP-
C1/ Akt) 和對照組(SCF + p EGFP-C1) 中c-kit 、Akt 及VEGF 的mRNA 和蛋白表達,并且隨著SCF 的濃度增高該
作用則越明顯( P lt; 0. 05) ; 與對照組相比較,實驗組c-kit mRNA 和蛋白的表達無明顯變化( P gt; 0. 05) ,而Akt 和
VEGF mRNA 和蛋白表達則明顯增強( P lt; 0. 01) 。結論　成功構建GFP/ Akt 融合基因表達載體在MSCs 中表達,
可誘導Akt 及VEGF mRNA 和蛋白的表達; SCF 可能通過PI3/ Akt 途徑刺激c2kit 、Akt 及VEGF mRNA 和蛋白
的表達。

引用本文： 霍鑫,唐海燕,孔宏亮,劉英,張宏偉,王欣,胡新華,張強. SCF 對體外轉染p EGFP2C1/ Akt 的骨髓間充質干細胞中c-kit 、Akt 及VEGF 表達的影響. 中國普外基礎與臨床雜志, 2008, 15(2): 116-121. doi: 復制

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9.	Datta SR,Brunet A,Greenberg ME.Cellular survival:a play in three Akts[J].Genes Dev,1999; 13(22):2905.
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12.	Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues[J].Science,1997; 276(5309):71.
13.	Kaio E,Tanaka S,Kitadai Y,et al.Clinical significance of angiogenic factor expression at the deepest invasive site of advanced colorectal carcinoma[J].Oncology,2003; 64(1):61.
14.	Furudoi A,Tanaka S,Haruma K,et al.Clinical significance of vascular endothelial growth factor C expression and angiogenesis at the deepest invasive site of advanced colorectal carcinoma[J].Oncology,2002; 62(2):157.
15.	Gerber HP,Condorelli F,Park J,et al.Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes.Flt-1,but not Flk-1/KDR,is up-regulated by hypoxia[J].J Biol Chem,1997; 272(38):23659.
16.	Langley KE,Bennett LG,Wypych J,et al.Soluble stem cell factor in human serum[J].Blood,1993; 81(3):656.
17.	Jin T,Nakatani H,Taguchi T,et al.STI571 (Glivec) suppresses the expression of vascular endothelial growth factor in the gastrointestinal stromal tumor cell line,GIST-T1[J].World J Gastroenterol,2006; 12(5):703.
18.	Persad S,Attwell S,Gray V,et al.Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase:critical roles for kinase activity and amino acids arginine 211 and serine 343[J].J Biol Chem,2001; 276(29):27462.
19.	Hemmings BA.Akt signaling:linking membrane events to life and death decisions[J].Science,1997; 275(5300):628.
20.	Downward J.Mechanisms and consequences of activation of protein kinase B/Akt[J].Curr Opin Cell Biol,1998; 10(2):262.
21.	Matsui T,Li L,Wu JC,et al.Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart[J].J Biol Chem,2002; 277(25):22896.
22.	Misteli T,Spector DL.Applications of the green fluorescent protein in cell biology and biotechnology[J].Nat Biotechnol,1997; 15(10):961.
23.	Bagli E,Stefaniotou M,Morbidelli L,et al.Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3′-kinase activity[J].Cancer Res,2004; 64(21):7936.

1. <><><><><><><> [1]Pereira RF,Halford KW,O’Hara MD,et al.Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone,cartilage,and lung in irradiated mice[J].Proc Natl Acad Sci USA,1995; 92(11):4857.
2. Azizi SA,Stokes D,Augelli BJ,et al.Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats--similarities to astrocyte grafts[J].Proc Natl Acad Sci USA,1998; 95(7):3908.
3. Ferrari G,Cusella-De Angelis G,Coletta M,et al.Muscle regeneration by bone marrow-derived myogenic progenitors[J].Science,1998; 279(5356):1528.
4. Lee K,Majumdar MK,Buyaner D,et al.Human mesenchymal stem cells maintain transgene expression during expansion and differentiation[J].Mol Ther,2001; 3(6):857.
5. Yarden Y,Kuang WJ,Yang-Feng T,et al.Human proto-oncogene c-kit:a new cell surface receptor tyrosine kinase for an unidentified ligand[J].EMBO J,1987; 6(11):3341.
6. Ashman LK.The biology of stem cell factor and its receptor C-kit[J].Int J Biochem Cell Biol,1999; 31(10):1037.
7. Zeng S,Xu Z,Lipkowitz S,et al.Regulation of stem cell factor receptor signaling by Cbl family proteins (Cbl-b/c-Cbl)[J].Blood,2005; 105(1):226.
8. Morales-Ruiz M,Fulton D,Sowa G,et al.Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt[J].Circ Res,2000; 86(8):892.
9. Datta SR,Brunet A,Greenberg ME.Cellular survival:a play in three Akts[J].Genes Dev,1999; 13(22):2905.
10. Ikehara S.A novel strategy for allogeneic stem cell transplantation:perfusion method plus intra-bone marrow injection of stem cells[J].Exp Hematol,2003; 31(12):1142.
11. Asahara T,Murohara T,Sullivan A,et al.Isolation of putative progenitor endothelial cells for angiogenesis[J].Science,1997; 275(5302):964.
12. Prockop DJ.Marrow stromal cells as stem cells for nonhematopoietic tissues[J].Science,1997; 276(5309):71.
13. Kaio E,Tanaka S,Kitadai Y,et al.Clinical significance of angiogenic factor expression at the deepest invasive site of advanced colorectal carcinoma[J].Oncology,2003; 64(1):61.
14. Furudoi A,Tanaka S,Haruma K,et al.Clinical significance of vascular endothelial growth factor C expression and angiogenesis at the deepest invasive site of advanced colorectal carcinoma[J].Oncology,2002; 62(2):157.
15. Gerber HP,Condorelli F,Park J,et al.Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes.Flt-1,but not Flk-1/KDR,is up-regulated by hypoxia[J].J Biol Chem,1997; 272(38):23659.
16. Langley KE,Bennett LG,Wypych J,et al.Soluble stem cell factor in human serum[J].Blood,1993; 81(3):656.
17. Jin T,Nakatani H,Taguchi T,et al.STI571 (Glivec) suppresses the expression of vascular endothelial growth factor in the gastrointestinal stromal tumor cell line,GIST-T1[J].World J Gastroenterol,2006; 12(5):703.
18. Persad S,Attwell S,Gray V,et al.Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase:critical roles for kinase activity and amino acids arginine 211 and serine 343[J].J Biol Chem,2001; 276(29):27462.
19. Hemmings BA.Akt signaling:linking membrane events to life and death decisions[J].Science,1997; 275(5300):628.
20. Downward J.Mechanisms and consequences of activation of protein kinase B/Akt[J].Curr Opin Cell Biol,1998; 10(2):262.
21. Matsui T,Li L,Wu JC,et al.Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart[J].J Biol Chem,2002; 277(25):22896.
22. Misteli T,Spector DL.Applications of the green fluorescent protein in cell biology and biotechnology[J].Nat Biotechnol,1997; 15(10):961.
23. Bagli E,Stefaniotou M,Morbidelli L,et al.Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3′-kinase activity[J].Cancer Res,2004; 64(21):7936.

《中國普外基礎與臨床雜志》

SCF 對體外轉染p EGFP2C1/ Akt 的骨髓間充質干細胞中c-kit 、Akt 及VEGF 表達的影響

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

《中國普外基礎與臨床雜志》

SCF 對體外轉染p EGFP2C1/ Akt 的骨髓間充質干細胞中c-kit 、Akt 及VEGF 表達的影響

摘要 全文 圖表 視頻 參考文獻 施引文獻 補充材料

Format

Content

摘要全文圖表視頻參考文獻施引文獻補充材料