Stem Cell Factor Enhances Expression of c-kit , Akt and VEGF in Mesenchymal Stem Cells Transfected by pEGFP-C1/ Akt Gene in Vit ro_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

1.Department of Geriatrics,The First Affiliated Hospital of China Medical University,Shenyang110001,China;;
2.Department of Endocrine,The General Hospital of Daqing Oil Field,Daqing163000， China;;
3.Department of Circulation,The First Affiliated Hospital of China Medical University;;
4.Department of Endocrine,The First Affiliated Hospital of China Medical UniversityCorresponding Author：ZHANG Qiang,E-mail:qzhang48@hotmail.com;

Corresponding author：

Keywords：

Bone mesenchymal stem cell ,Akt gene ,Stem cell factor ,Vascular endothelialgrowth factor

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Objective To construct green fluorescent protein (GFP)/Akt fusion gene vector for observing the expression and localization of GFP/Akt in rats bone marrow-derived mesenchymal stem cells （MSCs）. Stem cell factor （SCF） effected expression of c-kit, Akt and VEGF mRNA and protein in MSCs transfected by pEGFP-C1/Akt through PI3-Akt pathway.Methods Akt recombined GFP vector by restriction enzymes, MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP were evaluated by fluorescene microscopy. The expressions of c-kit, Akt and VEGF mRNA and protein were examined by RT-PCR and Western blot after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. SCF effected the expression of c-kit, Akt and VEGF mRNA and protein after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. Results Restriction endonuclease assay and sequence analysis verified that thesuccessfulconstructionoftherecombinantvectorpEGFP-C1/AktandefficienthighexpressionofpEGFP-C1/Akt fusion protein in the MSCs of rats. Under fluorescent microscence, green flurescence was seen homogeneously distributed in the entire cell of the cells transfected by the recombinant vector pEGFP-C1, and diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C1/Akt. The expression of Akt and VEGF mRNA and protein were significantly higher in MSCs transfected by pEGFP-C1/Akt (p lt;0.05). The expression of c-kit, Akt and VEGF mRNA and protein were significantly higher in experiment group (SCF+pEGFP-C1/Akt) and control group (SCF+pEGFP-C1), p lt;0.05. In experiment group， SCF stimulation enhanced expression of Akt and VEGF mRNA and protein (p lt;0.01). Conclusion GFP/Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of rats induces the expression of Akt and VEGF mRNA and protein. SCF stimulation enhanced expression of c-kit, Akt and VEGF mRNA and protein through PI3/Akt pathway.

Citation： HUO Xin,TANG Haiyan,KONG Hongliang,LIU Ying,ZHANG Hongwei,WANG Xin,HU Xinhua,ZHANG Qiang. Stem Cell Factor Enhances Expression of c-kit , Akt and VEGF in Mesenchymal Stem Cells Transfected by pEGFP-C1/ Akt Gene in Vit ro. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2008, 15(2): 116-121. doi: Copy

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16.	Langley KE,Bennett LG,Wypych J,et al.Soluble stem cell factor in human serum[J].Blood,1993; 81(3):656.
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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.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Stem Cell Factor Enhances Expression of c-kit , Akt and VEGF in Mesenchymal Stem Cells Transfected by pEGFP-C1/ Akt Gene in Vit ro

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