體外Akt基因轉染對骨髓間充質干細胞葡萄糖轉運載體-4表達和易位的影響_《中國普外基礎與臨床雜志》

作者：

邵磊 ¹ , 周書春 ¹ , 徐桂萍 ² ,  孔宏亮 ²

1.鄭州大學第二附屬醫院心臟內科（鄭州 450014）;;
2.遼寧省人民醫院心臟內科（沈陽 110016）;

關鍵詞：

大鼠骨髓間充質干細胞 Akt基因轉染缺氧氚標-脫氧葡萄糖攝取葡萄糖轉運載體-4

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目的探討葡萄糖轉運載體（GLUT）-4是否參與骨髓間充質干細胞（MSCs）的葡萄糖轉運以及Akt基因轉染提高MSCs耐缺氧能力是否與GLUT-4易位和表達有關。
方法將經Akt基因轉染和未轉染的MSCs均行常氧（5% CO2）和缺氧（94% N2、1%O2和5%CO2）37 ℃孵育8 h。放射同位素法檢測氚標-脫氧葡萄糖（3H-G）的攝取量，免疫細胞化學染色、Western blot和RT-PCR分別檢測GLUT-4的蛋白質和mRNA表達。
結果 ①缺氧轉染組的3H-G攝取量是缺氧非轉染組的（1.39±0.13）倍（P＜0.05），但仍低于常氧非轉染組（P＜0.05）。②MSCs在常氧或缺氧、Akt基因轉染或無轉染的條件下均可表達GLUT-4蛋白。與常氧非轉染組比較，缺氧非轉染組GLUT-4 mRNA和蛋白的表達水平明顯降低（P＜0.05）。③與缺氧非轉染組比較，缺氧轉染組的GLUT-4 mRNA〔（1.756±0.152）倍〕和蛋白〔細胞總GLUT-4蛋白（1.653±0.312）倍,細胞膜GLUT-4蛋白（2.041±0.258）倍〕的表達水平明顯提高（P＜0.05），且GLUT-4蛋白易位明顯；但與常氧非轉染組比較，其GLUT-4 mRNA和蛋白的表達水平仍較低（P＜0.05）。④MSCs的3H-G攝取量與細胞膜中GLUT-4蛋白的表達呈正相關（r=0.415,P=0.001）。
結論 GLUT-4可能參與MSCs的葡萄糖轉運，Akt基因提高MSCs耐缺氧能力可能與提高GLUT-4的表達和易位有關。

引用本文： 邵磊,周書春,徐桂萍,孔宏亮. 體外Akt基因轉染對骨髓間充質干細胞葡萄糖轉運載體-4表達和易位的影響. 中國普外基礎與臨床雜志, 2010, 17(1): 52-56. doi: 復制

1.	Ernens I, Goodfellow SJ, Innes F, et al. Hypoxic stress suppresses RNA polymerase Ⅲ recruitment and tRNA gene transcription in cardiomyocytes[Ｊ]. Nucleic Acids Res, 2006; 34(1): 286２94.
2.	Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is upregulated by hypoxia through a HIF1αdependent mechanism [Ｊ]. J Biol Chem, 2006; 281(14): 90309037.
3.	Wollert KC, Drexler H. Clinical applications of stem cells for the heart [Ｊ]. Circ Res, 2005; 96(2): 151163.
4.	Kong HL, Liu NN, Huo X, et al. Cell multiplication, apoptosis and pAkt protein expression of bone mesenchymal stem cells of rat under hypoxia environment [Ｊ]. JNMU, 2007; 21(4): 233239.
5.	孔宏亮, 劉寧寧, 霍鑫，等. 缺氧對大鼠骨髓間充質干細胞增殖、凋亡及葡萄糖攝取和Akt表達的影響 [Ｊ].中國現代醫學雜志， 2008; 18（10）： 13741382.
6.	孔宏亮, 劉寧寧, 張海，等. 缺氧環境下大鼠骨髓間充質干細胞的凋亡及Fas、FasL的表達 [Ｊ]. 中國組織化學與細胞化學雜志, 2007; 16(5): 538543.
7.	齊國先，孔宏亮，霍鑫，等. 缺氧環境下大鼠骨髓間充質干細胞凋亡相關蛋白和mRNA的表達 [Ｊ]. 中國組織化學與細胞化學雜志, 2008; 17(2): 138143.
8.	孔宏亮, 張利群, 曹善峰，等. Akt基因轉染對骨髓間充質干細胞缺氧時凋亡和增殖的影響 [Ｊ]. 中國組織化學與細胞化學雜志, 2008; 17(3): 225231.
9.	霍鑫，孔宏亮，胡新華，等. pEGFPC1/Akt體外轉染骨髓間充質干細胞后對血管內皮生長因子表達的影響 [Ｊ]. 中華實驗外科雜志, 2008; 25(2)： 149151.
10.	霍鑫, 馮金煒, 劉方峰, 等. pEGFPC1/Akt體外轉染骨髓間充質干細胞對后肢缺血大鼠血管生成的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2008; 15(6): 402407.
11.	霍鑫, 孔宏亮, 劉方峰, 等. 無血清條件對誘導轉染pEGFPC1/Akt的骨髓間充質干細胞凋亡的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2008; 15(7): 479483.
12.	霍鑫，唐海燕，孔宏亮，等. SCF對體外轉染pEGFPC1/Akt的骨髓間充質干細胞中ckit、Akt及VEGF表達的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2008; 15(2): 116121.
13.	Hemmings BA. Akt signaling: linking membrane events to life and death decisions [Ｊ]. Science, 1997; 275(5300): 628630.
14.	Downward J. Mechanisms and consequences of activation of protein kinase B/Akt [Ｊ]. Curr Opin Cell Biol, 1998; 10(2): 262267.
15.	Foster LJ, Li D, Randhawa VK, et al. Insulin accelerates interendosomal GLUT4 traffic via phosphatidylinositol 3kinase and protein kinase B [Ｊ]. J Biol Chem, 2001; 276(47): 4421244221.
16.	Sweeney G, Garg RR, Ceddia RB, et al. Intracellular delivery of phosphatidylinositol (3,4,5)trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake [Ｊ]. J Biol Chem, 2004; 279(31): 3223332242.
17.	Maffucci T, Brancaccio A, Piccolo E, et al. Insulin induces phosphatidylinositol3phosphate formation through TC10 activation [Ｊ]. EMBO J, 2003; 22(16): 41784189.
18.	JeBailey L, Rudich A, Huang X, et al. Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulininduced actin remodeling [Ｊ]. Mol Endocrinol, 2004; 18(2): 359372.
19.	Kupriyanova TA, Kandror KV. Akt2 binds to Glut4containing vesicles and phosphorylates their component proteins in response to insulin [Ｊ]. J Biol Chem, 1999; 274(3): 14581464.
20.	Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
21.	Chen X, AlHasani H, Olausson T, et al. Activity, phosphorylation state and subcellular distribution of GLUT4targeted Akt2 in rat adipose cells [Ｊ]. J Cell Sci, 2003; 116(Pt 17): 35113518.
22.	Kane S, Sano H, Liu SC, et al. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPaseactivating protein (GAP) domain [Ｊ]. J Biol Chem, 2002; 277(25): 2211522118.
23.	Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
24.	Welsh GI, Hers I, Berwick DC, et al. Role of protein kinase B in insulinregulated glucose uptake [Ｊ]. Biochem Soc Trans, 2005; 33(Pt 2): 346349.
25.	Berwick DC, Dell GC, Welsh GI, et al. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles[Ｊ]. J Cell Sci, 2004; 117(Pt 25): 59855993.

1. Ernens I, Goodfellow SJ, Innes F, et al. Hypoxic stress suppresses RNA polymerase Ⅲ recruitment and tRNA gene transcription in cardiomyocytes[Ｊ]. Nucleic Acids Res, 2006; 34(1): 286２94.
2. Ullah MS, Davies AJ, Halestrap AP. The plasma membrane lactate transporter MCT4, but not MCT1, is upregulated by hypoxia through a HIF1αdependent mechanism [Ｊ]. J Biol Chem, 2006; 281(14): 90309037.
3. Wollert KC, Drexler H. Clinical applications of stem cells for the heart [Ｊ]. Circ Res, 2005; 96(2): 151163.
4. Kong HL, Liu NN, Huo X, et al. Cell multiplication, apoptosis and pAkt protein expression of bone mesenchymal stem cells of rat under hypoxia environment [Ｊ]. JNMU, 2007; 21(4): 233239.
5. 孔宏亮, 劉寧寧, 霍鑫，等. 缺氧對大鼠骨髓間充質干細胞增殖、凋亡及葡萄糖攝取和Akt表達的影響 [Ｊ].中國現代醫學雜志， 2008; 18（10）： 13741382.
6. 孔宏亮, 劉寧寧, 張海，等. 缺氧環境下大鼠骨髓間充質干細胞的凋亡及Fas、FasL的表達 [Ｊ]. 中國組織化學與細胞化學雜志, 2007; 16(5): 538543.
7. 齊國先，孔宏亮，霍鑫，等. 缺氧環境下大鼠骨髓間充質干細胞凋亡相關蛋白和mRNA的表達 [Ｊ]. 中國組織化學與細胞化學雜志, 2008; 17(2): 138143.
8. 孔宏亮, 張利群, 曹善峰，等. Akt基因轉染對骨髓間充質干細胞缺氧時凋亡和增殖的影響 [Ｊ]. 中國組織化學與細胞化學雜志, 2008; 17(3): 225231.
9. 霍鑫，孔宏亮，胡新華，等. pEGFPC1/Akt體外轉染骨髓間充質干細胞后對血管內皮生長因子表達的影響 [Ｊ]. 中華實驗外科雜志, 2008; 25(2)： 149151.
10. 霍鑫, 馮金煒, 劉方峰, 等. pEGFPC1/Akt體外轉染骨髓間充質干細胞對后肢缺血大鼠血管生成的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2008; 15(6): 402407.
11. 霍鑫, 孔宏亮, 劉方峰, 等. 無血清條件對誘導轉染pEGFPC1/Akt的骨髓間充質干細胞凋亡的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2008; 15(7): 479483.
12. 霍鑫，唐海燕，孔宏亮，等. SCF對體外轉染pEGFPC1/Akt的骨髓間充質干細胞中ckit、Akt及VEGF表達的影響 [Ｊ]. 中國普外基礎與臨床雜志, 2008; 15(2): 116121.
13. Hemmings BA. Akt signaling: linking membrane events to life and death decisions [Ｊ]. Science, 1997; 275(5300): 628630.
14. Downward J. Mechanisms and consequences of activation of protein kinase B/Akt [Ｊ]. Curr Opin Cell Biol, 1998; 10(2): 262267.
15. Foster LJ, Li D, Randhawa VK, et al. Insulin accelerates interendosomal GLUT4 traffic via phosphatidylinositol 3kinase and protein kinase B [Ｊ]. J Biol Chem, 2001; 276(47): 4421244221.
16. Sweeney G, Garg RR, Ceddia RB, et al. Intracellular delivery of phosphatidylinositol (3,4,5)trisphosphate causes incorporation of glucose transporter 4 into the plasma membrane of muscle and fat cells without increasing glucose uptake [Ｊ]. J Biol Chem, 2004; 279(31): 3223332242.
17. Maffucci T, Brancaccio A, Piccolo E, et al. Insulin induces phosphatidylinositol3phosphate formation through TC10 activation [Ｊ]. EMBO J, 2003; 22(16): 41784189.
18. JeBailey L, Rudich A, Huang X, et al. Skeletal muscle cells and adipocytes differ in their reliance on TC10 and Rac for insulininduced actin remodeling [Ｊ]. Mol Endocrinol, 2004; 18(2): 359372.
19. Kupriyanova TA, Kandror KV. Akt2 binds to Glut4containing vesicles and phosphorylates their component proteins in response to insulin [Ｊ]. J Biol Chem, 1999; 274(3): 14581464.
20. Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
21. Chen X, AlHasani H, Olausson T, et al. Activity, phosphorylation state and subcellular distribution of GLUT4targeted Akt2 in rat adipose cells [Ｊ]. J Cell Sci, 2003; 116(Pt 17): 35113518.
22. Kane S, Sano H, Liu SC, et al. A method to identify serine kinase substrates. Akt phosphorylates a novel adipocyte protein with a Rab GTPaseactivating protein (GAP) domain [Ｊ]. J Biol Chem, 2002; 277(25): 2211522118.
23. Bae SS, Cho H, Mu J, et al. Isoformspecific regulation of insulindependent glucose uptake by Akt/protein kinase B [Ｊ]. J Biol Chem, 2003; 278(49): 4953049536.
24. Welsh GI, Hers I, Berwick DC, et al. Role of protein kinase B in insulinregulated glucose uptake [Ｊ]. Biochem Soc Trans, 2005; 33(Pt 2): 346349.
25. Berwick DC, Dell GC, Welsh GI, et al. Protein kinase B phosphorylation of PIKfyve regulates the trafficking of GLUT4 vesicles[Ｊ]. J Cell Sci, 2004; 117(Pt 25): 59855993.

《中國普外基礎與臨床雜志》

體外Akt基因轉染對骨髓間充質干細胞葡萄糖轉運載體-4表達和易位的影響

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《中國普外基礎與臨床雜志》

體外Akt基因轉染對骨髓間充質干細胞葡萄糖轉運載體-4表達和易位的影響

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