• 1.鄭州大學(xué)第二附屬醫(yī)院心臟內(nèi)科（鄭州 450014）;;
  • 2.遼寧省人民醫(yī)院心臟內(nèi)科（沈陽 110016）;

目的  探討葡萄糖轉(zhuǎn)運(yùn)載體（GLUT）-4是否參與骨髓間充質(zhì)干細(xì)胞（MSCs）的葡萄糖轉(zhuǎn)運(yùn)以及Akt基因轉(zhuǎn)染提高M(jìn)SCs耐缺氧能力是否與GLUT-4易位和表達(dá)有關(guān)。
方法  將經(jīng)Akt基因轉(zhuǎn)染和未轉(zhuǎn)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(biāo)-脫氧葡萄糖（3H-G）的攝取量，免疫細(xì)胞化學(xué)染色、Western blot和RT-PCR分別檢測GLUT-4的蛋白質(zhì)和mRNA表達(dá)。
結(jié)果  ①缺氧轉(zhuǎn)染組的3H-G攝取量是缺氧非轉(zhuǎn)染組的（1.39±0.13）倍（P＜0.05），但仍低于常氧非轉(zhuǎn)染組（P＜0.05）。②MSCs在常氧或缺氧、Akt基因轉(zhuǎn)染或無轉(zhuǎn)染的條件下均可表達(dá)GLUT-4蛋白。與常氧非轉(zhuǎn)染組比較，缺氧非轉(zhuǎn)染組GLUT-4 mRNA和蛋白的表達(dá)水平明顯降低（P＜0.05）。③與缺氧非轉(zhuǎn)染組比較，缺氧轉(zhuǎn)染組的GLUT-4 mRNA〔（1.756±0.152）倍〕和蛋白〔細(xì)胞總GLUT-4蛋白（1.653±0.312）倍,細(xì)胞膜GLUT-4蛋白（2.041±0.258）倍〕的表達(dá)水平明顯提高（P＜0.05），且GLUT-4蛋白易位明顯； 但與常氧非轉(zhuǎn)染組比較，其GLUT-4 mRNA和蛋白的表達(dá)水平仍較低（P＜0.05）。④MSCs的3H-G攝取量與細(xì)胞膜中GLUT-4蛋白的表達(dá)呈正相關(guān)（r=0.415,P=0.001）。
結(jié)論  GLUT-4可能參與MSCs的葡萄糖轉(zhuǎn)運(yùn)，Akt基因提高M(jìn)SCs耐缺氧能力可能與提高GLUT-4的表達(dá)和易位有關(guān)。

引用本文： 邵磊,周書春,徐桂萍,孔宏亮. 體外Akt基因轉(zhuǎn)染對骨髓間充質(zhì)干細(xì)胞葡萄糖轉(zhuǎn)運(yùn)載體-4表達(dá)和易位的影響. 中國普外基礎(chǔ)與臨床雜志, 2010, 17(1): 52-56. doi: 復(fù)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(zhì)干細(xì)胞增殖、凋亡及葡萄糖攝取和Akt表達(dá)的影響 [Ｊ].中國現(xiàn)代醫(yī)學(xué)雜志， 2008; 18（10）： 13741382.
6. 孔宏亮, 劉寧寧, 張海， 等. 缺氧環(huán)境下大鼠骨髓間充質(zhì)干細(xì)胞的凋亡及Fas、FasL的表達(dá) [Ｊ]. 中國組織化學(xué)與細(xì)胞化學(xué)雜志, 2007; 16(5): 538543.
7. 齊國先， 孔宏亮， 霍鑫， 等. 缺氧環(huán)境下大鼠骨髓間充質(zhì)干細(xì)胞凋亡相關(guān)蛋白和mRNA的表達(dá) [Ｊ]. 中國組織化學(xué)與細(xì)胞化學(xué)雜志, 2008; 17(2): 138143.
8. 孔宏亮, 張利群, 曹善峰， 等. Akt基因轉(zhuǎn)染對骨髓間充質(zhì)干細(xì)胞缺氧時(shí)凋亡和增殖的影響 [Ｊ]. 中國組織化學(xué)與細(xì)胞化學(xué)雜志, 2008; 17(3): 225231.
9. 霍鑫， 孔宏亮， 胡新華， 等. pEGFPC1/Akt體外轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞后對血管內(nèi)皮生長因子表達(dá)的影響 [Ｊ]. 中華實(shí)驗(yàn)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(zhuǎn)染骨髓間充質(zhì)干細(xì)胞對后肢缺血大鼠血管生成的影響 [Ｊ]. 中國普外基礎(chǔ)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(dǎo)轉(zhuǎn)染pEGFPC1/Akt的骨髓間充質(zhì)干細(xì)胞凋亡的影響 [Ｊ]. 中國普外基礎(chǔ)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(zhuǎn)染pEGFPC1/Akt的骨髓間充質(zhì)干細(xì)胞中ckit、Akt及VEGF表達(dá)的影響 [Ｊ]. 中國普外基礎(chǔ)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. 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. 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. 3. Wollert KC, Drexler H. Clinical applications of stem cells for the heart [Ｊ]. Circ Res, 2005; 96(2): 151163.
  4. 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. 5. 孔宏亮, 劉寧寧, 霍鑫， 等. 缺氧對大鼠骨髓間充質(zhì)干細(xì)胞增殖、凋亡及葡萄糖攝取和Akt表達(dá)的影響 [Ｊ].中國現(xiàn)代醫(yī)學(xué)雜志， 2008; 18（10）： 13741382.
  6. 6. 孔宏亮, 劉寧寧, 張海， 等. 缺氧環(huán)境下大鼠骨髓間充質(zhì)干細(xì)胞的凋亡及Fas、FasL的表達(dá) [Ｊ]. 中國組織化學(xué)與細(xì)胞化學(xué)雜志, 2007; 16(5): 538543.
  7. 7. 齊國先， 孔宏亮， 霍鑫， 等. 缺氧環(huán)境下大鼠骨髓間充質(zhì)干細(xì)胞凋亡相關(guān)蛋白和mRNA的表達(dá) [Ｊ]. 中國組織化學(xué)與細(xì)胞化學(xué)雜志, 2008; 17(2): 138143.
  8. 8. 孔宏亮, 張利群, 曹善峰， 等. Akt基因轉(zhuǎn)染對骨髓間充質(zhì)干細(xì)胞缺氧時(shí)凋亡和增殖的影響 [Ｊ]. 中國組織化學(xué)與細(xì)胞化學(xué)雜志, 2008; 17(3): 225231.
  9. 9. 霍鑫， 孔宏亮， 胡新華， 等. pEGFPC1/Akt體外轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞后對血管內(nèi)皮生長因子表達(dá)的影響 [Ｊ]. 中華實(shí)驗(yàn)外科雜志, 2008; 25(2)： 149151.
  10. 10. 霍鑫, 馮金煒, 劉方峰, 等. pEGFPC1/Akt體外轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞對后肢缺血大鼠血管生成的影響 [Ｊ]. 中國普外基礎(chǔ)與臨床雜志, 2008; 15(6): 402407.
  11. 11. 霍鑫, 孔宏亮, 劉方峰, 等. 無血清條件對誘導(dǎo)轉(zhuǎn)染pEGFPC1/Akt的骨髓間充質(zhì)干細(xì)胞凋亡的影響 [Ｊ]. 中國普外基礎(chǔ)與臨床雜志, 2008; 15(7): 479483.
  12. 12. 霍鑫， 唐海燕， 孔宏亮， 等. SCF對體外轉(zhuǎn)染pEGFPC1/Akt的骨髓間充質(zhì)干細(xì)胞中ckit、Akt及VEGF表達(dá)的影響 [Ｊ]. 中國普外基礎(chǔ)與臨床雜志, 2008; 15(2): 116121.
  13. 13. Hemmings BA. Akt signaling: linking membrane events to life and death decisions [Ｊ]. Science, 1997; 275(5300): 628630.
  14. 14. Downward J. Mechanisms and consequences of activation of protein kinase B/Akt [Ｊ]. Curr Opin Cell Biol, 1998; 10(2): 262267.
  15. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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.