缺氧对大鼠骨髓间充质干细胞增殖、凋亡及葡萄糖摄取和Akt表达的影响  

Cell multiplication, apoptosis, glucose intaking and Akt protein expression of bone marrow mesenchymal stem cells of rat under hypoxia environment

作　　者：孔宏亮[1] 刘宁宁[2] 霍鑫[3] 王勃[1] 张海鹏[4] 高明宇[1] 齐国先[1] 

机构地区：[1]中国医科大学附属第一医院心脏内科,辽宁沈阳110004 [2]中国医科大学附属第一医院眼科,辽宁沈阳110004 [3]中国医科大学附属第一医院普外三科,辽宁沈阳110004 [4]中国医科大学附属第一医院病理生理学教研室,辽宁沈阳110004

出　　处：《中国现代医学杂志》2008年第10期1374-1378,1382,共6页China Journal of Modern Medicine

摘　　要：目的探讨缺氧对大鼠骨髓间充质干细胞(MSCs)糖摄取、凋亡、增殖和Akt表达的影响。方法将接种的P3细胞置于94%N2、1%O2和5%CO2缺氧箱中37℃孵育不同时间点(常氧、缺氧0.5、1、2、4和8h)后分别取出,应用放射同位素法检测MSCs对氚标记-葡萄糖(3H-G)的摄取,应用Annexin V/PI双染法进行流式细胞仪(FCM)分析MSCs凋亡率(apoptotic rate,AR)和死亡率(dead rate,DR),应用MTT分析细胞增殖状态以及检测Akt蛋白表达。结果培养的P3骨髓单个核细胞CD29+、CD71+、CD44+、CD34-和5-aza诱导后表达TnT及骨诱导分化液诱导后细胞ALP+等证实其为骨髓MSCs;各缺氧时间点3H-G摄取量较缺氧前均显著性降低(P<0.01),而各缺氧时间点之间没有统计学意义(P>0.05);MSCs常氧、缺氧0.5、1、2、4、6和8h时的AR均较缺氧前显著增高(P<0.01)、DR亦显著增高(P<0.05),不过,随着缺氧时间延伸,AR显著增加(P<0.05),而DR没有统计学意义(P>0.05);缺氧不同时间点MTT法OD值较常氧(缺氧结束同步时间点)均显著性降低(P<0.01),并随缺氧时间延伸显著降低(P<0.05),不过,与常氧(缺氧8h开始同步时间点)相比,均显著升高(P<0.05);与常氧相比,各缺氧时间点Akt蛋白IOD均显著增强(P<0.05),而各缺氧时间点改变无统计学意义(P>0.05)。结论缺氧既可引起MSCs摄糖量下降、增殖滞缓,又可上调Akt蛋白表达和促进MSCs凋亡。[Objective] Aimed at elucidating whether cell multiplication, apoptosis, glucose intaking and akt protein expression of bone mesenchymal stem cells of rat et al were influenced by hypoxia environment ex vivo. [Methods] Passage 3 of bone marrow MSCs of wistar rat were cultured in culturing chamber with 94% N2, 1% O2, 5% CO2 at 37℃. At different hypoxia time points ie 0, 0.5, 1, 4 and 8 h, glucose uptaking was assayed by using radiation isotope ^3H-G, Apoptotic Rate （AR） and dead rate（DR） were analyzed by Flow cytometry （FCM） after Annexin V/PI staining, cell multiplication by MTT methods and Akt protein by immunocytochemistry and western blot. [Results] Assay for CD29^＋, CD44^＋, CD71^＋, CD34^-, Tn T^＋ after 5-azacytidine agent inducing and ALP＋ after bone differentiation agent inducing suggest these bone marrowderived cells were MSCs. The 3H-G intaking ratio（157±11）, （110±11）, （107±13）, （103±10）, （100±9） and （98±10） of MSCs at different hypoxia time points significantly decreased than normoxia（P〈0.01）, however, there is not statistical significance （P 〉0.05） for different hypoxia time points. The AR [（0.09%±2.03%）, （12.9%±1.72%）, （13.7%±2.26%）, （13.8±3.01）%, （14.1%±2.78%） and （14.7%±4.01%） at 0, 0.5, 1, 4 and 8 h, respectively, P 〈0.01] and DR [（0.04%±1.79%）, （0.93%±1.85%）, （3.11%±2.14%）, （4.09%±2.36%）, （4.72%±2.05%） and （4.91%±3.72%） at 0, 0.5, 1, 4 and 8 h, respectively, P 〈0.05] at different hypoxia time points significantly increased than normoxia. The AR further went up with time stretching （P 〈0.05）, however, there was not statistical significance （P 〉0.05） for the DR. OD value of MTT methods at different hypoxia time points significantly decreased than those of normoxia （P 〈0.01, synchronizing the end of hypoxia） and further aggravated, however, all significantly increased than those of normoxia （P 〈0.05, synchronizing the begin of hypoxia 8 h）. IOD of Akt

关 键 词：大鼠骨髓间充质干细胞 缺氧 摄3H-G量 细胞凋亡 AKT蛋白 

分 类 号：Q255[生物学—细胞生物学]