摘要
目的观察不同缺氧条件对小鼠骨髓基质细胞（BMSC）成分组成的影响。方法 Balb/c小鼠15只脱颈处死,取双侧股骨,分离BMSC接种于T25培养瓶中。将细胞随机分为对照组、缺氧1组、缺氧2组、氯化钴组。各组细胞分别以低密度（5×102/cm2）、中密度（1×104/cm2）、高密度（1.5×106/cm2）接种于培养基。对照组置于常氧条件（37℃、5%CO2、21%O2）培养,缺氧1组于2.5%~4%O2、5%CO2、91%~92.8%N2条件下培养,缺氧2组置于7%~8%O2、5%CO2、87%~88%N2条件下培养。氯化钴组在培养基中加入氯化钴100μΜ/L。各组细胞培养7 d后观察结果。光镜及透射电镜下观察BMSC细胞形态和超微结构变化,免疫组化法检测细胞中碱性磷酸酶（ALP）表达,RT-PCR法检测OCT4及内皮细胞相关基因（VEGF、FLT-1、FLK-1）表达。结果对照组以梭形成纤维样细胞为主,少有细胞融合现象;缺氧1、2组出现较多个体较大、扁平而宽阔的骰子样细胞;氯化钴组细胞呈圆形,个体较小。缺氧1组BMSC增殖率高于缺氧2组;中密度接种细胞增殖率最高;氯化钴组细胞增殖受到明显抑制。缺氧1、2组ALP染色阳性细胞多于对照组及氯化钴组,且缺氧1组高于缺氧2组（P均〈0.05）。缺氧1、2组OCT4基因表达受到抑制,仅对照组及氯化钴组OCT4基因表达阳性。VEGF在缺氧1、2组表达均为阴性,而在氯化钴组表达阳性;各组FLK-1基因表达均为阳性;FLT-1基因表达均为阴性。结论在2.5%~4%O2、1×104/cm2接种密度条件下,小鼠BMSC中一种分化程度较高、宽阔、扁平、ALP表达阳性的细胞比例进一步增高。
Objective To observe the effects of different hypoxic conditions on the cellular constituents of mice bone marrow stromal cells （BMSC）. Methods Fifteen Balb/c mice were taken off the neck to death, and bone marrow were collected from the femoral diaphysis. The isolated BMSC were plated in T25 culture flasks. The isolated bone marrow cells were randomly divided into control group, hypoxia 1 group, hypoxia 2 group, and COCl2 group. BMSC in each group were seeded at low density（ 1 × 10^2/cm2 ）, medium density（ 1 × 10^4/cm2 ）, and high density（ 1 x 106/cm2 ） in culture medium. The control group cell was cultured at normoxic conditions（37 ℃ ,5% CO2 ,21% 02 ）. The hypoxia 1 group cell was cultured at 2.5% ～ 4% 02,5% CO2 and 91% - 92.8% N2. The hypoxia 2 group cell was cultured at 7% - 8% 02 , 5% CO2, 88% - 87% N2, as well as 100 μmol/L COC12 was added in COCl2 group. The culture results were observed 7 days after seeding. The morphology and ultrastructure of BMSC were observed by light microscope and transmission electron micro- scope, and the expression of ALP was analyzed by immunohistochemistry. The OCT4 gene and endothelial cells relative genes （ VEGF, FLT1, FLK1 ） were detected by RT-PCR. Results The number of cells in hypoxic 1 group significantly increased compared to the control group and hypoxia 2 group, while COC12 inhibited the proliferation of BMSC. The mor- phology of BMSC in hypoxic group was wide, flat and big. The uhrastructure analysis showed that there were more vacuoles after under hypoxic conditions than the number under normoxic conditions. The proportion of ALP positive cells in BMSC under hypoxic culture increased compared to that under control group and hypoxia 2 group. However, the morphology of BMSC was small and round after COC12 treatment, and no ALP positive ceils existed in COC12 group. Furthermore, hypoxia inhibited the expression of OCT4 gene, and can not increase the expression of endothelial ceils relative genes such as VEGF, FLT1 and FLK1. Conclusion When plated at 1 × 10^4/cm^2 density under 2.5% to 4% 02 concentration, a kind of BMSC cell type, which was wide, flat, ALP positive and high differentiated cell increased in the proportion of BMSC.
出处
《山东医药》
CAS
北大核心
2015年第2期20-23,共4页
Shandong Medical Journal
基金
国家自然科学基金青年基金资助项目(81000196)
关键词
缺氧
骨髓基质细胞
细胞分化
hypoxia
bone marrow stromal cells
differentiation
