CAJ | 학술논문

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。
목적：관찰불동결양조건대소서골수기질세포（ BMSC）성분조성적영향。방법 Balb／c소서15지탈경처사，취쌍측고골，분리BMSC접충우T25배양병중。장세포수궤분위대조조、결양1조、결양2조、록화고조。각조세포분별이저밀도（5×102／cm2）、중밀도（1×104／cm2）、고밀도（1．5×106／cm2）접충우배양기。대조조치우상양조건（37℃、5％CO2、21％O2）배양，결양1조우2．5％～4％O2、5％CO2、91％～92．8％N2조건하배양，결양2조치우7％～8％O2、5％CO2、87％～88％N2조건하배양。록화고조재배양기중가입록화고100μΜ／L。각조세포배양7 d후관찰결과。광경급투사전경하관찰BMSC세포형태화초미결구변화，면역조화법검측세포중감성린산매（ALP）표체，RT-PCR법검측OCT4급내피세포상관기인（VEGF、FLT-1、FLK-1）표체。결과대조조이사형성섬유양세포위주，소유세포융합현상；결양1、2조출현교다개체교대、편평이관활적투자양세포；록화고조세포정원형，개체교소。결양1조BMSC증식솔고우결양2조；중밀도접충세포증식솔최고；록화고조세포증식수도명현억제。결양1、2조ALP염색양성세포다우대조조급록화고조，차결양1조고우결양2조（ P균＜0．05）。결양1、2조OCT4기인표체수도억제，부대조조급록화고조OCT4기인표체양성。 VEGF재결양1、2조표체균위음성，이재록화고조표체양성；각조FLK-1기인표체균위양성；FLT-1기인표체균위음성。결론재2．5％～4％O2、1×104／cm2접충밀도조건하，소서BMSC중일충분화정도교고、관활、편평、ALP표체양성적세포비례진일보증고。
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 T 25 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 ×102/cm2), medium density(1 ×104/cm2), and high density(1 ×106/cm2) in culture medium. The control group cell was cultured at normoxic conditions (37 ℃,5% CO2,21%O2).The hypoxia 1 group cell was cul-tured at 2.5%~4%O2 , 5%CO2 and 91%~92.8%N2 .The hypoxia 2 group cell was cultured at 7%~8%O2 , 5%CO2 , 88%~87%N2, as well as 100 μmol/L COCl2 was added in COCl 2 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 COCl2 inhibited the proliferation of BMSC .The mor-phology of BMSC in hypoxic group was wide , flat and big .The ultrastructure 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 COCl 2 treatment, and no ALP positive cells existed in COCl 2 group.Furthermore,hypoxia inhibited the expression of OCT 4 gene, and can not increase the expression of endothelial cells relative genes such as VEGF, FLT1 and FLK1.Conclusion When plated at 1 ×104/cm2 density under 2.5%to 4%O2 concentration, a kind of BMSC cell type , which was wide , flat, ALP positive and high differentiated cell increased in the proportion of BMSC .