CAJ | 학술논문

目的：探讨诱导大鼠骨髓单个核细胞向血管内皮细胞分化的方法和诱导的血管内皮细胞的生物学特性。方法采用Ficoll密度梯度离心法收集SD大鼠股骨及胫骨骨髓细胞中的单个核细胞，孵育至细胞汇合40％后分为两组：实验组在含10％胎牛血清（FBS）的M199培养液中添加诱导剂血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）；对照组加入含10％ FBS的M199培养液，继续培养、孵育。观察细胞生长形态特征，应用CCK8试剂盒检测细胞增殖，绘制细胞生长曲线，并计算细胞群体倍增时间。采用流式细胞仪检测细胞表面特异性标记物CD31、CD34和血管性假血友病因子（vWF），免疫组织荧光法检测vWF。结果实验组细胞培养6 d后贴壁细胞呈线性排列，10～12 d后细胞增大，周边为毛刺状，单个核位于中央，类铺路石状排列；对照组细胞呈梭形生长，4 d后细胞呈梭形、纺锤形或三角形。实验组细胞生长曲线呈M型，群体倍增时间为8 d （192 h ）；对照组细胞生长曲线呈S 型，群体倍增时间为5 d （120 h ）。实验组细胞表面特异性标记物 CD31、CD34、vWF 分别为296．％、502．％、982．％，对照组细胞表面特异性标记物CD31、CD34、vWF分别为06．％、19．％、53．％。实验组免疫荧光染色vWF阳性，对照组免疫荧光染色vWF阴性。结论 SD大鼠骨髓单个核细胞内添加诱导剂后能在体外向血管内皮细胞分化，并具有一定的生物学特性。
목적：탐토유도대서골수단개핵세포향혈관내피세포분화적방법화유도적혈관내피세포적생물학특성。방법채용Ficoll밀도제도리심법수집SD대서고골급경골골수세포중적단개핵세포，부육지세포회합40％후분위량조：실험조재함10％태우혈청（FBS）적M199배양액중첨가유도제혈관내피생장인자（VEGF）화감성성섬유세포생장인자（bFGF）；대조조가입함10％ FBS적M199배양액，계속배양、부육。관찰세포생장형태특정，응용CCK8시제합검측세포증식，회제세포생장곡선，병계산세포군체배증시간。채용류식세포의검측세포표면특이성표기물CD31、CD34화혈관성가혈우병인자（vWF），면역조직형광법검측vWF。결과실험조세포배양6 d후첩벽세포정선성배렬，10～12 d후세포증대，주변위모자상，단개핵위우중앙，류포로석상배렬；대조조세포정사형생장，4 d후세포정사형、방추형혹삼각형。실험조세포생장곡선정M형，군체배증시간위8 d （192 h ）；대조조세포생장곡선정S 형，군체배증시간위5 d （120 h ）。실험조세포표면특이성표기물 CD31、CD34、vWF 분별위296．％、502．％、982．％，대조조세포표면특이성표기물CD31、CD34、vWF분별위06．％、19．％、53．％。실험조면역형광염색vWF양성，대조조면역형광염색vWF음성。결론 SD대서골수단개핵세포내첨가유도제후능재체외향혈관내피세포분화，병구유일정적생물학특성。
Objective To discuss the methods of differentiation of rat bone marrow mononuclear cells to vascular endothelial cells and biological characteristics of vascular endothelial cells .Methods Ficoll density gradient centrifugation was used to collect mononuclear cells of SD rat femoral and tibial bone marrow cells ,which were incubated and combined to 40% ,then divided them into two groups .Experimental group :adding VEGF and bFGF revulsant in M 199 nutrient fluid of 10% FBS ;Control group :M199 nutri-ent fluid of 10% FBS werw added to foster and incubate ;and observ morphological characters of cell grow th by using CCK8 reagent kit to detect and draw grow th curvature and calculate multiplication time of cell group ,then flow cytometry instrument was used to detect specific markers CD31 ,CD34 and vWF on cell′s surface ,immunohistochemical fluorescence detection of vWF .Results Cells in experimental group were linear series after 6 days ,and enlarged after 10-12 days ,there were burrs in its rim ,mononuclear cell was in center and arranged like paving stones .Cells in control group were in a spindle shape ,5-8 later they were in a spindle or triangle shape ,growth curve of cells in experimental group was M type ,group doubling time was 8 days (192 hours) ,growth curve of cells in control group was S type ,group doubling time was 5 days (120 hours) .Expression rates of cell surface markers CD31 ,CD34 and vWF from the cells obtained in experimental group were 9 .6% ,50 .2% ,98 .2% ,Expression rates of cell surface markers CD31 ,CD34 and vWF in control group were 0 6.% ,1 9.% ,5 3.% .Immunofluorescence staining vWF in ex-perimental group was positive , immunofluorescence staining vWF in control group was negative . Conclusion SD rat bone marrow mononuclear cells after adding revulsant can be splited into vascular en-dothelial cell in vitro and possess certain biological characteristics .