CAJ | 학술논문

目的探讨人骨髓来源MSCs的分离、培养和生物学特性,为骨组织工程提供种子细胞.方法从捐赠者髂骨穿刺收取骨髓细胞,采用密度梯度离心和贴壁培养法进行培养和纯化BMSCs,取生长良好的P3或P4代BMSCs进行检测:①MTT法测定细胞增殖和生长曲线分析;②流式细胞仪检测BMSCs细胞表面抗原标记和细胞增殖周期;③免疫荧光分析细胞骨架;④细胞染色体核型分析;⑤成骨细胞诱导分化及检测.结果 ①利用密度梯度离心和贴壁筛选的方法分离人BMSCs,经传代后细胞形态呈梭形,均匀一致.②MTT法测定细胞增殖和生长曲线分析结果显示BMSCs在传代后经历潜伏期、对数生长期和平台期.③流式细胞仪检测显示所获得的BMSCs表面抗原标记高度一致.④BMSCs细胞周期检测结果为G0+G1期913%、G2期4.1%和S期4.6%.⑤微管微丝免疫荧光染色结果显示,培养的BMSCs具有良好的细胞骨架系统.⑥第7代的BMSCs染色体检测结果显示,被测标本有正常染色体数目(2n=46,XY),且未见染色体异常.⑦成骨诱导后,Von kossa和茜素红染色均呈阳性.结论从人骨髓中能分离出高度一致的BMSCs,这些细胞具有正常细胞骨架结构以及正常的人类染色体数目和形态,具有向成骨细胞分化能力,可作为骨组织工程的种子细胞.
목적 탐토인골수래원MSCs적분리、배양화생물학특성,위골조직공정제공충자세포.방법 종연증자가골천자수취골수세포,채용밀도제도리심화첩벽배양법진행배양화순화BMSCs,취생장량호적P3혹P4대BMSCs진행검측:①MTT법측정세포증식화생장곡선분석;②류식세포의검측BMSCs세포표면항원표기화세포증식주기;③면역형광분석세포골가;④세포염색체핵형분석;⑤성골세포유도분화급검측.결과 ①이용밀도제도리심화첩벽사선적방법분리인BMSCs,경전대후세포형태정사형,균균일치.②MTT법측정세포증식화생장곡선분석결과현시BMSCs재전대후경력잠복기、대수생장기화평태기.③류식세포의검측현시소획득적BMSCs표면항원표기고도일치.④BMSCs세포주기검측결과위G0+G1기913%、G2기4.1%화S기4.6%.⑤미관미사면역형광염색결과현시,배양적BMSCs구유량호적세포골가계통.⑥제7대적BMSCs염색체검측결과현시,피측표본유정상염색체수목(2n=46,XY),차미견염색체이상.⑦성골유도후,Von kossa화천소홍염색균정양성.결론 종인골수중능분리출고도일치적BMSCs,저사세포구유정상세포골가결구이급정상적인류염색체수목화형태,구유향성골세포분화능력,가작위골조직공정적충자세포.
Objective To investigate the methods of the isolation, cultivation and characteristic of mesenchymal stem cells derived from human bone marrow ( hBMSCs ), so as to pave the way for searching the new source of seed cells in bone tissue engineering.Methods Bone marrow were inspired from ilium of donators, mononuclear cells were isolated by density gradient centrifugation and cultured in vitro. The higher purity of BMSCs were obtained by keeping the adherent cells and removal of nonadherent cells repeatedly. P3 or P4 BMSC populations were collected and examined as follow ①drawing a growth curve of the BMSCs by MTT colorimetric assay; ② detecting expressed the surface marker expression and the growing circle of BMSCs by flow cytometric analysis; ③Cytoskeleton was detected by immunofluorescence; ④Karyotype of BMSCs was examined by Giemsa stain. ⑤ the efficiency of differentiation into osteoblast was assessed by related methods.Results The highly purified BMSCs were obtained by density gradient centrifugation and keeping the adherent cells derived from human bone marrow and the BMSCs showed long fusiform or spindle and fibroblast-like shaped after repeated passages. ?The growth curve by MTT displayed that every passage experienced incubation period, log phase and platform period. ?The cell surface markers assay with flow cytometry indicated highly uniform of surface markers. ?The cell circle assay displayed G0 + 1 stage was in high percentage ( about 91.3% ), but G2 and S stage were in low percentage ( about 4.1% and 4.6% respectively ). ? The cultured BMSCs possessed normal morphology and cytoskeleton. ?The cells of P7 had normal human chromosome number ( 46, XY ) and appearance. ⑦Under inductive medium in vitro, the BMSCs could be induced into osteoblasts which were positive when stained by Von kossa and alizarin red. Conclusions: The highly purity and uniform of MSCs derived from human bone marrow can be obtained These BMSCs possess normal morphology, cytoskeleton, and chromosome number, the ability of differentiation into osteoblasts, can be used as a new source of seed cells in bone tissue engineering.iform