中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
10期
1432-1435,后插二
,共5页
聂君%李劲松%王建军%胡少勃%江科%杨振华
聶君%李勁鬆%王建軍%鬍少勃%江科%楊振華
섭군%리경송%왕건군%호소발%강과%양진화
骨髓间充质干细胞%慢病毒%绿色荧光蛋白
骨髓間充質榦細胞%慢病毒%綠色熒光蛋白
골수간충질간세포%만병독%록색형광단백
Mesenchymal stem cells%Lentiviral vector%Green fluorescent protein
目的 建立大鼠骨髓间充质干细胞(MSCs)的分离和培养方法,观察绿色荧光蛋白(GFP)基因通过慢病毒载体感染MSCs的表达.方法 采用原代贴壁法获得骨髓MSCs,观察细胞形态和生长变化,流式细胞仪鉴定细胞表面标志,体外诱导MSCs向脂肪细胞和成骨细胞分化.采用含有增强型绿色荧光蛋白(EGFP)基因慢病毒载体感染培养的MSCs,比较细胞感染前后生物学特性.结果 原代贴壁筛选结合差异传代培养的MSCs表面标志阳性率分别为CD44 94.81%,CD90 99.53%,CD106 76.34%,MSCs在pH值稳定于7.2～7.4的环境可传20代.体外MSCs诱导分化后特异性染色显示脂质沉淀和骨结节,表达脂肪细胞和成骨细胞特异基因.慢病毒载体可有效感染大鼠MSCs,加入聚凝胺感染效率达到80%,EGFP在MSCs感染后1个月仍持续表达.结论 骨髓MSCs可长期培养,具有良好的多向分化能力.携带EGFP基因慢病毒载体能高效感染MSCs,EGFP可作为MSCs体内研究的示踪标记.
目的 建立大鼠骨髓間充質榦細胞(MSCs)的分離和培養方法,觀察綠色熒光蛋白(GFP)基因通過慢病毒載體感染MSCs的錶達.方法 採用原代貼壁法穫得骨髓MSCs,觀察細胞形態和生長變化,流式細胞儀鑒定細胞錶麵標誌,體外誘導MSCs嚮脂肪細胞和成骨細胞分化.採用含有增彊型綠色熒光蛋白(EGFP)基因慢病毒載體感染培養的MSCs,比較細胞感染前後生物學特性.結果 原代貼壁篩選結閤差異傳代培養的MSCs錶麵標誌暘性率分彆為CD44 94.81%,CD90 99.53%,CD106 76.34%,MSCs在pH值穩定于7.2～7.4的環境可傳20代.體外MSCs誘導分化後特異性染色顯示脂質沉澱和骨結節,錶達脂肪細胞和成骨細胞特異基因.慢病毒載體可有效感染大鼠MSCs,加入聚凝胺感染效率達到80%,EGFP在MSCs感染後1箇月仍持續錶達.結論 骨髓MSCs可長期培養,具有良好的多嚮分化能力.攜帶EGFP基因慢病毒載體能高效感染MSCs,EGFP可作為MSCs體內研究的示蹤標記.
목적 건립대서골수간충질간세포(MSCs)적분리화배양방법,관찰록색형광단백(GFP)기인통과만병독재체감염MSCs적표체.방법 채용원대첩벽법획득골수MSCs,관찰세포형태화생장변화,류식세포의감정세포표면표지,체외유도MSCs향지방세포화성골세포분화.채용함유증강형록색형광단백(EGFP)기인만병독재체감염배양적MSCs,비교세포감염전후생물학특성.결과 원대첩벽사선결합차이전대배양적MSCs표면표지양성솔분별위CD44 94.81%,CD90 99.53%,CD106 76.34%,MSCs재pH치은정우7.2～7.4적배경가전20대.체외MSCs유도분화후특이성염색현시지질침정화골결절,표체지방세포화성골세포특이기인.만병독재체가유효감염대서MSCs,가입취응알감염효솔체도80%,EGFP재MSCs감염후1개월잉지속표체.결론 골수MSCs가장기배양,구유량호적다향분화능력.휴대EGFP기인만병독재체능고효감염MSCs,EGFP가작위MSCs체내연구적시종표기.
Objective To establish a method of isolation and culture of the rat mesenchymal stem cells (MSCs) , and to investigate the expression of green fluorescent protein (GFP) gene carried by lentiviral vectors into MSCs. Methods MSCs, which were initially isolated from the bone marrow of rats, were cultured in vitro, isolated by trypsin digestion method, purified by adherence method and tested by flow cytomertry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was measured by reverse transcription-polymerase chain reaction (RT-PCR). MSCs were infected with lentiviral vectors. The results of the expression of GFP and infection efficiency were observed by fluorescence microscope. Results MSCs of high purity were obtained from bone marrow using adherence screening method. The stable pH of culture medium between 7.2-7.4 helped MSCs having subculture of 20 generations. MSCs typically expressed the antigens CD44 (94.81%) , CD90 (99.53% ) and CD106 (76.34%). They were negative for typical lymphocytic markers like CD45 ( 1.94% ) and CD11b ( 1.42% ) and for the early hematopoietic markers CD34 (0.04%). The infection rate of lentiviral vector in medium containing polybrene could reach 80%. The exogenous EGFP and multilineage potential of MSCs had no severe influences on each other. Conclusion Since the MSCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.
