CAJ | 학술논문

背景:骨髓间充质干细胞在骨髓中的含量极低,体外的长期培养过程中易丧失干细胞潜能以及体内移植后安全性等问题的存在,限制了骨髓间充质干细胞在临床上的广泛应用.目的:探索体外分离纯化、冻存大鼠骨髓间充质干细胞的最适方法,并观察以此方法培养的骨髓间充质干细胞移植体内后是否具有成瘤性.方法:分别采用差速贴壁结合24 h首次换液、24 h首次换液、48 h首次换液的方法纯化培养骨髓间充质干细胞,筛选最适纯化方法进行后续实验.配制含体积分数为10%,20%,30%,40%,50%胎牛血清的细胞冻存液冻存细胞,复苏后计算细胞存活率,测定复苏后细胞的生长曲线及成脂诱导能力.将第3,15代骨髓间充质干细胞进行裸鼠肌肉、肝脏局部注射体内移植,45 d后取注射部位行病理组织标本检查.结果与结论:差速贴壁结合24 h首次换液法所获得骨髓间充质干细胞纯度最高,而细胞增殖能力与其他两组无明显差别,因此选用该方法纯化骨髓间充质干细胞,然后进行传代培养;含体积分数为30%血清冻存液既可保证细胞活性与增殖能力,又可保证干细胞特性及多向分化潜能.骨髓间充质干细胞传至15代仍保持间充质干细胞特性,骨髓间充质干细胞移植入裸鼠肝脏45 d后仍可在肝脏局部存活,生长状态与体外培养相似,无异型性及向周围浸润生长,提示体外长时间培养15代以内的骨髓间充质干细胞可在裸鼠体内存活且无成瘤性.
배경:골수간충질간세포재골수중적함량겁저,체외적장기배양과정중역상실간세포잠능이급체내이식후안전성등문제적존재,한제료골수간충질간세포재림상상적엄범응용.목적:탐색체외분리순화、동존대서골수간충질간세포적최괄방법,병관찰이차방법배양적골수간충질간세포이식체내후시부구유성류성.방법:분별채용차속첩벽결합24 h수차환액、24 h수차환액、48 h수차환액적방법순화배양골수간충질간세포,사선최괄순화방법진행후속실험.배제함체적분수위10%,20%,30%,40%,50%태우혈청적세포동존액동존세포,복소후계산세포존활솔,측정복소후세포적생장곡선급성지유도능력.장제3,15대골수간충질간세포진행라서기육、간장국부주사체내이식,45 d후취주사부위행병리조직표본검사.결과여결론:차속첩벽결합24 h수차환액법소획득골수간충질간세포순도최고,이세포증식능력여기타량조무명현차별,인차선용해방법순화골수간충질간세포,연후진행전대배양;함체적분수위30%혈청동존액기가보증세포활성여증식능력,우가보증간세포특성급다향분화잠능.골수간충질간세포전지15대잉보지간충질간세포특성,골수간충질간세포이식입라서간장45 d후잉가재간장국부존활,생장상태여체외배양상사,무이형성급향주위침윤생장,제시체외장시간배양15대이내적골수간충질간세포가재라서체내존활차무성류성.
BACKGROUND:Bone marrow mesenchymal stem cel s are restricted in clinical application due to low contents in bone marrow, easy to lose stem cel potential during long-term in vitro culture, and unclear safety after in vivo transplantation. OBJECTIVE:To explore an optimal isolation, purification and cytopreservation method of rat bone marrow mesenchymal stem cel s and observe the tumorigenicity after implanting the in vitro cultured cel s into nude mice. METHODS:Bone marrow mesenchymal stem cel s were obtained from bone marrow cavity, purified and cutured by three different methods. The culture-expanded bone marrow mesenchymal stem cel s were cryopreserved with cytoprotectant solution containing different serum concentrations at-80 ℃. Fol owing recovery from cryopreservation, cel growth capacity and adiogenic capacity were determined. Passage 3 and 15 bone marrow mesenchymal stem cel s were injected into the muscle and liver of nude mice to observe the neoplasia in local site. RESULTS AND CONCLUTION:The purity of bone marrow mesenchymal stem cel s obtained by differential adhesion combined with 24-hour first exchange method was higher compared to other control groups, and there was no significant difference in cel proliferation capacity between groups. Bone marrow mesenchymal stem cel s cryopreserved with 30%serum could ensure the viability, proliferation ability and stem cel characterisitics and multi-differentiation potential. Bone marrow mesenchymal stem cel s might keep stem cel properties until to the 15th generation. After 45 days of transplantation to nude mice, no neoplasia was found in the local site. The muscle tissue had normal structure. Passage 15 bone marrow mesenchymal stem cel s could survive and proliferate in the liver with no hetermorphism and invasion to the surrounding liver tissue.