CAJ | 학술논문

背景:骨髓干细胞移植成功的定性指标是标记移植的细胞至组织器官后存活并发挥了正常的功能.目前细胞学研究方面采用的标记方法有酶联标记、氚胸腺嘧啶核苷标记、荧光标记和5-溴脱氧尿嘧啶核苷标记等.目的:观察5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞的特点.设计:单一样本观察.单位:华北煤炭医学院附属医院心胸外科.材料:实验于2003-07/2004-12在华北煤炭医学院中心实验室完成.取月龄3个月的日本大耳白兔6只,雌雄不限,体质量(3.00±0.25)kg.方法:①利用密度为1 073 g/L的Percoll分离骨髓的单核细胞,以含体积分数为0.1的胎牛血清的低糖Dulbecco's改良的Eagle's培养基培养与扩增间充质干细胞,纯度可达95%左右.②用5-溴脱氧尿嘧啶核·苷标记骨髓间充质干细胞24,48,72,96 h后的检测标记率(标记组);阴性对照为未经5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞;空白对照为以磷酸盐缓冲液或正常鼠血清替代一抗.主要观察指标:①3组各检测200个细胞,并在不同时间点观察.②5-溴脱氧尿嘧啶核苷标记的骨髓间充质干细胞免疫组织化学染色结果及细胞计数结果.结果:①标记组均见5-溴脱氧尿嘧啶核苷阳性细胞,5-溴脱氧尿嘧啶核苷阳性反应物位于细胞核,呈棕色、颗粒状或弥漫性分布;对照组未见阳性细胞.②标记组24,48,72,96 h 5-溴脱氧尿嘧啶核苷阳性细胞的数目分别为48±2,100±4,173±2,178±3,随着标记时间的延长,标记率逐渐增高,标记72 h后标记率在85%以上.③阴性对照和空白对照组各时间点均为0.结论:①用5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞的最佳时间是72 h.②标记后检测的敏感性好,在低倍镜中亦可见,适合于大块组织的定量研究.③结果表明5-溴脱氧尿嘧啶核苷标记骨髓间充质干细胞是可行的.
배경:골수간세포이식성공적정성지표시표기이식적세포지조직기관후존활병발휘료정상적공능.목전세포학연구방면채용적표기방법유매련표기、천흉선밀정핵감표기、형광표기화5-추탈양뇨밀정핵감표기등.목적:관찰5-추탈양뇨밀정핵감표기골수간충질간세포적특점.설계:단일양본관찰.단위:화북매탄의학원부속의원심흉외과.재료:실험우2003-07/2004-12재화북매탄의학원중심실험실완성.취월령3개월적일본대이백토6지,자웅불한,체질량(3.00±0.25)kg.방법:①이용밀도위1 073 g/L적Percoll분리골수적단핵세포,이함체적분수위0.1적태우혈청적저당Dulbecco's개량적Eagle's배양기배양여확증간충질간세포,순도가체95%좌우.②용5-추탈양뇨밀정핵·감표기골수간충질간세포24,48,72,96 h후적검측표기솔(표기조);음성대조위미경5-추탈양뇨밀정핵감표기골수간충질간세포;공백대조위이린산염완충액혹정상서혈청체대일항.주요관찰지표:①3조각검측200개세포,병재불동시간점관찰.②5-추탈양뇨밀정핵감표기적골수간충질간세포면역조직화학염색결과급세포계수결과.결과:①표기조균견5-추탈양뇨밀정핵감양성세포,5-추탈양뇨밀정핵감양성반응물위우세포핵,정종색、과립상혹미만성분포;대조조미견양성세포.②표기조24,48,72,96 h 5-추탈양뇨밀정핵감양성세포적수목분별위48±2,100±4,173±2,178±3,수착표기시간적연장,표기솔축점증고,표기72 h후표기솔재85%이상.③음성대조화공백대조조각시간점균위0.결론:①용5-추탈양뇨밀정핵감표기골수간충질간세포적최가시간시72 h.②표기후검측적민감성호,재저배경중역가견,괄합우대괴조직적정량연구.③결과표명5-추탈양뇨밀정핵감표기골수간충질간세포시가행적.
BACKGROUND: The indicant of successful transplanted bone marrow stem cells is that the labeled transplanted ceils can survive in the target organs and can their biological functions. Up to date, at cell level, there are several labeled methods such as enzyme-linked, 3H-TdR, fluorescent and 5-bromodexyuridine method, etc.OBJECTIVE: To observe the biological characteristics of bone marrowderived mesenchymal stem cells labeled with 5-bromodexyuridine. DESIGN: Observations in single kind of sample SETTING: Department of Thoracic Cardiac Surgery, Affiliated Hospital of North China Coal Medical College MATERIALS: The experiment was carried out in the Experimental Center of North China Coal Medical College from July, 2003 to November,2004 on six Japanese long-eared rabbits ,with the age of 3 months , of either gender and the body mass of (3.00±0.25)kg.METHODS:①Monocytes were isolated from the bone marrow with Percoll reagent of the concentration of 1 073 g/L. The mesenchymal stem cells were cultured and proliferated with Eagle's medium, which was improved by adding 10% low sugar Dulbecco's to fetal bovine serum, so that their purity could reach about 95%. Secondly, in the experimental groups, the percentage of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine was detected after 24 hours, 48 hours, 72 hours and 96hours, respectively. The negative-controlled group was composed of bone marrow-derived mesenchymal stem cells non-labeled with 5-bromodexyuridine while the blank-controlled group was made by substituting phosphate buffer or normal mice serum for primary antibody.MAIN OUTCOME MEASURES: In three different groups, 200 cells of each group were detected and observed at different time. Outcomes of the immunochemical stauning and counting of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were also determined.RESULTS: The positive bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were observed in all the experimental groups. The positive materials were brown, granular and dfiffusedly scattered in the nucleus while no positive cell was observed in the controlled groups. After 24 hours, 48 hours, 72 hours and 96 hours, the numbers of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine were 48±2, 100±4, 173:t:2, 178±3 respectively. The labeling percentage was raised gradually with the elongation of time. 72 hours later,the labeling percentage is above 85%. The numbers in negative-controlled and blank-controlled groups were all zero.CONCLUSION: The appropriate time of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuriding is 72 hours. The sensibility of post-labeling detection is high due to the results being observed in the low power microscope, which makes this method suitable to quantitative analysis in massive tissue. These results show that the method of bone marrow-derived mesenchymal stem cells labeled with 5-bromodexyuridine is feasible.