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背景:骨髓间充质干细胞是一种具有自我复制及多向分化潜能的干细胞,在体外可以向神经细胞、脂肪细胞、成骨细胞分化。目的:观察不同代数的成人骨髓间充质干细胞体外向神经元细胞转化的效率,为骨髓间充质干细胞应用于临床提供可靠的实验数据。设计:单一样本实验。单位:吉林大学中日联谊医院神经内科。对象:骨髓组织取自吉林大学第一医院骨科行脊柱融合术患者9例,男6例,女3例;患者均知情同意。方法:实验于2002-09/2003-02在吉林大学中日联谊医院中心实验室完成。对成人骨髓间充质干细胞的原代和传代培养。分为实验组和对照组,实验组又分:神经元烯醇化酶组,神经丝蛋白-M组,胶质纤维酸性蛋白组和尼氏染色组。采用β-巯基乙醇做为诱导剂,选用第2,4,6,8代成人骨髓间充质干细胞在体外诱导6h后,用细胞化学及免疫组织化学检测神经元细胞、星形胶质细胞标记蛋白的表达。主要观察指标:①成人骨髓间充质干细胞传代培养的生长曲线分析。②尼氏染色结果。③免疫组织化学染色结果。结果:①传代培养具有以下共性特征:传代培养潜伏期12～24h;传代培养对数增殖期约为7～10d,11～13d进入细胞生长平台期。②第2,4,6代成人骨髓间充质干细胞诱导后胞浆中均可见深蓝色的颗粒状尼氏体,第8代诱导6h后胞浆中未见明显的深蓝色尼氏体结构。③不同代数的成人骨髓间充质干细胞经诱导6h后均表达神经元烯醇化酶、神经丝蛋白-M,不表达胶质纤维酸性蛋白;第2,4,6代的阳性率无显著性差异(P>0.05),第8代与第2,4,6代的阳性率有显著性差异(P<0.05)。结论:β-巯基乙醇在体外可定向诱导成人骨髓间充质干细胞转化为神经元细胞,第2,4,6代的阳性率明显高于第8代。
BACKGROUND: Bone marrow mesenchymal stem cells are stem cells that have the potential of self-replication and multi-directional differentiation and can differentiate into nerve cells, adipocytes and osteoblasts in vitro. OBJECTIVE: To observe the efficiency of algebraic adult bone marrow mesenchymal stem cells (BMSCs) transformed into neurons in vitro and to provide reliable experimental data for clinical application of BMSCs. Design: Single sample experiment. Unit: Sino-Japanese Friendship Hospital, Jilin University, Department of Neurology. PARTICIPANTS: Myeloid tissue was obtained from 9 patients with spine fusion under orthopedic department of the First Hospital of Jilin University, 6 males and 3 females; all patients were informed consent. METHODS: The experiment was performed at the Central Laboratory of Sino-Japanese Friendship Hospital, Jilin University from September 2002 to February 2003. Primary and subculture of adult human bone marrow mesenchymal stem cells. Divided into experimental group and control group, the experimental group is divided into: neuron enolase group, neurofilament protein-M group, glial fibrillary acidic protein group and Nissl staining group. Using β-mercaptoethanol as an inducer, the second, fourth, sixth and eighth generations of human bone marrow-derived mesenchymal stem cells were selected for inducing in vitro for 6 h, then the cytochemistry and immunohistochemistry were used to detect neuronal cells, astrocyte markers Protein expression. MAIN OUTCOME MEASURES: ① Growth curve analysis of subcultured adult human bone marrow mesenchymal stem cells. ② Nissl staining results. ③ immunohistochemical staining results. Results: (1) The subculture had the following common features: The incubation period of subculture was 12-24 hours. The logarithmic proliferation stage of subculture was about 7-10 days and reached the plateau stage of 11-13 days. The second, the sixth and the sixth and the sixth generation adult human bone marrow mesenchymal stem cells can be seen in the cytoplasm after dark granular Niger body, after 8th generation of 6h after the cytoplasm no obvious dark blue Niusite structure. (3) The neuronal enolase, neurofilament protein-M, and glial fibrillary acidic protein were expressed in all algebraic adult BMSCs after 6h of induction; there was no significant difference in the 2nd, 4th, and 6th generation (P> 0.05). There was a significant difference between the 8th generation and the 2nd, 4th and 6th generations (P <0.05). CONCLUSION: β-Mercaptoethanol can induce adult BMSCs into neurons in vitro. The positive rates of the 2nd, 4th and 6th generation were significantly higher than those of the 8th generation.