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目的:以含有碱性成纤维细胞生长因子和二甲基亚砜的培养液对原代培养的成人骨骼肌干细胞进行诱导,探讨其转化为神经细胞的可行性。方法:实验于2004-06/12在军事医学科学院基础医学研究所完成。实验所用3例成人正常颞肌标本取自于开颅手术患者,由北京协和医院神经外科提供,患者知情同意。机械分离加混合消化液处理分离单细胞,体外培养及克隆纯化,培养至第3代时加入含有碱性成纤维细胞生长因子二甲基亚砜的培养液诱导分化,进行反转录-聚合酶链反应分析和免疫荧光细胞化学染色,观察诱导后细胞形态变化,同时结合普通光线在100倍下随机计数6个视野(大于100个细胞),计算阳性细胞的百分比。结果:①骨骼肌干细胞培养24h后见少量细胞贴壁呈短的梭形或棒状。培养第3天红细胞开始裂解成细胞碎屑并聚集、悬浮在培养基中,这些碎屑随着换液而逐渐消失。克隆培养的细胞大约于接种后两三周才能铺满孔底,胞体呈梭形,排列规整。②荧光显微镜下胞核蓝染,每个细胞都呈Desmin染色阳性,证明成人骨骼肌干细胞能够在体外进行克隆培养。③在含有碱性成纤维细胞生长因子和二甲基亚砜的培养液诱导下,骨骼肌干细胞可分化为形态上类似于神经元和星形胶质细胞的细胞。④反转录-聚合酶链反应分析证实诱导后的骨骼肌干细胞有神经丝蛋白和胶质纤维酸性蛋白基因表达。⑤免疫荧光细胞化学检测到诱导后的骨骼肌干细胞呈神经丝蛋白和胶质纤维酸性蛋白免疫反应阳性。⑥诱导后骨骼肌干细胞分化为神经丝蛋白阳性细胞的比例为(35.9±6.3)%,分化为胶质纤维酸性蛋白阳性细胞的比例为(43.7±5.3)%。结论:成人骨骼肌干细胞在体外经诱导分化后形态类似于神经元和星形胶质细胞,并且表达神经元标志物神经丝蛋白、星形胶质细胞标志物胶质纤维酸性蛋白,认为成人骨骼肌干细胞在体外可诱导分化为神经元和星形胶质细胞。
OBJECTIVE: To induce primary cultured adult skeletal muscle stem cells with medium containing basic fibroblast growth factor and dimethyl sulfoxide (DMSO) to explore the feasibility of transforming into neural cells. Methods: The experiment was performed at the Institute of Basic Medical Sciences, Academy of Military Medical Sciences from June to June 2004. Three adult normal temporomandibular specimens were obtained from patients undergoing craniotomy and were provided by Department of Neurosurgery, Peking Union Medical College Hospital. Patients were informed consent. Mechanical separation and mixed digestion solution were used to separate single cells, cultured in vitro and cloned and purified, cultured to the third generation, medium containing basic fibroblast growth factor dimethyl sulfoxide was added to induce differentiation, and reverse transcription-polymerase Chain reaction analysis and immunofluorescence cytochemistry to observe the changes of cell morphology after induction, combined with ordinary light at 100 times under the random counting of six fields (more than 100 cells), calculate the percentage of positive cells. Results: (1) Skeletal muscle stem cells showed a small spindle-like or rod-like appearance after 24h incubation. On day 3 of culturing, erythrocytes begin to lyse and accumulate into cellular debris, suspended in the culture medium, and these fragments gradually disappear as fluid is changed. Cloned cultured cells about two to three weeks after inoculation to fill the bottom of the hole, cell body was spindle-shaped, arranged in order. ② Under the fluorescence microscope, the nuclei were stained blue, and each cell showed positive Desmin staining, which proved that adult skeletal muscle stem cells could be cultured in vitro. ③ In the medium containing basic fibroblast growth factor and dimethyl sulfoxide, skeletal muscle stem cells can differentiate into cells that are morphologically similar to neurons and astrocytes. ④ Reverse transcription - polymerase chain reaction analysis confirmed the induced skeletal muscle stem cells have neurofilament and glial fibrillary acidic protein gene expression. ⑤ immunofluorescence cytochemistry detected skeletal muscle stem cells after induction of neurofilament protein and glial fibrillary acidic protein immunopositive. ⑥ The percentage of skeletal muscle stem cells differentiated into neurofilament positive cells was (35.9 ± 6.3)% and the percentage of differentiated into glial fibrillary acidic protein positive cells was (43.7 ± 5.3)%. CONCLUSIONS: Adult skeletal muscle stem cells are morphologically similar to neurons and astrocytes after being induced to differentiate in vitro and express the neuron marker neurofilament protein and astrocyte marker glial fibrillary acidic protein, which are considered as adult skeletal Muscle stem cells can be induced to differentiate into neurons and astrocytes in vitro.