【摘要】 目的:探讨碱性成纤维细胞生长因子和神经生长因子联合诱导成年大鼠骨髓基质细胞分化为神经细胞的可能性和条件,为神经细胞的再生和脊髓内移植提供实验基础。方法:实验于2005-07/10在锦州医学院附属第一医院骨科实验室完成。选取清洁级、鼠龄1个月SD大鼠1只,拉颈处死后无菌条件下取出股骨,除去骨周围的肌肉组织,剪开骨两端,显露骨髓腔,以IMDM培养液从一端冲洗骨髓腔,将骨髓冲到平皿中,进行骨髓基质细胞的分离培养,传至第5代的骨髓基质细胞用于实验。诱导前24h先用含1μg/L碱性成纤维细胞生长因子及体积分数为0.2的胎牛血清的DMFM培养液培养,以促进细胞分裂,然后用神经生长因子作诱导剂,观察细胞形态的变化,并采用免疫组织化学法检测诱导后细胞表达神经丝蛋白、神经元烯醇化酶等特异性标志物情况。结果:①原代和传代培养时大鼠骨髓基质细胞的生长情况:原代培养2～4d细胞处于静止状态。第五六天可见有贴壁细胞开始伸展为椭圆型、短梭型、长梭型等,这些细胞即为骨髓间充质干细胞。至第14天骨髓基质细胞基本铺满瓶底,此时即可传代。传代后2～4d第1代细胞基本处于静止状态,第5天开始缓慢增殖,约在第8天长满瓶底,传代周期为六七天。②碱性成纤维细胞生长因子与神经生长因子联合诱导后骨髓基质细胞的形态变化:经碱性成纤维细胞生长因子预诱导2h后,骨髓基质细胞形态无明显变化,但胞体变大。神经生长因子诱导12h后,部分细胞分化,胞体向胞核收缩呈锥形,类似神经细胞,细胞突起之间呈渐进性变化,但无明显增殖现象。③大鼠骨髓基质细胞分化后的鉴定和转化率的测定:神经样细胞的胞体与突起,神经元烯醇化酶(γ-γ)和神经丝蛋白染色呈强阳性,而未分化的骨髓基质细胞为阴性。定量计数分析表达神经元烯醇化酶(γ-γ)为(69.6±3.8)%,表达神经丝蛋白为(71.3±3.3)%。结论:碱性成纤维细胞生长因子和神经生长因子联合应用能将骨髓基质细胞诱导成神经元样细胞,预诱导24h后撤离二者可增加细胞转化率,促进诱导后的细胞成熟,为细胞移植治疗脊髓损伤提供一种高效种子细胞生产方法。更多还原

【Abstract】 AIM To probe into the possibility and conditions of combination of basic fibroblast growth factorbFGF and nerve growth factor for inducing adult rat bone marrow stromal cellsBMSCs to differentiate into neurons so as to provide experimental basis for regeneration of nerve cell and transplantation of bone marrow. METHODS This experiment was conducted at the laboratory for Orthopaedic Department First Hospital Affiliated to Jinzhou Medical College from July to October 2005. One 1-month SD rat of clean degree was chosen. Femur was taken out under aseptic condition after the rat was put to death then muscular tissue around the bone was removed. Two ends of the bone were cut open and bone marrow cavity was exposed and then was washed from one end with IMDM culture medium. The bone marrow was washed out to the flat plate for isolation of BMSCs. The 5th generation BMSCs were used for the experiment. DMEM culture medium containing 1 μg/L bFGF and 0.2 volume fraction of fetal bovine serum was used to promote cell division within 24 hours before induction then nerve growth factor was added as inductor . The morphological change of the cells was observed. Neurofilament protein and neuron-specific enolase and other specific markers expressed by cells were detected with immunohistochemical method after induction. RESULTS ① Growth of BMSCs of the rats at primary and generative culture Cells were in quiescent state after primary culture for 2 to 4 days. On the 5th to 6th days the adhered cells became ellipse short-shuttle shape long-shuttle shape and so on and these were BMSCs. Till the 14th day BMSCs covered the bottom of bottle and passage could begin at this time. On the day 2 to 4 days after passage cells of the 1st generation were basically in quiescent state proliferation began slowly from the 5th day. Cells covered the bottom of bottle on the 8th day and passage period was about 6 to 7 days. ② Morphological change of BMSCs after induction of bFGF and nerve growth factor After pre-induction of bFGF  there was no obvious change of morphological change of BMSCs but the cell body became big. After induction of nerve growth factor for 12 hours partial cells differentiated cell body shrinked to nucleus presenting taper similar to nerve cell. Progressive change appeared among the mutations but there was no obvious proliferation. ③ Identification after differentiation of BMSCs and the determination of transformation efficiency Cell body and mutation neuron-specific enolase γ-γ and neurofilament protein of nerve-like cells was positive in staining while that of non-differentiated BMSCs was negative. Quantitative count analysis showed (69.6±3.8)% was neuron-specific enolase γ-γ and (71.3±3.3)% was neurofilament protein. CONCLUSION The BMSCs can differentiate into neuron-like cells by the induction of basic fibroblast growth factor and nerve growth factor. After pre-induction for 24 hours we removed the two factors and found that they could increase the transforming efficiency and promote the cellular maturity. This method may provide a high-efficiency way to produce seed cells in cell transplantation for treatment of spinal injury.更多还原