全反式维甲酸诱导大鼠骨髓间充质干细胞向神经细胞分化后对T细胞增殖的抑制

背景:研究表明骨髓间充质干细胞能抑制T淋巴细胞的增殖活化,发挥负性免疫调节作用.目的:利用全反式维甲酸体外定向诱导大鼠骨髓间充质干细胞向神经元样细胞分化,并探讨分化细胞对T细胞增殖的作用.设计、时间及地点:细胞学体外观察,于2008-03在广东医学院完成.材料:4周龄雄性SD大鼠2只,由广东医学院动物中心提供.新鲜健康人血由广东医学院检验科提供.方法:通过贴壁法分离大鼠骨髓间充质干细胞,体外传代扩增.取传至5代的骨髓间充质干细胞,加入含0.3 mg/L全反式维甲酸、体积分数为10%胎牛血清的LG-DMEM液进行预诱导,24 h后吸去预诱导液,加入含0.6 mg/L全反式维甲酸的LG-DMEM液向神经元样细胞诱导分化.取新鲜健康人血制备反应细胞,大鼠骨髓间充质干细胞作为刺激细胞,进行单向混合淋巴细胞反应实验,设立4组:对照组,单纯反应细胞100 μL,细胞浓度1×109L-1;实验1组:1×104神经元样细胞+100 μL反应细胞:实验2组:1×105神经元样细胞+100 μL反应细胞:实验3组:1×106神经元样细胞+100 μL反应细胞.主要观察指标:诱导分化后细胞形态变化及神经细胞鉴定,单向混合淋巴细胞反应结果.结果:加入诱导液50 min后,光镜下骨髓间充质干细胞呈典型的核周体形态,3 h后大多数细胞转变为双极或多极神经元细胞样形态,出现胞体和突起.免疫细胞化学染色结果显示,大部分细胞呈神经元特异性烯醇化酶及巢蛋白阳性表达,而胶质纤维酸性蛋白呈阴性表达.与对照组比较,各实验组放射性核素每分钟闪烁数值均明显减少(P<0.05),且各实验组间比较差异亦有显著性意义(P<0.05),随着加入细胞数量的增多,抑制作用越明显.结论:全反式维甲酸可在体外诱导大鼠骨髓间充质干细胞分化为神经元样细胞,分化后的神经元样细胞能够抑制人淋巴细胞增殖,并呈剂量依赖性增加.

All-trans retinoic acid-induced nerve cell differentiation of rat bone marrow mesenchymal stem cells inhibits T lymphocyte proliferation

BACKGROUND:Several studies have demonstrated that bone marrow mesenchymal stem cells (BMSCs) inhibit the proliferation and activation of T lymphocytes, exerting a role of negative immunological regulation. OBJECTIVE: To induce rat BMSCs to differentiate into neuron-like cells in vitro using all-trans retinoic acid and to investigate the effects of differentiated cells on T lymphocyte proliferation.DESIGN, TIME AND SETTING: A cytological in vitro observation was performed in Guangdong Medical College in March 2008.MATERIALS: Two male 4-week-old Sprague Dawley rats were provided by the Laboratory Animal Center of Guangdong Medical College. Fresh healthy human blood was provided by the Department of Clinical Laboratory, Guangdong Medical College. METHODS: Rat BMSCs were isolated by adherent method and were passaged in vitro. Cells of passage 5 were pre-induced with Low glucose-dulbecco's modified eagle's medium (LG-DMEM) containing 0.3 mg/L all-trans retinoic acid and 10% fetal bovine serum. Twenty-four hours later, aforementioned LG-DMEM was discarded and LG-DMEM containing 0.6 mg/L all-trans retinoic acid was added to induce cells differentiation into neuron-like cells. Fresh healthy human blood was taken to prepare responding cells. Rat BMSCs, as stimulator cells, were included for one-way mixed lymphocyte reaction. Four groups were designated. Control group: 100 μL of responding cells at a concentration of 1×109/L; Experimental group 1:1×104 neuron-like cells + 100 μL of responding cells; Experimental group 2:1×105 neuron-like cells + 100 μL of responding cells; Experimental group 3:1×106 neuron-like cells + 100 μL of responding cells.MAIN OUTCOME MEASURES: Morphological observation of induce-differentiated cells, identification of nerve cells, and one-way mixed lymphocyte reaction results.RESULTS: Fifty minutes after addition of inducing medium, under the optical microscope, BMSCs exhibited a typical morphology of perikaryon. Three hours later, most of cells became into bipolar or multipolar neuron-like cell appearance with cell bodies and processes. Immunocytochemical staining results showed that majority of cells exhibited neuron specific enolase- and nestin-positive expression and glial fibrillary acidic protein-negative expression. Compared with the control group, radionuclide counts per minute were significantly reduced in each experimental group (P<0.05). There was significant difference in radionuclide counts per minute between experimental groups (P<0.05). With increasing BMSCs amount, the inhibitory effects on T lymphocyte proliferation were more evident.CONCLUSION: AII-trans retinoic acid can induce neuron-like cell differentiation of rat BMSCs in vitro. The neuron-like cells can inhibit human T lymphocyte proliferation in a dose-dependent manner.