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目的 BMSCs具有多向分化潜能,在不同培养条件下能够向多种细胞分化,但年龄对BMSCs分化的影响尚不明确。探讨雪旺细胞(Schwann cells,SCs)形成的体外微环境对不同年龄大鼠BMSCs向神经元和少突胶质细胞分化的影响。方法从幼年(1月龄)Wistar大鼠预变性坐骨神经中分离纯化SCs,从幼年(1月龄)、成年(6月龄)、老年(12月龄)Wistar大鼠骨髓中提取BMSCs,均行免疫荧光鉴定。将第3代SCs与PKH26标记的第2代BMSCs在双层培养皿内等体积、等密度共培养,根据BMSCs来源不同将实验分为3组:A、B、C组SCs分别与幼年(1月龄)、成年(6月龄)、老年(12月龄)大鼠BMSCs共培养。倒置相差显微镜下观察分化过程中BMSCs形态变化,免疫荧光染色检测共培养后第7天BMSCs分化表达神经特异性烯醇化酶(neuron-specifi c enolase,NSE)和磷脂碱性蛋白(myelin basicprotein,MBP)的情况,ELISA法检测各组细胞共培养0、3、6、9、12 d培养液上清神经调节蛋白1(neuregulin 1,NRG1)的表达。结果实验成功分离并培养SCs和BMSCs,免疫荧光鉴定SCs呈S-100阳性表达,BMSCs呈CD29阳性表达、CD44阳性表达、CD90阳性表达。细胞共培养第7天倒置相差显微镜观察示,A组BMSCs胞体回缩,呈圆形或锥形,并带有突起,形态类似神经组织细胞;B、C组大部分BMSCs形态无明显改变。细胞共培养第7天免疫荧光染色示,A、B、C组BMSCs的NSE阳性表达率分别为22.39%±2.86%、12.89%±1.78%和2.69%±0.80%,MBP阳性表达率分别为16.13%±2.39%、6.33%±1.40%和0.92%±0.17%,各组间NSE阳性表达率和MBP阳性表达率比较差异均有统计学意义(P<0.05)。ELISA法检测示,A组细胞培养液上清中NRG1含量于细胞共培养后逐渐增多,且呈时间依赖性增加;细胞共培养6、9、12 d,A组NRG1含量高于B、C组,B组高于C组,比较差异均有统计学意义(P<0.05)。结论不同年龄大鼠BMSCs与SCs共培养均可向神经元和少突胶质细胞分化,但分化能力随年龄增加而降低,SCs分泌的多种生长因子可能是诱导BMSCs分化的重要因素。
Purpose BMSCs have multidirectional differentiation potential and can differentiate into many kinds of cells under different culture conditions. However, the influence of age on the differentiation of BMSCs is not yet clear. To investigate the effect of in vitro microenvironment formed by Schwann cells (SCs) on the differentiation of BMSCs to neurons and oligodendrocytes in rats of different ages. Methods The SCs were isolated and purified from the pre-differentiated sciatic nerve of Wistar rats. The BMSCs were isolated from the bone marrow of Wistar rats in young (1 month old), adult (6 months old) and old (12 months old) Immunofluorescence identification. The 3rd generation SCs and PKH26 labeled second generation BMSCs were co-cultured in equal volume in two-layer petri dishes. According to the different sources of BMSCs, the experiment was divided into three groups: SCs in groups A, B and C were compared with juvenile Month old), adult (6 month old), old (12 month old) rat BMSCs were co-cultured. The morphological changes of BMSCs during differentiation were observed under an inverted phase contrast microscope. The expression of neuron-specific enolase (NSE) and myelin basic protein (MBP) on the 7th day after co-culture were detected by immunofluorescence staining. ). The expression of neuregulin 1 (NRG1) in culture supernatants of 0, 3, 6, 9 and 12 days in each group was detected by ELISA. Results The SCs and BMSCs were successfully isolated and cultured. The Ss-100 positive cells were identified by immunofluorescence. The BMSCs were positive for CD29, positive for CD44 and positive for CD90. On the 7th day of co-culture, the phase contrast microscopy showed that the cell bodies of group A were contracted, round or conical, with protrusions and morphology similar to that of nerve tissue cells. The morphology of most BMSCs in group B and C did not change significantly. On day 7 of co-culture, immunofluorescence staining showed that the positive expression rates of NSE in BMSCs of groups A, B and C were 22.39% ± 2.86%, 12.89% ± 1.78% and 2.69% ± 0.80% respectively, and the positive rates of MBP were 16.13 % ± 2.39%, 6.33% ± 1.40% and 0.92% ± 0.17%, respectively. There was significant difference in NSE positive expression rate and MBP positive expression rate among all groups (P <0.05). ELISA assay showed that the content of NRG1 in group A cell culture supernatant gradually increased in a time-dependent manner and increased in time-dependent manner. The cells were co-cultured for 6, 9 and 12 days. The content of NRG1 in group A was higher than that in group B and C , B group was higher than C group, the difference was statistically significant (P <0.05). Conclusion BMSCs co-cultured with SCs can differentiate into neurons and oligodendrocytes in different age groups. However, the differentiation ability of BMSCs decreases with increasing age. Many growth factors secreted by SCs may be important factors in inducing BMSCs differentiation.