体外诱导兔骨髓间充质干细胞分化为类许旺细胞的初步研究

Primary Study on the Mesenchymal Stem Cells from Rabbits' Marrow Differentiated to Schwann-like Cells in Vitro

目的:探索将兔骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)在体外定向诱导分化为类许旺细胞(Schwann Cells,SC)有效的诱导方法。方法:(1)中国白兔5只,穿刺抽取股骨大转子骨髓3mL,用密度为1.073g/mL的percoll淋巴分离液行密度梯度分离,吸取中间白色膜状细胞层,接种在加有10%FCS的DMEM培养液的塑料培养瓶中,观察细胞形态并传代。(2)MSCs传至第3代,实验组加入加有0.5mmolβ-巯基乙醇、20ng/mL全反式黄酸、2.5μmol Forskolin、5ng/mL bFGF、20ng/mLPDGF、100ng/mL HRG的培养液培养24h,然后换用10%FBS的DMEM培养液培养24h,同样方法再诱导一次。对照组不加诱导剂,用10%FCS的DMEM培养液培养。观察两组MSCs的细胞形态,7d后用抗S-100、GFAP和P75抗体做免疫组织化学染色来鉴定诱导后MSCs。结果:MSCs在体外培养以梭形细胞为主,细胞平行排列或旋涡状生长,胞浆丰富,核大,核染色质细,有些核仁明显。细胞经多次传代后,呈长梭型。加入诱导剂后有少量细胞死亡,诱导后的细胞免疫组化阳性。诱导后细胞S-100、GFAP、P75免疫组化染色阳性率分别为74.9%、83%、70%。结论:兔MSCs可以在体外培养、增殖,并经β-巯基乙醇、全反式黄酸、Forskolin、bFGF、PDGF、HRG联合诱导分化为类SC。

Objective：To search for a more efficient method to induce mesenchymal stem cells （MSCs） differentiating into schwann-liked cells in vitro. Methods： （ 1 ） Approximate 3 mL of bone marrow was taken suction from five rabbits＇reater trochanter of femur by sterile operations, and delaminated by means of density gradient separation in 1. 073 g/mL percoll lymphocytes separating medium. The cells in white- intermedial- membrane layer were imbibed carefully, and then seeded in plastic culture flasks with growth medium, which were Dulbecco modified eagle medium （DMEM） containing 10% fetal calf serum （FCS） , and the cell shapes were observed day by day. （2） The cells at third passages were cultured in a differentiated medium containing 0.5mmol β-mercaptoethanol （BME）, 20ng/mL retinoic acid, 2.5 μmol forskolin, 5ng/mL basic fibroblast, growth factor （bFGF） , 20ng/mL platelet-derived growth factor（PDGF） and 100ng/mL heregulin （HRG） for 24 hours. Then the cells were further cultured in the growth medium for 24 hours. After that, the medium were replaced with differentiated medium for 24 hours, and eventually retrieved to the growth medium with changing fresh every 2 days. In control group, except for differentiation medium, the culture of MSCs was in the same manner of experimental group. The shapes of MSCs in both groups were observed by inverted microscope. After seven days, these cells were identified by immunocytochemistry staining with anti-S-100, P75 and GFAP antibodies. Results ： Most of MSCs cultured in vitro present as spindle, were similar to the shapes of fibroblasts, and lined up parallelly or turbulently. These cells possesed abundant endochylema, karyomegaly, fine nuclear chromatin, and sometimes obvious chromatospherites. A few MSCs died after culturing with differentiation medium. The undifferentiated MSCs were negative of immunocytochemistry stain, however, the MSCs induced by differentiation medium were positive for anti-S100, GFAP, and P75 antibodies, with positive ratios were 74.9%, 83% and 70% respectively in the experimental group. Conclusion：Our findings indicate that MSCs of rabbits could be cultured, proliferated and differentiated to schwann-liked cells by induction of BME, retinoic acid, forskolin, bFGF, PDGF and HRG.