CAJ | 학술논문

背景:骨髓间充质干细胞诱导分化为神经细胞,是神经系统疾病细胞治疗的有效手段,但目前的时效尚不完善.目的:应用神经发育音猬因子诱导恒河猴骨髓间充质干细胞向神经样细胞分化.方法:应用经典的维甲酸方案与音猬因子方案两种方法诱导恒河猴骨髓间充质干细胞分化为神经样细胞,采用密度梯度离心法分离培养恒河猴骨髓间充质干细胞,倒置相差显微镜观察生长情况,MTT法测定细胞生长曲线,流式细胞仪鉴定细胞表型,免疫组织化学鉴定分化细胞标志,透射电镜和扫描电镜观察分化细胞超微结构.结果与结论:体外分离培养的恒河猴骨髓间充质干细胞,经流式细胞仪表型鉴定,具有较高均一性.通过音猬因子诱导方案诱导处理7 d后,分化细胞多数表现为NSE、NF-M、Tau和Nestin染色阳性,经图像统计分析发现经音猬因子诱导方案神经干细胞标志物Nestin阳性率显著高于维甲酸诱导方案(P＜0.01),另一方面经维甲酸诱导方案诱导的细胞表现GFAP阳性率高于音猬因子诱导方案,差异具有显著性意义(P＜0.05).提示音猬因子诱导方案是一种诱导恒河猴骨髓间充质干细胞向神经样细胞分化的有效途径.
배경:골수간충질간세포유도분화위신경세포,시신경계통질병세포치료적유효수단,단목전적시효상불완선.목적:응용신경발육음위인자유도항하후골수간충질간세포향신경양세포분화.방법:응용경전적유갑산방안여음위인자방안량충방법유도항하후골수간충질간세포분화위신경양세포,채용밀도제도리심법분리배양항하후골수간충질간세포,도치상차현미경관찰생장정황,MTT법측정세포생장곡선,류식세포의감정세포표형,면역조직화학감정분화세포표지,투사전경화소묘전경관찰분화세포초미결구.결과여결론:체외분리배양적항하후골수간충질간세포,경류식세포의표형감정,구유교고균일성.통과음위인자유도방안유도처리7 d후,분화세포다수표현위NSE、NF-M、Tau화Nestin염색양성,경도상통계분석발현경음위인자유도방안신경간세포표지물Nestin양성솔현저고우유갑산유도방안(P＜0.01),령일방면경유갑산유도방안유도적세포표현GFAP양성솔고우음위인자유도방안,차이구유현저성의의(P＜0.05).제시음위인자유도방안시일충유도항하후골수간충질간세포향신경양세포분화적유효도경.
BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)differentiating into neural cells is an effective way of cell therapy of nervous system disease.However,the methods used nowadays still need to be improved.OBJECTIVE:To induce the differentiation of rhesus monkey BMSCs into neuron-like cells by using sonic hedgehog factor.METHODS:Rhesus monkey BMSCs differentiating into neuron-like cells was induced by typical retinoic acid and sonic hedgehog factor.Rhesus monkey BMSCs were isolated and cultured by density gradient centrifugation method.Cell growth was observed under an inverted phase contrast microscope and cell growth curve was determined using MTT assay.Flow cytometry was performed to characterize the phenotype of BMSCs,and immunohistochemistry was utilized to assess differentiated cells.Ultra-structure of the differentiated cells was observed by transmission and scanning electron microscopes.RESULTS AND CONCLUSION:Rhesus monkey BMSCs cultured in vitro were identified by flow cytometry,with high homogenicity.Following sonic hedgehog factor disposal for 7 days,differentiated cells were mainly positive for neurone specific enolase,neurofilament protein,Tau and glial fibrillary acidic protein(GFAP).Image statistical analysis found that in sonic hedgehog factor scheme,neural stem cells marker Neetin positive rate was significantly higher compared with the rstinoic acid scheme(P＜0.01).GFAP-positive rate was greater in the retinoic acid scheme than in the sonic hedgehog factor scheme(P＜0.05).Results indicated that sonic hedgehog factor scheme is an effective pathway of rhesus monkey BMSC differentiation into neuron-like cells.