CAJ | 학술논문

目的观察激活态雪旺细胞( ASCs)分泌神经营养因子对诱导人脐血间充质干细胞(HUCBMSCs)神经方向分化的作用.方法采用双酶消化组织块法结合机械分离法分离培养ASCs；Ficoll法分离得到人脐血单个核细胞(HCMNCs)并经贴壁培养、分离后获得HUCBMSCs.采用全反式微甲酸+碱性成纤维细胞生长因子(bFGF)+表皮生长因子(EGF)(对照组)、ASCs培养基半量换液法(实验组).应用免疫组织化学、蛋白印迹法( Western blot)半定量、荧光实时聚合酶链反应( Real-time PCR)[神经纤维200 (NF200)、胶质纤维酸性蛋白(GFAP)、髓磷脂碱性蛋白(MBP)]定量分析等方法在诱导培养后1、2、3、4周进行综合评价HUCBMSCs诱导分化,对实验结果进行统计学分析.结果免疫组织化学染色结果证实HUCBMSCs在ASCs和全反式微甲酸及bFGF、EGF的作用下向神经源性细胞分化.不同诱导时间细胞阳性率、Western blot半定量、Real-time PCR定量分析结果显示差异有统计学意义(P＜0.05),而同一时间内两种诱导方法比较差异无统计学意义(P＞0.05).结论 ASCs可以分泌多种神经营养因子并促进HUCBMSCs向神经源性细胞分化,与传统全反式微甲酸诱导方法比较更简单.
목적 관찰격활태설왕세포( ASCs)분비신경영양인자대유도인제혈간충질간세포(HUCBMSCs)신경방향분화적작용.방법 채용쌍매소화조직괴법결합궤계분리법분리배양ASCs；Ficoll법분리득도인제혈단개핵세포(HCMNCs)병경첩벽배양、분리후획득HUCBMSCs.채용전반식미갑산+감성성섬유세포생장인자(bFGF)+표피생장인자(EGF)(대조조)、ASCs배양기반량환액법(실험조).응용면역조직화학、단백인적법( Western blot)반정량、형광실시취합매련반응( Real-time PCR)[신경섬유200 (NF200)、효질섬유산성단백(GFAP)、수린지감성단백(MBP)]정량분석등방법재유도배양후1、2、3、4주진행종합평개HUCBMSCs유도분화,대실험결과진행통계학분석.결과 면역조직화학염색결과증실HUCBMSCs재ASCs화전반식미갑산급bFGF、EGF적작용하향신경원성세포분화.불동유도시간세포양성솔、Western blot반정량、Real-time PCR정량분석결과현시차이유통계학의의(P＜0.05),이동일시간내량충유도방법비교차이무통계학의의(P＞0.05).결론 ASCs가이분비다충신경영양인자병촉진HUCBMSCs향신경원성세포분화,여전통전반식미갑산유도방법비교경간단.
Objective To observe the capability of human umbilical cord blood mesenchymal stem cells (HUCBMSCs) to differentiate into neurogenic cells in vitro under the impact of activated Schwann cells (ASCs)-derived neuronotrophins.Methods ASCs were cultured by using the dual-enzyme digestion method combined with mechanical separation.HUCBMSCs were cultured through isolating HCMNCs by Ficoll' s way and then plastic-adherent culture.Two methods were used for HUCBMSCs culture:all-trans retinoic acid + bFGF + EGF ( control group),1/2 DMEM + ASCs culture ( medium after ASCs cultured for 2 days,experimental group).Immunohistochemistry,Western blotting,and semi-quantitative and realtime polymerase chain reaction (PCR,NF200,GFAP,MBP) quantitative analysis were used to evaluate neurogenic differentiation of HUCBMSCs.Results The results of immunohistochemistry staining,such as NF200,GFAP and MBP,confirmed that HUCBMSCs could differentiate into neurogenic cells under the effect of ASCs and RA + bFGF + EGF.The statistical analysis of Western blotting and RT-PCR ( NF200,GFAP,MBP ) indicated that there existed significant difference between two groups.Conclusion HUCBMSCs can proliferate and differentiate into neurogenic cells under the effect of numerous neurotrophic factors from ASCs in vitro.