小鼠肌源性干细胞的分离、培养及其分化能力检测  

Isolation and culture of muscle derived stem cells from murine skeletal muscle and the test of their differentiation capacity

作　　者：谭云鹤[1] 种铁[1] 郭子宽[2] 甘为民[1] 

机构地区：[1]西安交通大学医学院第二附属医院泌尿外科,陕西西安710004 [2]军事医学科学院放射与辐射医学研究所实验血液学研究室,北京100850

出　　处：《西安交通大学学报（医学版）》2011年第6期668-671,共4页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的探讨小鼠肌源性干细胞(MDSCs)的原代培养方法及其分化潜能,为肌性泌尿组织的细胞修复、治疗奠定基础。方法运用中性蛋白酶(DispaseⅡ)、胰蛋白酶及Ⅰ型胶原酶联合消化法分离小鼠MDSCs,并运用差速贴壁法进行纯化。倒置显微镜下观察细胞形态,RT-PCR法鉴定MyoD及Desmin的表达,同时对分离的细胞进行成骨、成脂等分化诱导并鉴定。结果运用中性蛋白酶可很好地消化组织,差速贴壁法能得到纯度较高的MDSCs;RT-PCR检测发现MDSCs不表达MyoD以及Desmin,提示其为不同于肌卫星细胞的肌源性干细胞;成骨诱导检测碱性磷酸酶(ALP)染色(+),成脂诱导油红O染色(+);其可自体分化诱导为心肌样细胞,免疫荧光法检测心肌肌钙蛋白(cTnI)呈现阳性结果。结论利用此分离方法可获得纯度较高且具有较强分化潜能的肌源性干细胞,有助于进一步进行组织工程研究。Objective To explore the method for primary culture of mouse muscle derived stem cells(MDSCs) and evaluate their differentiation ability so as to lay foundation for urethral sphincter regeneration and treatment.Methods MDSCs were isolated by the digestive juice containing dispase Ⅱ and collagenase Ⅰ and were purified by preplate technique.The cells were observed by inverted microscope and identified by RT-PCR assay.Then the differentiation of osteoblasts and adipoblasts and cardiomyocytes-like was detected.Results Highly purified MDSCs were obtained and showed MyoD(-) and Desmin(-),which indicated that MDSCs were different from muscle satellite cells.After induction,ALP staining and red oil O staining were positive and cardiomyocytes-like expressed anti-cTnI by spontaneous induction.Conclusion MDSCs can be obtained and have powerful pluripotency with this method.These cells could be used for tissue engineering.

关 键 词：肌源性干细胞 中性蛋白酶 差速贴壁 原代培养 分化 心肌样细胞 鉴定 MYOD DESMIN 

分 类 号：R694.6[医药卫生—泌尿科学]