悬浮组织块法分离培养sd大鼠脂肪干细胞的试验研究.pdfVIP

刘摇 琴等摇 悬浮组织块法分离培养SD大鼠脂肪干细胞的实验研究摇 第8期 ·1197 · doi:10.3969/ j.issn.1000鄄484X.2017.08.015 悬浮组织块法分离培养SD大鼠脂肪干细胞的实验研究 刘摇 琴摇 王丽平摇 陈摇 芳摇 李晓明摇 朱以良摇 张摇 宜 (中国人民解放军武汉总医院医学实验科,武汉 430070) 摇 摇 中图分类号摇 R392郾33摇 摇 文献标志码摇 A摇 摇 文章编号摇 1000鄄484X(2017)08鄄1197鄄04 [摘摇 要]摇 目的:建立SD大鼠脂肪干细胞悬浮组织块分离培养法。 方法:取正常SD大鼠股沟处脂肪组织,用悬浮组织 块法和组织块贴壁法分离培养SD大鼠脂肪干细胞,观察细胞的生长状况及形态特征。 取第3代对数期细胞,CCK鄄8法绘制其 生长曲线;免疫荧光法检测其表面抗原CD29、CD44、CD45 的表达情况;分别用成脂、成骨诱导培养液进行诱导分化,油红O、 茜素红染色定性鉴定。 结果:两种方法体外培养得到的SD大鼠脂肪干细胞形态为梭形纤维样,生长力旺盛,生长曲线为典型 的“S冶型;免疫荧光法检测显示第3代细胞强表达CD29、CD44,CD45表达阴性;细胞成脂诱导培养 14 d后,油红O染色为阳 性;成骨诱导培养14 d后,茜素红染色为阳性。 结论:悬浮组织块法可以分离培养出SD大鼠脂肪干细胞,方法简便,为体外分 离培养SD大鼠脂肪干细胞提供了一种新的方法。 [关键词]摇 悬浮组织块法;SD大鼠脂肪干细胞;分离;培养 Anexperimentalstudyofisolationof SDratadipose鄄derivedstemcellsusingsus鄄 pended explant culture method LIU Qin,WANG Li鄄Ping,CHEN Fang,LI Xiao鄄Ming,ZHU Yi鄄Liang,ZHANG Yi郾Department of Medical Experiments,Wuhan General Hospital of Chinese People忆s LiberationArmy,Wuhan430070,China [Abstract]摇 Objective:To isolate SD rat adipose鄄derived stem cells(ASCs)by suspended explant culture method郾Methods: The healthy rat inguinal fat pads were obtained郾The SD rat ASCs were isolated by suspended explant culture method and explant adherent culture method郾The growth status and morphology were observed郾The growth curve and cell surface markers CD29,CD44, CD45 of the 3rd passage cells were analyzed respectively by CCK鄄8,immunocytochemistry;the 3rd passage cells were induced individually by adipogenic differentiation medium and osteogenic differentiation medium,and cellswere examined by oil red O staining and alizarin red staining郾Results:The SD rat ASCs obtained by the two methods exhibited a spindle鄄shaped appearance and could rapidly expand郾Thecell growthcurvesweretypical of S type郾Immunocytochemistry analysisrevealedthatthethirdpassageof SDrat ASCswere positive for CD29,CD44,but were negative for CD45郾SD rat ASCs were positive for oil red O stain