肝素联合层黏连蛋白对大鼠脂肪间充质干细胞向运动神经元分化的影响  

Differentiation of rat adipose derived mesenchymal stem cells into motoneurons induced by heparin combined with laminin

作　　者：姚克[1] 李德华[1] 刘学元[1] 房艳[1] 

机构地区：[1]辽宁医学院解剖学教研室,辽宁锦州121000

出　　处：《中国现代医学杂志》2015年第22期11-17,共7页China Journal of Modern Medicine

摘　　要：目的研究单独或联合应用肝素(heparin)与层黏连蛋白(laminin)对大鼠脂肪间充质干细胞(AD-MSCs)向运动神经元分化的影响。方法用酶消化法分离纯化SD大鼠AD-MSCs,取传至第3代的AD-MSCs,检测表面抗原CD31、CD44、CD49d及CD106的表达情况,MTT法检测增殖活性。根据处理因素不同为4组:空白对照组,不加任何诱导剂;L组(laminin组),加入1μg/ml层黏连蛋白;H组(heparin组),加入50μg/ml肝素;HL组(heparin+laminin组),加入50μg/ml肝素和1μg/ml层黏连蛋白。细胞诱导后第15天,观察细胞形态的变化;采用免疫细胞化学方法检测神经元特异性烯醇化酶(NSE)、神经胶质纤维酸性蛋白(GFAP)、Hb9和Ch AT的表达,比较各组抗体的阳性率。诱导后3、6、9、12和15 d分别收集各组细胞,ELISA法检测细胞内Ach的含量。结果原代细胞为贴壁生长的长梭形细胞,流式细胞仪检测结果显示,CD44高表达,CD49d低表达,CD31、CD106阴性。MTT法结果表明AD-MSCs经多次传代培养后,其增殖能力没有明显降低。空白对照组和L组细胞诱导后形态变化不明显,NSE阳性表达率均很低,两者之间无明显差别。H组和HL组细胞诱导第15天后具有神经元样形态;NSE的阳性表达率比其他两组明显增多。H组和HL组细胞诱导后可见Hb9表达阳性,阳性率分别为为6.89%和30.47%,Ch AT阳性率分别为10.83%和39.36%,其他两组无Hb9和Ch AT表达,各组的GFAP均低表达。ELISA检测结果显示,对照组和L组诱导后细胞不表达乙酰胆碱(Ach),而H和HL组自诱导后第9天起表达Ach,且随着诱导时间的延长逐渐增加,到第15天时分别达到1.27和4.98ng/ml。结论肝素联合层黏连蛋白能显著促进大鼠AD-MSCs向运动神经元分化。[Objective] To study the effects of heparin alone or combined with laminin on the differentiation of the rat adipose derived mesenchymal stem cells （AD-MSCs） into the motoneurons, and to provide the theoretical ba- sis for further clinical application as seed cells. [Methods] AD-MSCs of rat were extracted and purified by the en- zyme digestion method. The expressions of the surface antigen CD31, CD44, CD49d, CD106 were detected. Prolifer- ation activity was detected by MTT method. Four groups were classified according to the different components in medium： Control group; L group, 1 μg/ml laminin; H group, 50 μg/ml heparin; HL group, 1 μg/ml laminin and 50 μg/ml heparin. On the 15th day after being induced, the morphologic changes of the cells were observed. The ex- pressions of NSE, GFAP, Hb9 and ChAT were detected via immunocytochemical methods, and the expressional rates of positive cells in each group were compared. At 3, 6, 9, 12 and 15 days following induction, cells were separately collected from each group. Acetylcholine content was measured using an enzyme-linked immunosorbent assay kit. [Results] Primary cells adherent to the culture flask were spindle-shaped in morphology. CD44 expression was high, CD49d expression was low, CD31 and CD106 was negative. MTF results showed that the proliferation capacity of AD-MSCs was not decreased after they were repeatedly subcultured. In the control and L group, there were no obvious changes in morphology after being induced, and the expressional rates of NSE positive cells were both very low, with no difference between them. In H and HL group, the cells took on a typical neuronal morphology after be- ing induced for 15 days. The expressional rates of NSE positive cells were both higher than those in other two groups. In H and HL group, the expressional rates of Hb9 was 6.89% and 30.47% respectively, while the expressional rates of ChAT was 10.83% and 39.36%. There wasn＇t expression of Hb9 and ChAT in other two groups. ELISA results revealed that acetylcho

关 键 词：脂肪间充质干细胞 肝素 层黏连蛋白 运动神经元 诱导 

分 类 号：R394.2[医药卫生—医学遗传学]