胎盘间充质干细胞诱导为神经细胞的方法比较  

Comparison of methods of inducing neuroblasts from placental mesenchymal stem cells

作　　者：杨建成 王燕 金毅然[2] 李家辉 马晓娜[2] 马海滨[2] 梁雪云[2] Yang Jiancheng;Wang Yan;Jin Yiran;Li Jiahui;Ma Xiaona;Ma Haibin;Liang Xueyun(School of Clinical Medicine,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Ningxia Human Stem Cell Research Institution,General Hospital of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学临床医学院,银川750004 [2]宁夏医科大学总医院宁夏人类干细胞研究所,银川750004

出　　处：《神经解剖学杂志》2021年第2期196-202,共7页Chinese Journal of Neuroanatomy

基　　金：宁夏自然科学基金(2019AAC03177)。

摘　　要：目的:比较DMEM/F12、高糖DMEM和Neurobasal-A培养基(NA)3种培养基对胎盘间充质干细胞(PMSCs)诱导成神经细胞的效果。方法:分别采用DMEM/F12组(DMEM/F12)、高糖DMEM组(DMEM)和Neurobasal-A组(NA)培养基诱导培养P3代人类PMSCs,比较各组诱导生成的神经球数量及其直径评价不同方法诱导后形成的神经细胞球的增殖能力;利用免疫荧光染色鉴定诱导成的神经球的巢蛋白(nestin)和生长相关蛋白43(GAP-43)的表达;利用Western Blot检测细胞神经丝蛋白(NF)表达量;分别采用由DMEM/F12、高糖DMEM和Neurobasal-A培养基诱导获得的神经细胞的条件培养基对由H_(2)O_(2)氧化损伤的神经元细胞系HT22细胞进行培养后,利用免疫荧光染色观察各组细胞caSpaSe-3的表达评价不同方法诱导成的神经细胞对氧化损伤的神经元的治疗效果。结果:采用3种不同诱导培养基将PMSCs培养24 h后,各组均有神经球出现;诱导形成的神经样细胞的nestin和GAP-43表达为阳性;NF在诱导形成的神经样细胞中的表达水平明显高于未诱导的Normal组的表达水平(P<0.05);分别采用3种由不同诱导方法获得的神经细胞的条件培养基对氧化损伤的HT22细胞进行培养后,阳性表达caspase-3的HT22细胞数目明显减少(P<0.05)。结论:3种诱导培养基均可以有效地将PMSCs诱导成神经细胞,诱导形成的神经细胞对氧化损伤的神经元具有一定的促进修复作用。Objective: To compare the effects of three kinds of culture mediums,which compounded by either DMEM/F12,DMEM with 4. 5 g/L glucose or Neurobasal-A medium( NA),on the neuroblasts induction from placental mesenchymal stem cells( Normal group). Methods: Either DMEM/F12( DMEM/F12 group) or DMEM with 4. 5 g/L glucose( DMEM group) or Neurobasal-A( NA group) medium was used to culture human PMSCs. The number of induced neurosphere were counted and the diameter of induced neurosphere were measured in each group,by which to evaluate the proliferation ability of induced neurospheres in each group;Immunofluorescence staining was used to identify the protein expression of nestin and growth-associated protein 43( GAP-43) in the induced neurospheres from each group;Western Blot was used to detect protein expression level of neurofilament( NF). The conditioned medium of neurospheres from each group was used to culture the neuronal cell line HT22 cells,which treated by H2O2 in advance,and then the protein expression of caspase-3 was checked by immunofluorescence staining to evaluate the therapeutic effect of induced neurosphere on oxidatively damaged neurons. Results: The neurospheres appeared in each group after cultured 24 h with different induction medium;The positive expressions of nestin and GAP-43 were observed in each group;The expression level of NF in the neurosphere from either DMEM/F12 group or DMEM group or NA group was higher than that of in the PMSCs group( P < 0. 05);The number of caspase-3 positive cells of the oxidatively damaged HT22 was significantly reduced( P < 0. 05) after cultured with the conditioned medium from the induced neurosphere in each group. Conclusion: All of these three methods could effectively induce placental mesenchymal stem cells into neural stem cells,and the induced cells are of antioxidative effects.

关 键 词：胎盘间充质干细胞 神经细胞 诱导方法 抗氧化作用 

分 类 号：R743.3[医药卫生—神经病学与精神病学]