中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
37期
6019-6023
,共5页
赵玲%赵红斌%潘茜%李根%王九娜%唐俊杰
趙玲%趙紅斌%潘茜%李根%王九娜%唐俊傑
조령%조홍빈%반천%리근%왕구나%당준걸
干细胞%骨髓干细胞%红景天苷%骨髓间充质干细胞%Ca2+/CaM信号通路%神经细胞%国家自然科学基金
榦細胞%骨髓榦細胞%紅景天苷%骨髓間充質榦細胞%Ca2+/CaM信號通路%神經細胞%國傢自然科學基金
간세포%골수간세포%홍경천감%골수간충질간세포%Ca2+/CaM신호통로%신경세포%국가자연과학기금
bone marrow%mesenchymal stem cells%rhodiola%neurons%calcium signaling
背景：课题组前期研究表明，红景天苷能诱导骨髓间充质干细胞向神经元样细胞定向分化，Ca2+信号是实现其生物学信号传导的重要途径之一目的：探讨Ca 2+/CaM 信号通路在红景天苷诱导骨髓间充质干细胞向神经细胞定向分化中的作用及机制。方法：实验分为空白对照组和红景天苷诱导组，红景天苷诱导组将不同质量浓度红景天苷(5，10，20，50，100 mg/L)作用骨髓间充质干细胞24 h和100 mg/L红景天苷作用骨髓间充质干细胞12，24，48，72 h。采用Western blot方法分别检测红景天苷诱导骨髓间充质干细胞后神经标志分子MAP2和Ca 2+/CaM信号通路中关键蛋白CaM、CaMKⅡ的表达水平。另外实验设阻断剂组，分别加Ca2+信号通路特异性阻断剂：500μmol/L EGTA (细胞外Ca2+螯合剂)、1 mmol/L Nifedipine (L型Ca2+通道阻断剂)、10 mmol/L LY294002(PI3K抑制剂)分别作用细胞30 min后，再加入100 mg/L红景天苷作用细胞24 h，采用Western blot方法检测阻断Ca2+/CaM信号通路后NSE、CaM的表达情况。结果与结论：①红景天苷诱导后，MAP2的表达上调(P<0.01)，说明红景天苷可诱导骨髓间充质干细胞分化为神经细胞。②不同质量浓度红景天苷诱导骨髓间充质干细胞24 h后，10 mg/L红景天苷组CaM、CaMKⅡ的表达与其他诱导组比较显著上调(P<0.01)；同一质量浓度红景天苷诱导72 h后CaM、CaMKⅡ表达明显下调(P<0.01)。③阻断细胞外Ca 2+和PI3K信号通路后，NSE与CaM的表达水平较红景天苷诱导组上调(P<0.05)。结果表明，红景天苷通过抑制Ca2+/CaM信号通路实现骨髓间充质干细胞向神经细胞定向分化。
揹景：課題組前期研究錶明，紅景天苷能誘導骨髓間充質榦細胞嚮神經元樣細胞定嚮分化，Ca2+信號是實現其生物學信號傳導的重要途徑之一目的：探討Ca 2+/CaM 信號通路在紅景天苷誘導骨髓間充質榦細胞嚮神經細胞定嚮分化中的作用及機製。方法：實驗分為空白對照組和紅景天苷誘導組，紅景天苷誘導組將不同質量濃度紅景天苷(5，10，20，50，100 mg/L)作用骨髓間充質榦細胞24 h和100 mg/L紅景天苷作用骨髓間充質榦細胞12，24，48，72 h。採用Western blot方法分彆檢測紅景天苷誘導骨髓間充質榦細胞後神經標誌分子MAP2和Ca 2+/CaM信號通路中關鍵蛋白CaM、CaMKⅡ的錶達水平。另外實驗設阻斷劑組，分彆加Ca2+信號通路特異性阻斷劑：500μmol/L EGTA (細胞外Ca2+螯閤劑)、1 mmol/L Nifedipine (L型Ca2+通道阻斷劑)、10 mmol/L LY294002(PI3K抑製劑)分彆作用細胞30 min後，再加入100 mg/L紅景天苷作用細胞24 h，採用Western blot方法檢測阻斷Ca2+/CaM信號通路後NSE、CaM的錶達情況。結果與結論：①紅景天苷誘導後，MAP2的錶達上調(P<0.01)，說明紅景天苷可誘導骨髓間充質榦細胞分化為神經細胞。②不同質量濃度紅景天苷誘導骨髓間充質榦細胞24 h後，10 mg/L紅景天苷組CaM、CaMKⅡ的錶達與其他誘導組比較顯著上調(P<0.01)；同一質量濃度紅景天苷誘導72 h後CaM、CaMKⅡ錶達明顯下調(P<0.01)。③阻斷細胞外Ca 2+和PI3K信號通路後，NSE與CaM的錶達水平較紅景天苷誘導組上調(P<0.05)。結果錶明，紅景天苷通過抑製Ca2+/CaM信號通路實現骨髓間充質榦細胞嚮神經細胞定嚮分化。
배경：과제조전기연구표명，홍경천감능유도골수간충질간세포향신경원양세포정향분화，Ca2+신호시실현기생물학신호전도적중요도경지일목적：탐토Ca 2+/CaM 신호통로재홍경천감유도골수간충질간세포향신경세포정향분화중적작용급궤제。방법：실험분위공백대조조화홍경천감유도조，홍경천감유도조장불동질량농도홍경천감(5，10，20，50，100 mg/L)작용골수간충질간세포24 h화100 mg/L홍경천감작용골수간충질간세포12，24，48，72 h。채용Western blot방법분별검측홍경천감유도골수간충질간세포후신경표지분자MAP2화Ca 2+/CaM신호통로중관건단백CaM、CaMKⅡ적표체수평。령외실험설조단제조，분별가Ca2+신호통로특이성조단제：500μmol/L EGTA (세포외Ca2+오합제)、1 mmol/L Nifedipine (L형Ca2+통도조단제)、10 mmol/L LY294002(PI3K억제제)분별작용세포30 min후，재가입100 mg/L홍경천감작용세포24 h，채용Western blot방법검측조단Ca2+/CaM신호통로후NSE、CaM적표체정황。결과여결론：①홍경천감유도후，MAP2적표체상조(P<0.01)，설명홍경천감가유도골수간충질간세포분화위신경세포。②불동질량농도홍경천감유도골수간충질간세포24 h후，10 mg/L홍경천감조CaM、CaMKⅡ적표체여기타유도조비교현저상조(P<0.01)；동일질량농도홍경천감유도72 h후CaM、CaMKⅡ표체명현하조(P<0.01)。③조단세포외Ca 2+화PI3K신호통로후，NSE여CaM적표체수평교홍경천감유도조상조(P<0.05)。결과표명，홍경천감통과억제Ca2+/CaM신호통로실현골수간충질간세포향신경세포정향분화。
BACKGROUND:Our previous studies have shown that salidroside can induce bone marrow mesenchymal stem cells directly into neuron-like cells, and Ca2+signal is one important way to achieve its biological signal transduction. OBJECTIVE:To investigate the role and mechanism of the calcium/calmodulin (Ca 2+/CaM) signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into nerve cells. METHODS:Bone marrow mesenchymal stem cells were divided into two groups:control groups and salidroside groups. Salidroside groups were induced with different concentrations of salidroside (5, 10, 20, 50 and 100 mg/L) for 24 hours and 100 mg/L salidroside was added to culture cells for different time (12, 24, 48 and 72 hours). Western blot assay was used to detect the expression levels of neural cellmarker, microtubule-associated protein 2, and the important protein of Ca2+/CaM signaling pathway:CaM and calmodulin dependent kinase II (CaMK II). Then Ca2+/CaM signaling pathway specific blockers were applied to cells respectively for 30 minutes, including 500 μmol/L EGTA (Ca 2+chelator), 1 mmol/L Nifedipine(L-type Ca2+channel blocker) and 10 mmol/L LY294002 (PI3K inhibitor). Then, 100 mg/L salidroside was added and cultured for 24 hours. Western blot assay was used to detect the expression of neuron-specific enolase and CaM in the Ca2+/CaM signaling pathway. RESULTS AND CONCLUSION:(1) After inducing with salidroside, the expression of microtubule-associated protein 2 were upregulated (P<0.01), indicating that salidrosid can induce the neuronal differentiation of bone marrow mesenchymal stem cells. (2) After different concentrations of salidrosid induced bone marrow mesenchymal stem cells for 24 hours, the expressions of CaM and CaMK II were significantly upregulated in the 10 mg/L group ( P<0.01);For the 100 mg/L salidrosid that was added for cellinduction for different time, the expressions of CaM and CaMK II were significantly downregulated in 72-hour group (P<0.01). (3) After blocking extracellular Ca2+and PI3K signaling pathway, the expressions of neuron-specific enolase and CaM were higher than those in salidrosid groups (P<0.05). These results suggest that salidrosid can induce bone marrow mesenchymal stem cellto directly differentiate into nerve cells by inhibiting the Ca2+/CaM signaling pathway.
