中国组织工程研究与临床康复
中國組織工程研究與臨床康複
중국조직공정연구여림상강복
JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH
2008年
29期
5793-5796
,共4页
柴立辉%吴素霞%陈宗德%曹孟德%马远方
柴立輝%吳素霞%陳宗德%曹孟德%馬遠方
시립휘%오소하%진종덕%조맹덕%마원방
多巴胺神经元%骨髓间充质干细胞%分化%脑源性神经营养因子%高效液相色谱
多巴胺神經元%骨髓間充質榦細胞%分化%腦源性神經營養因子%高效液相色譜
다파알신경원%골수간충질간세포%분화%뇌원성신경영양인자%고효액상색보
背景:研究人骨髓间充质干细胞定向分化为多巴胺神经元及分化后细胞的功能特征,对细胞移植治疗包括帕金森病在内的神经精神性疾病具有重要临床意义.目的:采用脑源性神经营养因子、forskolin及多巴胺联合诱导人骨髓间充质干细胞向多巴胺神经元定向分化,观察诱导产生的多巴胺神经元的结构和功能.设计:随机对照观察.单位:郑州大学及河南大学.材料:实验于2005-03/2006-09在郑州大学和河南大学实验室完成.实验用骨髓取自15名健康志愿者.脑源性神经营养因子、多巴胺、forskolin及单克隆抗体酪氨酸羟化酶为美国Sigma公司产品.方法:①通过密度梯度离心获取人骨髓中的单个核细胞,贴壁培养纯化骨髓间充质干细胞.采用50 μmol/L 脑源性神经营养因子,10 μ mol/Lforskolin和10 μmol/L多巴胺联合对骨髓间充质干细胞进行诱导.②诱导2周后应用电子显微镜观察细胞超微结构;免疫细胞化学染色检测神经元烯醇化酶(NSE)和酪氨酸羟化酶(TH)的表达:采用RT-PCR检测多巴胺神经元分化过程中转录因子Nmr1,Ptx3,Lam1b及酪氨酸羟化酶mRNA表达;同时以未诱导的细胞为对照,应用高效液相色谱检测细胞多巴胺的释放水平.主要观察指标:①细胞超微结构.②NAE和TH的表达变化.③细胞内关键转录因子及酪氨酸羟化酶mRNA变化.④细胞释放多巴胺情况.结果:①细胞超微结构:诱导2周后细胞胞浆中有大量密集的呈扁平囊状的粗面内质网及其间的一些游离核糖体以及神经微丝的形成.②NSE和TH表达的变化:免疫细胞化学染色结果表明诱导分化后NSE和TH阳性细胞的表达随诱导时间的延长逐渐增高(P<0.001).③细胞内酪氨酸羟化酶及关键转录因子变化:RT-PCR结果显示细胞均可表达Nurr1,Ptx3,Lmx1b和酪氨酸羟化酶的mRNA.④细胞释放多巴胺情况:诱导2周后的细胞多巴胺释放水平高于未经诱导的细胞,差异有统计学意义(P<0.05).结论:脑源性神经营养因子、forskolin及多巴胺可在体外诱导人骨髓间充质干细胞向多巴胺神经元分化,并具有多巴胺神经元的结构和功能特征.
揹景:研究人骨髓間充質榦細胞定嚮分化為多巴胺神經元及分化後細胞的功能特徵,對細胞移植治療包括帕金森病在內的神經精神性疾病具有重要臨床意義.目的:採用腦源性神經營養因子、forskolin及多巴胺聯閤誘導人骨髓間充質榦細胞嚮多巴胺神經元定嚮分化,觀察誘導產生的多巴胺神經元的結構和功能.設計:隨機對照觀察.單位:鄭州大學及河南大學.材料:實驗于2005-03/2006-09在鄭州大學和河南大學實驗室完成.實驗用骨髓取自15名健康誌願者.腦源性神經營養因子、多巴胺、forskolin及單剋隆抗體酪氨痠羥化酶為美國Sigma公司產品.方法:①通過密度梯度離心穫取人骨髓中的單箇覈細胞,貼壁培養純化骨髓間充質榦細胞.採用50 μmol/L 腦源性神經營養因子,10 μ mol/Lforskolin和10 μmol/L多巴胺聯閤對骨髓間充質榦細胞進行誘導.②誘導2週後應用電子顯微鏡觀察細胞超微結構;免疫細胞化學染色檢測神經元烯醇化酶(NSE)和酪氨痠羥化酶(TH)的錶達:採用RT-PCR檢測多巴胺神經元分化過程中轉錄因子Nmr1,Ptx3,Lam1b及酪氨痠羥化酶mRNA錶達;同時以未誘導的細胞為對照,應用高效液相色譜檢測細胞多巴胺的釋放水平.主要觀察指標:①細胞超微結構.②NAE和TH的錶達變化.③細胞內關鍵轉錄因子及酪氨痠羥化酶mRNA變化.④細胞釋放多巴胺情況.結果:①細胞超微結構:誘導2週後細胞胞漿中有大量密集的呈扁平囊狀的粗麵內質網及其間的一些遊離覈糖體以及神經微絲的形成.②NSE和TH錶達的變化:免疫細胞化學染色結果錶明誘導分化後NSE和TH暘性細胞的錶達隨誘導時間的延長逐漸增高(P<0.001).③細胞內酪氨痠羥化酶及關鍵轉錄因子變化:RT-PCR結果顯示細胞均可錶達Nurr1,Ptx3,Lmx1b和酪氨痠羥化酶的mRNA.④細胞釋放多巴胺情況:誘導2週後的細胞多巴胺釋放水平高于未經誘導的細胞,差異有統計學意義(P<0.05).結論:腦源性神經營養因子、forskolin及多巴胺可在體外誘導人骨髓間充質榦細胞嚮多巴胺神經元分化,併具有多巴胺神經元的結構和功能特徵.
배경:연구인골수간충질간세포정향분화위다파알신경원급분화후세포적공능특정,대세포이식치료포괄파금삼병재내적신경정신성질병구유중요림상의의.목적:채용뇌원성신경영양인자、forskolin급다파알연합유도인골수간충질간세포향다파알신경원정향분화,관찰유도산생적다파알신경원적결구화공능.설계:수궤대조관찰.단위:정주대학급하남대학.재료:실험우2005-03/2006-09재정주대학화하남대학실험실완성.실험용골수취자15명건강지원자.뇌원성신경영양인자、다파알、forskolin급단극륭항체락안산간화매위미국Sigma공사산품.방법:①통과밀도제도리심획취인골수중적단개핵세포,첩벽배양순화골수간충질간세포.채용50 μmol/L 뇌원성신경영양인자,10 μ mol/Lforskolin화10 μmol/L다파알연합대골수간충질간세포진행유도.②유도2주후응용전자현미경관찰세포초미결구;면역세포화학염색검측신경원희순화매(NSE)화락안산간화매(TH)적표체:채용RT-PCR검측다파알신경원분화과정중전록인자Nmr1,Ptx3,Lam1b급락안산간화매mRNA표체;동시이미유도적세포위대조,응용고효액상색보검측세포다파알적석방수평.주요관찰지표:①세포초미결구.②NAE화TH적표체변화.③세포내관건전록인자급락안산간화매mRNA변화.④세포석방다파알정황.결과:①세포초미결구:유도2주후세포포장중유대량밀집적정편평낭상적조면내질망급기간적일사유리핵당체이급신경미사적형성.②NSE화TH표체적변화:면역세포화학염색결과표명유도분화후NSE화TH양성세포적표체수유도시간적연장축점증고(P<0.001).③세포내락안산간화매급관건전록인자변화:RT-PCR결과현시세포균가표체Nurr1,Ptx3,Lmx1b화락안산간화매적mRNA.④세포석방다파알정황:유도2주후적세포다파알석방수평고우미경유도적세포,차이유통계학의의(P<0.05).결론:뇌원성신경영양인자、forskolin급다파알가재체외유도인골수간충질간세포향다파알신경원분화,병구유다파알신경원적결구화공능특정.
BACKGROUND: It is of significance to study the direct differentiation of human bone marrow mesenchymal stem cells (hBMSCs) into dopaminergic neurons and their functional characteristics for the treatment of neuropsychiatric disease such as Parkinson's disease with cell transplantation.OBJECTIVE: To induce the direct differentiation of hBMSCs into dopaminergic neurons by brain-derived neurotrophic factor (BDNF), forskolin, and dopamine, and to investigate the structural and functional characteristics of dopaminergic neurons. DESIGN: Randomized controlled observation.SETTING: Zhengzhou University and Henan University.MATERIALS: This study was performed in the laboratories of Zhengzhou University and Henan University from March 2005 to September 2006. Bone marrow was extracted from 15 healthy volunteers. All subjects provided the informed consent, and the experiment was approved by the local ethics committee. BDNF, dopamine, forskolin, and monoclonal antibody tyrosine hydroxylase were provided by Sigma Company, USA.METHODS: Mononuclear cells were obtained from human bone marrow by density gradient centrifugation, and BMSCs were purified with adherent culture. BMSCs were induced by BDNF (50_ μ mol/L), forskolin (10 μ mol/L), and dopamine (10 μ mol/L). After two weeks, electron microscope was adopted to observe cellular ultrastructure; immunocytochemical staining was adopted to observe the expression of NSE and TH proteins, and RT-PCR was adopted to detect expressions of Nurr1, Ptx3, Lmx1b, tyrosine hydroxylase mRNA during the differentiation into dopaminergic neurons. Moreover, non-induced cells were collected as the control group, and level of dopamine release was measured by high performance liquid chromatogram.MAIN OUTCOME MEASURES: Cellular ultramicrostructure; changes in the expression of NSE and TH protein; released level of dopamine.RESULTS: Cellular ultramicrostructure: After two weeks, a great quantity of concentrated cystiform-shaped rough endoplasmic reticulum, a lot of free ribosome, and some neurofilament appeared in cytoplasm. Changes in the expression of NSE and TH proteins: immunocytochemistry staining indicated the ratio of NSE-positive and TH-positive neural cells was significantly increased in different stages after induction (P<0.001). Changes of key transcription factors and tyrosine hydroxylase mRNA: RT-PCR indicated that all cells could express Nurr1, Ptx3, Lmx1b, and tyrosine hydroxylase mRNA. Level of dopamine release: After two weeks, level of dopamine release in the induced cells was significantly higher than that in the non-induced cells (P < 0.05).CONCLUSION: BDNF, forskolin, and dopamine can induce hBMSCs to differentiate into cells that have the structural and functional characteristics of dopaminergic neurons in vitro.
