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
16期
3171-3174
,共4页
王振宇%佟雷%季丽莉%赵久红%唐源远
王振宇%佟雷%季麗莉%趙久紅%唐源遠
왕진우%동뢰%계려리%조구홍%당원원
神经干细胞%大鼠海马%表皮生长因子%脑源性神经生长因子%分化
神經榦細胞%大鼠海馬%錶皮生長因子%腦源性神經生長因子%分化
신경간세포%대서해마%표피생장인자%뇌원성신경생장인자%분화
背景:目前神经干细胞的定向分化是神经干细胞研究的热点.脑源性神经生长因子作为诱导剂可否诱导神经干细胞分化为神经元.目的:观察在表皮生长因子培养条件下,不同质量浓度脑源性神经生长因子对成年大鼠海马神经干细胞向神经元分化的影响.设计:对比实验.单位:中国医科大学人体解剖学教研室.材料:实验于2007-08在中国医科大学神经解剖研究室完成.提取成年SD大鼠海马神经干细胞,无血清技术培养.清洁级成年SD大鼠3只,体质量200～250 g,由中国医科大学实验动物部提供,实验过程中对动物的处置符合动物伦理学标准.实验过程中应用的表皮生长因子、脑源性神经生长因子均由R&D公司提供.方法:①无菌条件下分离大鼠海马组织,用含碱性成纤维生长因子、表皮生长因子、B27的无血清细胞培养技术体外培养神经干细胞.②取第4代神经干细胞,利用有限稀释法进行单细胞克隆培养.将单细胞克隆传代后的克隆球细胞进行神经干细胞的鉴定.克隆球细胞行巢蛋白免疫细胞化学染色,诱导分化1周后进行神经元特异性烯醇化酶、胶质纤维酸性蛋白免疫细胞化学染色.③单细胞克隆的神经干细胞脑源性神经生长因子诱导分化实验,根据脑源性神经生长因子质量浓度不同分成0 μg/L浓度的对照组和50、100、150、200μg/L浓度的4个实验组,各组的培养液中均加入表皮生长因子,诱导分化1周后进行神经元特异性烯醇化酶免疫细胞化学染色,检测神经干细胞向神经元分化情况.主要观察指标:神经干细胞分化为神经元的比例.结果:①神经干细胞免疫细胞化学染色结果显示,单细胞克隆培养后克隆球细胞表达巢蛋白,诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白均呈阳性表达.②50,100μg/L组脑源性神经生长因子组神经干细胞分化为神经元比例明显高于对照组、150,200 μg/L脑源性神经生长因子组组(t=2.502～5.025,P < 0.05);50,100 μg/L脑源性神经生长因子组之间神经干细胞分化为神经元的比例差异不显著 (P > 0.05);对照组、150,200 μg/L组3组间神经干细胞分化为神经元比例差异不显著(P > 0.05)结论:在20 μg/L 表皮生长因子培养条件下,脑源性神经生长因子促使神经干细胞向神经元分化的较佳质量浓度为50 μg/L.
揹景:目前神經榦細胞的定嚮分化是神經榦細胞研究的熱點.腦源性神經生長因子作為誘導劑可否誘導神經榦細胞分化為神經元.目的:觀察在錶皮生長因子培養條件下,不同質量濃度腦源性神經生長因子對成年大鼠海馬神經榦細胞嚮神經元分化的影響.設計:對比實驗.單位:中國醫科大學人體解剖學教研室.材料:實驗于2007-08在中國醫科大學神經解剖研究室完成.提取成年SD大鼠海馬神經榦細胞,無血清技術培養.清潔級成年SD大鼠3隻,體質量200～250 g,由中國醫科大學實驗動物部提供,實驗過程中對動物的處置符閤動物倫理學標準.實驗過程中應用的錶皮生長因子、腦源性神經生長因子均由R&D公司提供.方法:①無菌條件下分離大鼠海馬組織,用含堿性成纖維生長因子、錶皮生長因子、B27的無血清細胞培養技術體外培養神經榦細胞.②取第4代神經榦細胞,利用有限稀釋法進行單細胞剋隆培養.將單細胞剋隆傳代後的剋隆毬細胞進行神經榦細胞的鑒定.剋隆毬細胞行巢蛋白免疫細胞化學染色,誘導分化1週後進行神經元特異性烯醇化酶、膠質纖維痠性蛋白免疫細胞化學染色.③單細胞剋隆的神經榦細胞腦源性神經生長因子誘導分化實驗,根據腦源性神經生長因子質量濃度不同分成0 μg/L濃度的對照組和50、100、150、200μg/L濃度的4箇實驗組,各組的培養液中均加入錶皮生長因子,誘導分化1週後進行神經元特異性烯醇化酶免疫細胞化學染色,檢測神經榦細胞嚮神經元分化情況.主要觀察指標:神經榦細胞分化為神經元的比例.結果:①神經榦細胞免疫細胞化學染色結果顯示,單細胞剋隆培養後剋隆毬細胞錶達巢蛋白,誘導分化後神經元特異性烯醇化酶、膠質纖維痠性蛋白均呈暘性錶達.②50,100μg/L組腦源性神經生長因子組神經榦細胞分化為神經元比例明顯高于對照組、150,200 μg/L腦源性神經生長因子組組(t=2.502～5.025,P < 0.05);50,100 μg/L腦源性神經生長因子組之間神經榦細胞分化為神經元的比例差異不顯著 (P > 0.05);對照組、150,200 μg/L組3組間神經榦細胞分化為神經元比例差異不顯著(P > 0.05)結論:在20 μg/L 錶皮生長因子培養條件下,腦源性神經生長因子促使神經榦細胞嚮神經元分化的較佳質量濃度為50 μg/L.
배경:목전신경간세포적정향분화시신경간세포연구적열점.뇌원성신경생장인자작위유도제가부유도신경간세포분화위신경원.목적:관찰재표피생장인자배양조건하,불동질량농도뇌원성신경생장인자대성년대서해마신경간세포향신경원분화적영향.설계:대비실험.단위:중국의과대학인체해부학교연실.재료:실험우2007-08재중국의과대학신경해부연구실완성.제취성년SD대서해마신경간세포,무혈청기술배양.청길급성년SD대서3지,체질량200～250 g,유중국의과대학실험동물부제공,실험과정중대동물적처치부합동물윤리학표준.실험과정중응용적표피생장인자、뇌원성신경생장인자균유R&D공사제공.방법:①무균조건하분리대서해마조직,용함감성성섬유생장인자、표피생장인자、B27적무혈청세포배양기술체외배양신경간세포.②취제4대신경간세포,이용유한희석법진행단세포극륭배양.장단세포극륭전대후적극륭구세포진행신경간세포적감정.극륭구세포행소단백면역세포화학염색,유도분화1주후진행신경원특이성희순화매、효질섬유산성단백면역세포화학염색.③단세포극륭적신경간세포뇌원성신경생장인자유도분화실험,근거뇌원성신경생장인자질량농도불동분성0 μg/L농도적대조조화50、100、150、200μg/L농도적4개실험조,각조적배양액중균가입표피생장인자,유도분화1주후진행신경원특이성희순화매면역세포화학염색,검측신경간세포향신경원분화정황.주요관찰지표:신경간세포분화위신경원적비례.결과:①신경간세포면역세포화학염색결과현시,단세포극륭배양후극륭구세포표체소단백,유도분화후신경원특이성희순화매、효질섬유산성단백균정양성표체.②50,100μg/L조뇌원성신경생장인자조신경간세포분화위신경원비례명현고우대조조、150,200 μg/L뇌원성신경생장인자조조(t=2.502～5.025,P < 0.05);50,100 μg/L뇌원성신경생장인자조지간신경간세포분화위신경원적비례차이불현저 (P > 0.05);대조조、150,200 μg/L조3조간신경간세포분화위신경원비례차이불현저(P > 0.05)결론:재20 μg/L 표피생장인자배양조건하,뇌원성신경생장인자촉사신경간세포향신경원분화적교가질량농도위50 μg/L.
BACKGROUND:The differentiation of neural stem cells (NSCs) is a hot research. Whether brain-derived neurotrophic factors (BDNF) can induce the differentiation of NSCs into neurons has not been detailed studied. OBJECTIVE:To investigate the effect of different concentrations of BDNF on the differentiation of NSCs from adult rat hippocampus into neurons in the medium with epidermal growth factor. DESIGN:A controlled study. SETTING:Department of Human Anatomy, China Medical University. MATERIALS:The experiment was performed at the Neurotomia Laboratory of China Medical University in August 2007. NSCs isolated from hippocampus of adult SD rats were inoculated in a serum-free medium. Three clean adult SD rats (200-250 g each) were provided by Laboratory Animal Department of China Medical University. Dispositions to the rats were consistent with ethical standards of animals. Epidermal growth factor (EGF) and BDNF were bought from R&D Company. METHODS:①NSCs obtained from rat hippocampus were cultured in the serum-free medium containing basic fibroblast growth factor (bFGF), EGF and B27. ②Single cell clone was obtained via limited dilution for the fourth passage of cells. Identification of NSCs for the passage of cells from monoclonal spheres was performed by Nestin immunocytochemistry. Neurons and astroglial cells were identified by immunocytochemistry for neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) a week after differentiation. ③Differentiation for the monoclonal spheres was done to appraisal the proportion of neurons. NSCs were divided for several groups containing 0 μg/L, 50 μg/L, 100 μg/L, 150 μg/L, and 200 μg/L groups according to the doses of BDNF. EGF was added in the media of each group. Immunocytochemistry for NSE was done a week later to count the positive cells. MAIN OUTCOME MEASURES:Proportion of neurons differentiated from NSCs.RESULTS:①NSCs immunocytochemical staining showed that Nestin was positive in monoclonal spheres, and NSE and GFAP were positive in differentiated cells. ②The proportion of differentiation from NSCs into neurons was significantly higher in groups treated with 50 μg/L and 100 μg/L BDNF compared to other groups（t=2.502-5.025, P < 0.05）, but there was no significant difference between 50 μg/L and 100 μg/L BDNF groups（P > 0.05）. Moreover, no significant difference was detected among 0 μg/L, 150 μg/L and 200 μg/L BDNF groups (P > 0.05). CONCLUSION:The optimal concentration of BDNF is 50 μg/L for differentiation from NSCs into neurons while the concentration of EGF is 20 μg/L.
