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
2007年
24期
4856-4860
,共5页
高明勇%肖建德%李振宇%闫洪印%白润涛%韩漫夫
高明勇%肖建德%李振宇%閆洪印%白潤濤%韓漫伕
고명용%초건덕%리진우%염홍인%백윤도%한만부
神经干细胞%分化%神经元%胶质细胞源性神经营养因子%转化生长因子β1
神經榦細胞%分化%神經元%膠質細胞源性神經營養因子%轉化生長因子β1
신경간세포%분화%신경원%효질세포원성신경영양인자%전화생장인자β1
背景:胶质细胞源性神经营养因子与转化生长因子β1同属于转化生长因子β超家族,两者在哺乳动物中枢与外周神经系统发育、分化及细胞周期的调节中扮演重要角色.目的:观察胶质细胞源性神经营养因子联合转化生长因子β1体外诱导脊髓源性神经干细胞的分化,并与单一因子诱导培养液比较.设计:对照观察.单位:南方医科大学附属深圳医院中心实验室.材料:选用10只清洁级孕16 d的SD大鼠,由华中科大同济医学院动物中心提供.主要试剂及材料:DMEM/F12,B27(GIBCO);碱性成纤维细胞生长因子,EGF,胶质细胞源性神经营养因子,转化生长因子-β1(PeproTech);胎牛血清(Hyclone);神经巢蛋白(nestin)多抗(武汉博士德);胶质纤维酸性蛋白(GFAP)多抗,神经丝蛋白(NF-200)单抗(Sigma).方法:实验于2005-10/2006-09在南方医科大学附属深圳医院中心实验室完成.无菌条件下分离大鼠胚胎,进行脊髓源性神经干细胞的分离培养.实验分①基础培养基组:含青霉素、链霉素、两性霉素B及体积分数为0.02 B27添加液的DMEM/F12.原代培养1周后,获取体外稳定增殖的鼠脊髓源性神经干细胞克隆.②对照组:在基础培养基中加入体积分数为O.1的胎牛血清.③碱性成纤维细胞生长因子组.④转化生长因子β1组.⑤胶质细胞源性神经营养因子+转化生长因子β1组.换用诱导培养基,即分别在基础培养基中加入20 μg/L碱性成纤维细胞生长因子、2 μg/L转化生长因子β1、10 μg/L胶质细胞源性神经营养因子+2 μg/L转化生长因子β1.①光镜观察在不同因子诱导情况下脊髓源性神经干细胞的分化情况.②免疫细胞化学染色标记检测神经元与星状胶质细胞的表达.③通过荧光激发流式细胞仪分选技术对分化细胞计数,检测不同诱导环境下神经干细胞分化为神经元及星状胶质细胞的阳性百分率.主要观察指标:①各组细胞分化的形态学特点.②各组神经干细胞免疫组织化学检测结果.③各组神经干细胞分化为神经元及星状胶质细胞的阳性百分率.结果:①细胞分化形态:分化早期可见神经球贴壁,大量细胞向四周爬出,1周后迁徙的大部分细胞完全贴壁,完成分化过程.在细胞密度较低区域可辨认出神经元样细胞、星状胶质样细胞、寡突胶质样细胞.②免疫组织化学检测结果:对照组、转化生长因子β1组存在大量胶质纤维酸性蛋白染色阳性的星状胶质细胞;而碱性成纤维细胞生长因子组及胶质细胞源性神经营养因子+转化生长因子β1组分化的神经元细胞数量较多,神经丝蛋白染色阳性.③神经元及星状胶质细胞的阳性百分比:诱导1周后,胶质细胞源性神经营养因子+转化生长因子β1组神经元阳性百分比高于血清对照组、碱性成纤维细胞生长因子组及转化生长因子β1组(x2=24.15,19.56,25.32,P＜0.05～0.01),星状胶质细胞阳性百分比低于血清对照组及转化生长因子β1组(x2=24.45,23.79,P＜0.01).结论:体外胶质细胞源性神经营养因子与转化生长因子β1的联合诱导有利于脊髓源性神经干细胞向神经元的分化,说明两者具有协同作用.
揹景:膠質細胞源性神經營養因子與轉化生長因子β1同屬于轉化生長因子β超傢族,兩者在哺乳動物中樞與外週神經繫統髮育、分化及細胞週期的調節中扮縯重要角色.目的:觀察膠質細胞源性神經營養因子聯閤轉化生長因子β1體外誘導脊髓源性神經榦細胞的分化,併與單一因子誘導培養液比較.設計:對照觀察.單位:南方醫科大學附屬深圳醫院中心實驗室.材料:選用10隻清潔級孕16 d的SD大鼠,由華中科大同濟醫學院動物中心提供.主要試劑及材料:DMEM/F12,B27(GIBCO);堿性成纖維細胞生長因子,EGF,膠質細胞源性神經營養因子,轉化生長因子-β1(PeproTech);胎牛血清(Hyclone);神經巢蛋白(nestin)多抗(武漢博士德);膠質纖維痠性蛋白(GFAP)多抗,神經絲蛋白(NF-200)單抗(Sigma).方法:實驗于2005-10/2006-09在南方醫科大學附屬深圳醫院中心實驗室完成.無菌條件下分離大鼠胚胎,進行脊髓源性神經榦細胞的分離培養.實驗分①基礎培養基組:含青黴素、鏈黴素、兩性黴素B及體積分數為0.02 B27添加液的DMEM/F12.原代培養1週後,穫取體外穩定增殖的鼠脊髓源性神經榦細胞剋隆.②對照組:在基礎培養基中加入體積分數為O.1的胎牛血清.③堿性成纖維細胞生長因子組.④轉化生長因子β1組.⑤膠質細胞源性神經營養因子+轉化生長因子β1組.換用誘導培養基,即分彆在基礎培養基中加入20 μg/L堿性成纖維細胞生長因子、2 μg/L轉化生長因子β1、10 μg/L膠質細胞源性神經營養因子+2 μg/L轉化生長因子β1.①光鏡觀察在不同因子誘導情況下脊髓源性神經榦細胞的分化情況.②免疫細胞化學染色標記檢測神經元與星狀膠質細胞的錶達.③通過熒光激髮流式細胞儀分選技術對分化細胞計數,檢測不同誘導環境下神經榦細胞分化為神經元及星狀膠質細胞的暘性百分率.主要觀察指標:①各組細胞分化的形態學特點.②各組神經榦細胞免疫組織化學檢測結果.③各組神經榦細胞分化為神經元及星狀膠質細胞的暘性百分率.結果:①細胞分化形態:分化早期可見神經毬貼壁,大量細胞嚮四週爬齣,1週後遷徙的大部分細胞完全貼壁,完成分化過程.在細胞密度較低區域可辨認齣神經元樣細胞、星狀膠質樣細胞、寡突膠質樣細胞.②免疫組織化學檢測結果:對照組、轉化生長因子β1組存在大量膠質纖維痠性蛋白染色暘性的星狀膠質細胞;而堿性成纖維細胞生長因子組及膠質細胞源性神經營養因子+轉化生長因子β1組分化的神經元細胞數量較多,神經絲蛋白染色暘性.③神經元及星狀膠質細胞的暘性百分比:誘導1週後,膠質細胞源性神經營養因子+轉化生長因子β1組神經元暘性百分比高于血清對照組、堿性成纖維細胞生長因子組及轉化生長因子β1組(x2=24.15,19.56,25.32,P＜0.05～0.01),星狀膠質細胞暘性百分比低于血清對照組及轉化生長因子β1組(x2=24.45,23.79,P＜0.01).結論:體外膠質細胞源性神經營養因子與轉化生長因子β1的聯閤誘導有利于脊髓源性神經榦細胞嚮神經元的分化,說明兩者具有協同作用.
배경:효질세포원성신경영양인자여전화생장인자β1동속우전화생장인자β초가족,량자재포유동물중추여외주신경계통발육、분화급세포주기적조절중분연중요각색.목적:관찰효질세포원성신경영양인자연합전화생장인자β1체외유도척수원성신경간세포적분화,병여단일인자유도배양액비교.설계:대조관찰.단위:남방의과대학부속심수의원중심실험실.재료:선용10지청길급잉16 d적SD대서,유화중과대동제의학원동물중심제공.주요시제급재료:DMEM/F12,B27(GIBCO);감성성섬유세포생장인자,EGF,효질세포원성신경영양인자,전화생장인자-β1(PeproTech);태우혈청(Hyclone);신경소단백(nestin)다항(무한박사덕);효질섬유산성단백(GFAP)다항,신경사단백(NF-200)단항(Sigma).방법:실험우2005-10/2006-09재남방의과대학부속심수의원중심실험실완성.무균조건하분리대서배태,진행척수원성신경간세포적분리배양.실험분①기출배양기조:함청매소、련매소、량성매소B급체적분수위0.02 B27첨가액적DMEM/F12.원대배양1주후,획취체외은정증식적서척수원성신경간세포극륭.②대조조:재기출배양기중가입체적분수위O.1적태우혈청.③감성성섬유세포생장인자조.④전화생장인자β1조.⑤효질세포원성신경영양인자+전화생장인자β1조.환용유도배양기,즉분별재기출배양기중가입20 μg/L감성성섬유세포생장인자、2 μg/L전화생장인자β1、10 μg/L효질세포원성신경영양인자+2 μg/L전화생장인자β1.①광경관찰재불동인자유도정황하척수원성신경간세포적분화정황.②면역세포화학염색표기검측신경원여성상효질세포적표체.③통과형광격발류식세포의분선기술대분화세포계수,검측불동유도배경하신경간세포분화위신경원급성상효질세포적양성백분솔.주요관찰지표:①각조세포분화적형태학특점.②각조신경간세포면역조직화학검측결과.③각조신경간세포분화위신경원급성상효질세포적양성백분솔.결과:①세포분화형태:분화조기가견신경구첩벽,대량세포향사주파출,1주후천사적대부분세포완전첩벽,완성분화과정.재세포밀도교저구역가변인출신경원양세포、성상효질양세포、과돌효질양세포.②면역조직화학검측결과:대조조、전화생장인자β1조존재대량효질섬유산성단백염색양성적성상효질세포;이감성성섬유세포생장인자조급효질세포원성신경영양인자+전화생장인자β1조분화적신경원세포수량교다,신경사단백염색양성.③신경원급성상효질세포적양성백분비:유도1주후,효질세포원성신경영양인자+전화생장인자β1조신경원양성백분비고우혈청대조조、감성성섬유세포생장인자조급전화생장인자β1조(x2=24.15,19.56,25.32,P＜0.05～0.01),성상효질세포양성백분비저우혈청대조조급전화생장인자β1조(x2=24.45,23.79,P＜0.01).결론:체외효질세포원성신경영양인자여전화생장인자β1적연합유도유리우척수원성신경간세포향신경원적분화,설명량자구유협동작용.
BACKGROUND: Glial cell-derived neurotrophic factor (GDNF) and transforming growth factor-beta 1 (TGF-β1)co-subordinate to TGF-β family. Both of them play very important roles in the development and differentiation of central and peripheral nervous system, and regulation of cell cycle in mammals.OBJECTIVE: To observe the differentiation of spinal cord-derived neural stem cells(NSCs) induced by GDNF combined with TGF-β1, and make a comparison of differentiation results with GDNF or TGF-β1 culture fluid.DESIGN: Controlled observation.SETTING: Central Laboratory, Shenzhen Hospital Affiliated to Southern Medical University.MATERIALS: Ten SD rats of clean grade, which were at conception for 16 days, were provided by the Experimental Animal Center, Tongji Medical College, Huazhong University of Science and Technplogy. Main reagents and materials:DMEM/F12,B27(GIBCO); basic fibroblast growth factor (bFGF), GDNF; TGF-β1(PeproTech);fetal bovine serum (FBS,Hyclone); nestin multiple antibody (Boster, Wuhan); glial fibrillary acidic protein (GFAP) multiple antibody; neurofilament protein (NF-200) monoclonal antibody (Sigma).METHODS: This experiment was carried out in the Central Laboratory, Shenzhen Hospital Affiliated to Southern Medcial University between October 2005 and September 2006. Under the aseptic condition, rat fetus was isolated for isolation and culture of spinal cord-derived neural stem cells. In this study, five groups were divided: basal medium group, control group, bFGF group, TGF-β1 group, GDNF+ TGF-β1 group. In the basal medium group, DMEM/F12 containing penicillin,streptomycin, amphotericin (AMPH) B and 0.02 volume fraction of B27 annex solution. At 1 week after primary culture, rat spinal cord-derived NSC clones proliferated in vitro stably were harvested. In the control group, 0.1 volume fraction of FBS was added into basal medium. In the later three groups, induced medium was exchanged, i.e. 20 μg/L bFGF, 2 μg/L TGF-β1, and 10 μg/L GDNF+2 μg/L TGF-β1 were added into the basal medium, respectively. ①The differentiation of spinal cord-derived NSCs induced by different factors were observed under the optical microscope. ②The expressions of neurons and astrocytes were detected by immunocytochemical staining labeling. ③ The differentiated cells were counted by sorting technique by means of fluorescence excitation flow cytometer, and the percentage of NSCs differentiating into neurons and astrocytes were detected under the different induction environments.MAIN OUTCOME MEASURES: ① Morphological feature of cell differentiation in each group. ② Immunohistochemical detection of NSCs in each group. ③ The percentage of NSCs differentiating into neurons and astrocytes in each group.RESULTS: ① Cell morphology during differentiation: At the early stage of differentiation, lots of cells creeped to all the directions, and one week later, most of the migrated cells adhered to the wall entirely. Neuron-like cells, astrocyte-like cells and oligodendrocyte-like cells could be identified in the low-density cell region. ②Immunohistochemical detection results: A lot of GFAP- positive astrocytes were found in the control group and TGF-β1 group; Many differentiated neurons and NF-200 staining positive were found in the bFGF group and GDNF+ TGF-β1 group. ③Percentage of stained neuron and astrocyte: at one week of induction, the percentage of stained neurons was higher in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.15,19.56,25.32,P ＜ 0.05-0.01), and the percentage of stained astrocytes was lower in the GDNF+ TGF-β1 group than in the control group, bFGF group and TGF-β1 group (x2=24.45,23.79,P ＜ 0.01 ).CONCLUSION: The combined in vitro induction of GDNF and TGF-β1 contributes to the neuronal differentiation of spinal cord-derived NSCs, indicating that both of them have synergistic effect.
