成人脂肪基质细胞体外诱导分化神经元的电生理功能和细胞凋亡研究
发布时间：2019-06-28 13:55
【摘要】：目的 体外分离、培养成人脂肪基质细胞,研究β-巯基乙醇诱导分化神经元的电生理功能和诱导过程中神经元的凋亡。 方法 1.参照Zuk、叶长青等分离培养成人脂肪基质细胞的方法进行细胞培养,消化收集第3-6代的成人脂肪基质细胞,制成细胞爬片,待细胞生长至70%～80%融合时,应用β-巯基乙醇诱导其向神经元细胞分化,按诱导时间不同将其分为诱导前组、预诱导组、诱导1h组、诱导3h组、诱导5h组、诱导8h组。应用倒置相差显微镜观察诱导分化后细胞形态变化。 2.应用MTT法检测成人脂肪基质细胞诱导后细胞的生长状态。 3.应用免疫细胞化学法检测诱导前后各组细胞的神经前体细胞标志物神经巢蛋白、成熟神经元标志物神经元特异性微管相关蛋白-2、神经元特异性烯醇化酶及神经胶质细胞标志物胶质纤维酸性蛋白的表达。 4.应用透射电镜观察成人脂肪基质细胞诱导分化的神经前体细胞、成熟神经元及发生凋亡细胞的超微结构特征。 5.应用激光共聚焦显微镜测定成人脂肪基质细胞诱导前及诱导5h时细胞膜电位。 6.应用DNA末端转移酶介导的原位缺口末端标记(TUNEL)法检测诱导后1h、3h、5h、8h组的细胞凋亡率。 7.图像采集及统计学处理:免疫细胞化学、TUNEL法阳性细胞率的计算采用每高倍光镜下随机计数100个细胞及阳性表达细胞,每张切片计数5次,计数3张切片(共计数15次),计算出各神经细胞标志物阳性细胞表达率的平均值;酶联免疫检测仪测定成人脂肪基质细胞诱导后细胞的OD值,每组设6个复孔,每个样本重复测3次,取平均OD值;H7500透射电镜观察细胞超微结构;Olympus激光共聚焦扫描显微镜观察细胞膜电位荧光强度的变化。实验数据采用EXCEL 2003建库,应用SPSS13.0统计软件包进行统计分析,同一组内不同时间点组间均数之间采用单因素方差分析(SNK-q检验),所得计量资料均以均数±标准差表示,以P=0.05为检验水准。 结果 1.原代培养的成人脂肪基质细胞于24h时已贴壁,呈类圆形、短梭形,48h时细胞呈长梭形,可见少量细胞伸出突起,类似于成纤维细胞,7-10d时可见大量呈漩涡状排列的长梭形细胞。传代12h细胞贴壁,24h时细胞呈成纤维细胞样形态,传至3-6代时,细胞形态均匀一致,基本去除了杂质细胞,细胞增生活跃。预诱导24h,个别细胞的形态发生变化,伸出短突起,细胞周围可见光晕,正式诱导1h,细胞核变大变圆,部分细胞伸出轴突样长突起,诱导3h,细胞胞质折光性增强,胞体周围可见明显光晕,诱导5h时细胞呈典型的神经元形态,细胞伸出较多的长突起,末端出现多级分支,且部分细胞的突起连接成网状,诱导8h时,细胞形态与5h比无明显变化,但此后细胞逐渐开始死亡,可见部分细胞脱离培养瓶瓶壁而浮起,至12 h左右大多数细胞已经死亡。 2. MTT测定结果示成人脂肪基质细胞在诱导分化过程中,细胞总数在预诱导组明显低于诱导前组(P0.05),诱导1h组细胞数量明显低于预诱导组(P0.05),诱导3h组、诱导5h组和诱导1h组相比,细胞数量无明显差异(P0.05),此时细胞生长较平稳,但诱导8h组的细胞数量低于诱导5h组(P0.05)。 3.体外培养的成人脂肪基质细胞诱导分化3h时神经前体细胞标志物—神经巢蛋白表达达高峰,为(86.25±4.82)%;诱导分化至5h时神经巢蛋白表达明显下降至诱导前以下水平;诱导5h时成熟神经元标志物—神经元特异性微管相关蛋白-2、神经元特异性烯醇化酶的阳性细胞表达率均达高峰,分别为(77.69±1.53)%,(85.92±3.07)%;诱导5h和8h组间无明显差异(P0.05),其余各组间差异均具有统计学意义(P0.05)。星形胶质细胞的标志物—胶质纤维酸性蛋白在诱导前后各时间点均未见表达。 4.透射电镜观察诱导分化3h时的神经前体细胞的超微结构,可见细胞表面突起较多,胞质中细胞器丰富,细胞核大,核/浆比例大,双层核膜且有较多核孔,常染色质多,异染色质少;观察诱导分化5h的神经元细胞可见胞浆中特异性细胞器—尼氏体;同时电镜下也观察到诱导分化5h时具有典型凋亡超微结构特征的细胞。 5.膜电位检测结果示诱导分化5h神经元有较高的静息膜电位,高钾刺激后产生迅速的去极化。 6.随着诱导反应时间延长,存活细胞数量逐渐减少,在此过程中细胞凋亡率明显上升(P0.05)。 结论 1.诱导分化5h神经元具有发育成熟的K+通道,高钾刺激后产生迅速的去极化。 2.诱导分化神经元存活时间短,随诱导反应时间延长细胞凋亡率呈上升趋势。
[Abstract]:Purpose In vitro separation and culture of adult fat matrix cells, the study of the electrophysiological function and the role of the neurons in the induction process of the induction-induced differentiation of the antigen-base ethanol Death. The method comprises the following steps of: carrying out cell culture on the method for separating and culturing the adult fat matrix cells with reference to Zuk and leaves and the like, and digesting and collecting the adult fat matrix cells of the third to sixth generation, and making the cells to grow to 70-8; In the fusion of 0%, the differentiation of the neurons was induced by the application of 1-1-base ethanol, and it was divided into the pre-induction group, the pre-induction group and the induction 1h group according to the induction time, and the group was induced for 3h, and the group was induced for 5 h. The induction of differentiation after induction of differentiation was observed with an inverted phase-contrast microscope. Cytological changes.2. After the induction of adult fat matrix cells by MTT method, 3. The cell growth status of the cells was detected by immunocytochemical method. Microtubule-associated protein-2, neuron-specific enolase and glial cell marker colloid 4. The expression of the fiber-acidic protein.4. The neural precursor cells, mature neurons and the generation of mature neurons induced by adult fat matrix cells were observed by transmission electron microscopy. Ultrastructural features of apoptotic cells. 6. The end-end labeling (TUNEL) of the in-situ notch mediated by the DNA-terminal transferase was used to detect the cell membrane potential. Apoptosis rate of cells in 3 h,5 h, and 8 h group.7. Image collection and statistical treatment: The calculation of the positive cell rate of the immune cell and the TUNEL method was 100 cells and positive expression cells at random under each high-power light microscope, and the count of each section was counted. 5 times, counting three slices (15 times in total), calculating the average value of the expression rate of the positive cell of each nerve cell marker, and measuring the OD value of the cell after the adult fat matrix cell is induced by the enzyme-linked immunodetector, and each group is provided with 6 complex holes, each sample is repeatedly tested for 3 times, and the average OD value is taken; The observation of the ultrastructure of the cells by the H7500 transmission electron microscope; Olysmus laser confocal scanning The changes of the fluorescence intensity of the cell membrane potential were observed by the microscope. The data of the experiment was built in EXCEL 2003, and the statistical analysis was carried out by using the SPSS13.0 statistical software package. The single-factor analysis of variance (SNK-q test) was used between the mean numbers of different time point groups in the same group, and the measured data were marked with the average number of time points. quasi-poor The results were as follows:1. The primary cultured adult fat matrix cells were attached at 24 h, and the cells were in the form of circular, short and fusiform, and the cells were in a long shuttle shape at 48 h, and a small number of cells were seen to protrude, similar to the fibroblasts,7. At-10 days, a large number of long-shuttle-shaped cells in a vortex-like arrangement were observed. The cells were cultured for 12 h, and the cells were in fibroblast-like form at 24 h and were transferred to 3-6 generations. Uniform, basically remove the impurity cells, the cell proliferation is active. The pre-induction of 24h, the form of individual cells changes, the short protrusion is extended, the visible light in the periphery of the cell is halo, the cell nucleus becomes larger and the nucleus becomes round, and the part of the cells extend out of the axon-like long protrusion, and the induction is induced for 3 h. At the time of the induction of 5 h, the cells showed a typical neuronal form, the cells extended to a large number of long protrusions, the end had a multi-stage branch, and the projections of some of the cells were connected into a net, and the cell morphology was induced at 8 h. There was no significant change in the ratio of 5 h, but after that the cells gradually began to die and part of the cells were isolated from the culture flask 2. The results of MTT assay showed that the total number of cells in adult adipocytes was significantly lower in the pre-induction group than in the pre-induction group (P0.05), and the number of cells in the induction group was significantly lower than that of the pre-induction group (P 0.05). There was no significant difference in the number of cells (P0.05). The number of cells in the 8-h group was lower than that in the induction group (P0.05). The expression of the neuron-specific enolase-specific microtubule-related protein-2 and the neuron-specific enolase in the mature neuron-specific microtubule-related protein-2 at the time of differentiation to 5 h reached the peak, respectively (77.69-1.53)% (85.92-3.0). 7)%; no significant difference between the induction and 8h groups (P0.05) 5) There was a significant difference between the other groups (P0.05). 4. The ultrastructure of the neural precursor cells at 3 h induced by the transmission electron microscope (TEM) showed that the surface of the cells was more prominent, the organelles in the cytoplasm were abundant, the nucleus was large, and the nucleus/ The proportion of the pulp is large, the double-layer nuclear membrane has more nuclear pores, the euchromatin is much, and the heterochromatin is few; the specific organelles of the cytoplasm are observed in the neuronal cells which are induced to differentiate for 5 hours; and simultaneously, the electron microscope Cells with typical apoptotic ultrastructure were also observed at 5 h of induction of differentiation.5. The results of the membrane potential test indicated that the induction of differentiation 5-hour neurons have higher resting membrane potential and rapid depolarizing after high-potassium stimulation. fine The number of cells decreased gradually, and the cell apoptosis rate in this process was significantly increased (P0.05). Conclusion 1 And the induced differentiation of the 5-h neurons has a mature K + channel, and the high-potassium stimulation generates a rapid depolarization.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329.28
本文编号：2507349
[Abstract]:Purpose In vitro separation and culture of adult fat matrix cells, the study of the electrophysiological function and the role of the neurons in the induction process of the induction-induced differentiation of the antigen-base ethanol Death. The method comprises the following steps of: carrying out cell culture on the method for separating and culturing the adult fat matrix cells with reference to Zuk and leaves and the like, and digesting and collecting the adult fat matrix cells of the third to sixth generation, and making the cells to grow to 70-8; In the fusion of 0%, the differentiation of the neurons was induced by the application of 1-1-base ethanol, and it was divided into the pre-induction group, the pre-induction group and the induction 1h group according to the induction time, and the group was induced for 3h, and the group was induced for 5 h. The induction of differentiation after induction of differentiation was observed with an inverted phase-contrast microscope. Cytological changes.2. After the induction of adult fat matrix cells by MTT method, 3. The cell growth status of the cells was detected by immunocytochemical method. Microtubule-associated protein-2, neuron-specific enolase and glial cell marker colloid 4. The expression of the fiber-acidic protein.4. The neural precursor cells, mature neurons and the generation of mature neurons induced by adult fat matrix cells were observed by transmission electron microscopy. Ultrastructural features of apoptotic cells. 6. The end-end labeling (TUNEL) of the in-situ notch mediated by the DNA-terminal transferase was used to detect the cell membrane potential. Apoptosis rate of cells in 3 h,5 h, and 8 h group.7. Image collection and statistical treatment: The calculation of the positive cell rate of the immune cell and the TUNEL method was 100 cells and positive expression cells at random under each high-power light microscope, and the count of each section was counted. 5 times, counting three slices (15 times in total), calculating the average value of the expression rate of the positive cell of each nerve cell marker, and measuring the OD value of the cell after the adult fat matrix cell is induced by the enzyme-linked immunodetector, and each group is provided with 6 complex holes, each sample is repeatedly tested for 3 times, and the average OD value is taken; The observation of the ultrastructure of the cells by the H7500 transmission electron microscope; Olysmus laser confocal scanning The changes of the fluorescence intensity of the cell membrane potential were observed by the microscope. The data of the experiment was built in EXCEL 2003, and the statistical analysis was carried out by using the SPSS13.0 statistical software package. The single-factor analysis of variance (SNK-q test) was used between the mean numbers of different time point groups in the same group, and the measured data were marked with the average number of time points. quasi-poor The results were as follows:1. The primary cultured adult fat matrix cells were attached at 24 h, and the cells were in the form of circular, short and fusiform, and the cells were in a long shuttle shape at 48 h, and a small number of cells were seen to protrude, similar to the fibroblasts,7. At-10 days, a large number of long-shuttle-shaped cells in a vortex-like arrangement were observed. The cells were cultured for 12 h, and the cells were in fibroblast-like form at 24 h and were transferred to 3-6 generations. Uniform, basically remove the impurity cells, the cell proliferation is active. The pre-induction of 24h, the form of individual cells changes, the short protrusion is extended, the visible light in the periphery of the cell is halo, the cell nucleus becomes larger and the nucleus becomes round, and the part of the cells extend out of the axon-like long protrusion, and the induction is induced for 3 h. At the time of the induction of 5 h, the cells showed a typical neuronal form, the cells extended to a large number of long protrusions, the end had a multi-stage branch, and the projections of some of the cells were connected into a net, and the cell morphology was induced at 8 h. There was no significant change in the ratio of 5 h, but after that the cells gradually began to die and part of the cells were isolated from the culture flask 2. The results of MTT assay showed that the total number of cells in adult adipocytes was significantly lower in the pre-induction group than in the pre-induction group (P0.05), and the number of cells in the induction group was significantly lower than that of the pre-induction group (P 0.05). There was no significant difference in the number of cells (P0.05). The number of cells in the 8-h group was lower than that in the induction group (P0.05). The expression of the neuron-specific enolase-specific microtubule-related protein-2 and the neuron-specific enolase in the mature neuron-specific microtubule-related protein-2 at the time of differentiation to 5 h reached the peak, respectively (77.69-1.53)% (85.92-3.0). 7)%; no significant difference between the induction and 8h groups (P0.05) 5) There was a significant difference between the other groups (P0.05). 4. The ultrastructure of the neural precursor cells at 3 h induced by the transmission electron microscope (TEM) showed that the surface of the cells was more prominent, the organelles in the cytoplasm were abundant, the nucleus was large, and the nucleus/ The proportion of the pulp is large, the double-layer nuclear membrane has more nuclear pores, the euchromatin is much, and the heterochromatin is few; the specific organelles of the cytoplasm are observed in the neuronal cells which are induced to differentiate for 5 hours; and simultaneously, the electron microscope Cells with typical apoptotic ultrastructure were also observed at 5 h of induction of differentiation.5. The results of the membrane potential test indicated that the induction of differentiation 5-hour neurons have higher resting membrane potential and rapid depolarizing after high-potassium stimulation. fine The number of cells decreased gradually, and the cell apoptosis rate in this process was significantly increased (P0.05). Conclusion 1 And the induced differentiation of the 5-h neurons has a mature K + channel, and the high-potassium stimulation generates a rapid depolarization.
【学位授予单位】：河北联合大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329.28
【参考文献】
相关期刊论文 前10条
1 廖杰,于力方,葛学铭;细胞膜电位的动态监测及其在银杏内酯研究中的应用[J];标记免疫分析与临床;2005年03期
2 张宏伟,张亚卓,张海霞,陈晓光,王晓良,于春江;星形胶质细胞诱导成年大鼠骨髓基质细胞分化前后的电生理功能鉴定[J];中华神经医学杂志;2004年05期
3 田增民,刘爽,李士月,赵全军,尹丰,郝秋星,周英;人神经干细胞临床移植治疗帕金森病[J];第二军医大学学报;2003年09期
4 张迅轶;仵敏娟;刘厚奇;;人皮下脂肪来源脂肪干细胞的分离培养及鉴定[J];第二军医大学学报;2007年08期
5 郑汉巧,欧阳静萍,余峰,黄卫华,李银萍,吴珂,庞代文;脂肪组织干细胞分化为神经元样细胞研究[J];武汉大学学报(医学版);2003年04期
6 刘文兰,张颖;细胞凋亡检测方法——TUNEL[J];解剖科学进展;1999年04期
7 赵耀东;张天一;黄强;王爱东;董军;兰青;秦正红;顾晓松;;体外培养的人源神经干-祖细胞超微结构分析[J];解剖学报;2008年05期
8 高丹宇,滕弘,朱晓峰;神经干细胞移植对Alzheimer病大鼠脑形态及动物行为学影响[J];黑龙江医药科学;2003年05期
9 刘斌;董静;李建民;刘宁;王瑞敏;张晋霞;李世英;陈贵良;;人脂肪组织来源的神经干细胞移植对大鼠局灶性脑缺血再灌注后细胞凋亡及Bcl-2、Bax蛋白表达的影响[J];临床神经病学杂志;2010年01期
10 曾荣;胡资兵;郭伟韬;林颢;孙欣;魏劲松;吴少科;;依达拉奉体外诱导大鼠骨髓基质干细胞分化为神经元样细胞的电生理研究[J];现代实用医学;2009年03期
，本文编号：2507349
本文链接：https://www.wllwen.com/xiyixuelunwen/2507349.html
最近更新
教材专著
