成人脂肪基質(zhì)細(xì)胞體外誘導(dǎo)分化神經(jīng)元的電生理功能和細(xì)胞凋亡研究
發(fā)布時(shí)間：2019-06-28 13:55
【摘要】：目的 體外分離、培養(yǎng)成人脂肪基質(zhì)細(xì)胞,研究β-巰基乙醇誘導(dǎo)分化神經(jīng)元的電生理功能和誘導(dǎo)過(guò)程中神經(jīng)元的凋亡。 方法 1.參照Z(yǔ)uk、葉長(zhǎng)青等分離培養(yǎng)成人脂肪基質(zhì)細(xì)胞的方法進(jìn)行細(xì)胞培養(yǎng),消化收集第3-6代的成人脂肪基質(zhì)細(xì)胞,制成細(xì)胞爬片,待細(xì)胞生長(zhǎng)至70%～80%融合時(shí),應(yīng)用β-巰基乙醇誘導(dǎo)其向神經(jīng)元細(xì)胞分化,按誘導(dǎo)時(shí)間不同將其分為誘導(dǎo)前組、預(yù)誘導(dǎo)組、誘導(dǎo)1h組、誘導(dǎo)3h組、誘導(dǎo)5h組、誘導(dǎo)8h組。應(yīng)用倒置相差顯微鏡觀察誘導(dǎo)分化后細(xì)胞形態(tài)變化。 2.應(yīng)用MTT法檢測(cè)成人脂肪基質(zhì)細(xì)胞誘導(dǎo)后細(xì)胞的生長(zhǎng)狀態(tài)。 3.應(yīng)用免疫細(xì)胞化學(xué)法檢測(cè)誘導(dǎo)前后各組細(xì)胞的神經(jīng)前體細(xì)胞標(biāo)志物神經(jīng)巢蛋白、成熟神經(jīng)元標(biāo)志物神經(jīng)元特異性微管相關(guān)蛋白-2、神經(jīng)元特異性烯醇化酶及神經(jīng)膠質(zhì)細(xì)胞標(biāo)志物膠質(zhì)纖維酸性蛋白的表達(dá)。 4.應(yīng)用透射電鏡觀察成人脂肪基質(zhì)細(xì)胞誘導(dǎo)分化的神經(jīng)前體細(xì)胞、成熟神經(jīng)元及發(fā)生凋亡細(xì)胞的超微結(jié)構(gòu)特征。 5.應(yīng)用激光共聚焦顯微鏡測(cè)定成人脂肪基質(zhì)細(xì)胞誘導(dǎo)前及誘導(dǎo)5h時(shí)細(xì)胞膜電位。 6.應(yīng)用DNA末端轉(zhuǎn)移酶介導(dǎo)的原位缺口末端標(biāo)記(TUNEL)法檢測(cè)誘導(dǎo)后1h、3h、5h、8h組的細(xì)胞凋亡率。 7.圖像采集及統(tǒng)計(jì)學(xué)處理:免疫細(xì)胞化學(xué)、TUNEL法陽(yáng)性細(xì)胞率的計(jì)算采用每高倍光鏡下隨機(jī)計(jì)數(shù)100個(gè)細(xì)胞及陽(yáng)性表達(dá)細(xì)胞,每張切片計(jì)數(shù)5次,計(jì)數(shù)3張切片(共計(jì)數(shù)15次),計(jì)算出各神經(jīng)細(xì)胞標(biāo)志物陽(yáng)性細(xì)胞表達(dá)率的平均值;酶聯(lián)免疫檢測(cè)儀測(cè)定成人脂肪基質(zhì)細(xì)胞誘導(dǎo)后細(xì)胞的OD值,每組設(shè)6個(gè)復(fù)孔,每個(gè)樣本重復(fù)測(cè)3次,取平均OD值;H7500透射電鏡觀察細(xì)胞超微結(jié)構(gòu);Olympus激光共聚焦掃描顯微鏡觀察細(xì)胞膜電位熒光強(qiáng)度的變化。實(shí)驗(yàn)數(shù)據(jù)采用EXCEL 2003建庫(kù),應(yīng)用SPSS13.0統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)分析,同一組內(nèi)不同時(shí)間點(diǎn)組間均數(shù)之間采用單因素方差分析(SNK-q檢驗(yàn)),所得計(jì)量資料均以均數(shù)±標(biāo)準(zhǔn)差表示,以P=0.05為檢驗(yàn)水準(zhǔn)。 結(jié)果 1.原代培養(yǎng)的成人脂肪基質(zhì)細(xì)胞于24h時(shí)已貼壁,呈類圓形、短梭形,48h時(shí)細(xì)胞呈長(zhǎng)梭形,可見(jiàn)少量細(xì)胞伸出突起,類似于成纖維細(xì)胞,7-10d時(shí)可見(jiàn)大量呈漩渦狀排列的長(zhǎng)梭形細(xì)胞。傳代12h細(xì)胞貼壁,24h時(shí)細(xì)胞呈成纖維細(xì)胞樣形態(tài),傳至3-6代時(shí),細(xì)胞形態(tài)均勻一致,基本去除了雜質(zhì)細(xì)胞,細(xì)胞增生活躍。預(yù)誘導(dǎo)24h,個(gè)別細(xì)胞的形態(tài)發(fā)生變化,伸出短突起,細(xì)胞周圍可見(jiàn)光暈,正式誘導(dǎo)1h,細(xì)胞核變大變圓,部分細(xì)胞伸出軸突樣長(zhǎng)突起,誘導(dǎo)3h,細(xì)胞胞質(zhì)折光性增強(qiáng),胞體周圍可見(jiàn)明顯光暈,誘導(dǎo)5h時(shí)細(xì)胞呈典型的神經(jīng)元形態(tài),細(xì)胞伸出較多的長(zhǎng)突起,末端出現(xiàn)多級(jí)分支,且部分細(xì)胞的突起連接成網(wǎng)狀,誘導(dǎo)8h時(shí),細(xì)胞形態(tài)與5h比無(wú)明顯變化,但此后細(xì)胞逐漸開始死亡,可見(jiàn)部分細(xì)胞脫離培養(yǎng)瓶瓶壁而浮起,至12 h左右大多數(shù)細(xì)胞已經(jīng)死亡。 2. MTT測(cè)定結(jié)果示成人脂肪基質(zhì)細(xì)胞在誘導(dǎo)分化過(guò)程中,細(xì)胞總數(shù)在預(yù)誘導(dǎo)組明顯低于誘導(dǎo)前組(P0.05),誘導(dǎo)1h組細(xì)胞數(shù)量明顯低于預(yù)誘導(dǎo)組(P0.05),誘導(dǎo)3h組、誘導(dǎo)5h組和誘導(dǎo)1h組相比,細(xì)胞數(shù)量無(wú)明顯差異(P0.05),此時(shí)細(xì)胞生長(zhǎng)較平穩(wěn),但誘導(dǎo)8h組的細(xì)胞數(shù)量低于誘導(dǎo)5h組(P0.05)。 3.體外培養(yǎng)的成人脂肪基質(zhì)細(xì)胞誘導(dǎo)分化3h時(shí)神經(jīng)前體細(xì)胞標(biāo)志物—神經(jīng)巢蛋白表達(dá)達(dá)高峰,為(86.25±4.82)%;誘導(dǎo)分化至5h時(shí)神經(jīng)巢蛋白表達(dá)明顯下降至誘導(dǎo)前以下水平;誘導(dǎo)5h時(shí)成熟神經(jīng)元標(biāo)志物—神經(jīng)元特異性微管相關(guān)蛋白-2、神經(jīng)元特異性烯醇化酶的陽(yáng)性細(xì)胞表達(dá)率均達(dá)高峰,分別為(77.69±1.53)%,(85.92±3.07)%;誘導(dǎo)5h和8h組間無(wú)明顯差異(P0.05),其余各組間差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。星形膠質(zhì)細(xì)胞的標(biāo)志物—膠質(zhì)纖維酸性蛋白在誘導(dǎo)前后各時(shí)間點(diǎn)均未見(jiàn)表達(dá)。 4.透射電鏡觀察誘導(dǎo)分化3h時(shí)的神經(jīng)前體細(xì)胞的超微結(jié)構(gòu),可見(jiàn)細(xì)胞表面突起較多,胞質(zhì)中細(xì)胞器豐富,細(xì)胞核大,核/漿比例大,雙層核膜且有較多核孔,常染色質(zhì)多,異染色質(zhì)少;觀察誘導(dǎo)分化5h的神經(jīng)元細(xì)胞可見(jiàn)胞漿中特異性細(xì)胞器—尼氏體;同時(shí)電鏡下也觀察到誘導(dǎo)分化5h時(shí)具有典型凋亡超微結(jié)構(gòu)特征的細(xì)胞。 5.膜電位檢測(cè)結(jié)果示誘導(dǎo)分化5h神經(jīng)元有較高的靜息膜電位,高鉀刺激后產(chǎn)生迅速的去極化。 6.隨著誘導(dǎo)反應(yīng)時(shí)間延長(zhǎng),存活細(xì)胞數(shù)量逐漸減少,在此過(guò)程中細(xì)胞凋亡率明顯上升(P0.05)。 結(jié)論 1.誘導(dǎo)分化5h神經(jīng)元具有發(fā)育成熟的K+通道,高鉀刺激后產(chǎn)生迅速的去極化。 2.誘導(dǎo)分化神經(jīng)元存活時(shí)間短,隨誘導(dǎo)反應(yīng)時(shí)間延長(zhǎng)細(xì)胞凋亡率呈上升趨勢(shì)。
[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.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329.28
本文編號(hào)：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.
【學(xué)位授予單位】：河北聯(lián)合大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329.28
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 廖杰,于力方,葛學(xué)銘;細(xì)胞膜電位的動(dòng)態(tài)監(jiān)測(cè)及其在銀杏內(nèi)酯研究中的應(yīng)用[J];標(biāo)記免疫分析與臨床;2005年03期
2 張宏偉,張亞卓,張海霞,陳曉光,王曉良,于春江;星形膠質(zhì)細(xì)胞誘導(dǎo)成年大鼠骨髓基質(zhì)細(xì)胞分化前后的電生理功能鑒定[J];中華神經(jīng)醫(yī)學(xué)雜志;2004年05期
3 田增民,劉爽,李士月,趙全軍,尹豐,郝秋星,周英;人神經(jīng)干細(xì)胞臨床移植治療帕金森病[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2003年09期
4 張迅軼;仵敏娟;劉厚奇;;人皮下脂肪來(lái)源脂肪干細(xì)胞的分離培養(yǎng)及鑒定[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2007年08期
5 鄭漢巧,歐陽(yáng)靜萍,余峰,黃衛(wèi)華,李銀萍,吳珂,龐代文;脂肪組織干細(xì)胞分化為神經(jīng)元樣細(xì)胞研究[J];武漢大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年04期
6 劉文蘭,張穎;細(xì)胞凋亡檢測(cè)方法——TUNEL[J];解剖科學(xué)進(jìn)展;1999年04期
7 趙耀東;張?zhí)煲?黃強(qiáng);王愛(ài)東;董軍;蘭青;秦正紅;顧曉松;;體外培養(yǎng)的人源神經(jīng)干-祖細(xì)胞超微結(jié)構(gòu)分析[J];解剖學(xué)報(bào);2008年05期
8 高丹宇,滕弘,朱曉峰;神經(jīng)干細(xì)胞移植對(duì)Alzheimer病大鼠腦形態(tài)及動(dòng)物行為學(xué)影響[J];黑龍江醫(yī)藥科學(xué);2003年05期
9 劉斌;董靜;李建民;劉寧;王瑞敏;張晉霞;李世英;陳貴良;;人脂肪組織來(lái)源的神經(jīng)干細(xì)胞移植對(duì)大鼠局灶性腦缺血再灌注后細(xì)胞凋亡及Bcl-2、Bax蛋白表達(dá)的影響[J];臨床神經(jīng)病學(xué)雜志;2010年01期
10 曾榮;胡資兵;郭偉韜;林顥;孫欣;魏勁松;吳少科;;依達(dá)拉奉體外誘導(dǎo)大鼠骨髓基質(zhì)干細(xì)胞分化為神經(jīng)元樣細(xì)胞的電生理研究[J];現(xiàn)代實(shí)用醫(yī)學(xué);2009年03期
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