成人脂肪基質(zhì)細胞體外誘導分化神經(jīng)元的電生理功能和細胞凋亡研究
發(fā)布時間：2019-06-28 13:55
【摘要】：目的 體外分離、培養(yǎng)成人脂肪基質(zhì)細胞,研究β-巰基乙醇誘導分化神經(jīng)元的電生理功能和誘導過程中神經(jīng)元的凋亡。 方法 1.參照Zuk、葉長青等分離培養(yǎng)成人脂肪基質(zhì)細胞的方法進行細胞培養(yǎng),消化收集第3-6代的成人脂肪基質(zhì)細胞,制成細胞爬片,待細胞生長至70%～80%融合時,應用β-巰基乙醇誘導其向神經(jīng)元細胞分化,按誘導時間不同將其分為誘導前組、預誘導組、誘導1h組、誘導3h組、誘導5h組、誘導8h組。應用倒置相差顯微鏡觀察誘導分化后細胞形態(tài)變化。 2.應用MTT法檢測成人脂肪基質(zhì)細胞誘導后細胞的生長狀態(tài)。 3.應用免疫細胞化學法檢測誘導前后各組細胞的神經(jīng)前體細胞標志物神經(jīng)巢蛋白、成熟神經(jīng)元標志物神經(jīng)元特異性微管相關蛋白-2、神經(jīng)元特異性烯醇化酶及神經(jīng)膠質(zhì)細胞標志物膠質(zhì)纖維酸性蛋白的表達。 4.應用透射電鏡觀察成人脂肪基質(zhì)細胞誘導分化的神經(jīng)前體細胞、成熟神經(jīng)元及發(fā)生凋亡細胞的超微結構特征。 5.應用激光共聚焦顯微鏡測定成人脂肪基質(zhì)細胞誘導前及誘導5h時細胞膜電位。 6.應用DNA末端轉移酶介導的原位缺口末端標記(TUNEL)法檢測誘導后1h、3h、5h、8h組的細胞凋亡率。 7.圖像采集及統(tǒng)計學處理:免疫細胞化學、TUNEL法陽性細胞率的計算采用每高倍光鏡下隨機計數(shù)100個細胞及陽性表達細胞,每張切片計數(shù)5次,計數(shù)3張切片(共計數(shù)15次),計算出各神經(jīng)細胞標志物陽性細胞表達率的平均值;酶聯(lián)免疫檢測儀測定成人脂肪基質(zhì)細胞誘導后細胞的OD值,每組設6個復孔,每個樣本重復測3次,取平均OD值;H7500透射電鏡觀察細胞超微結構;Olympus激光共聚焦掃描顯微鏡觀察細胞膜電位熒光強度的變化。實驗數(shù)據(jù)采用EXCEL 2003建庫,應用SPSS13.0統(tǒng)計軟件包進行統(tǒng)計分析,同一組內(nèi)不同時間點組間均數(shù)之間采用單因素方差分析(SNK-q檢驗),所得計量資料均以均數(shù)±標準差表示,以P=0.05為檢驗水準。 結果 1.原代培養(yǎng)的成人脂肪基質(zhì)細胞于24h時已貼壁,呈類圓形、短梭形,48h時細胞呈長梭形,可見少量細胞伸出突起,類似于成纖維細胞,7-10d時可見大量呈漩渦狀排列的長梭形細胞。傳代12h細胞貼壁,24h時細胞呈成纖維細胞樣形態(tài),傳至3-6代時,細胞形態(tài)均勻一致,基本去除了雜質(zhì)細胞,細胞增生活躍。預誘導24h,個別細胞的形態(tài)發(fā)生變化,伸出短突起,細胞周圍可見光暈,正式誘導1h,細胞核變大變圓,部分細胞伸出軸突樣長突起,誘導3h,細胞胞質(zhì)折光性增強,胞體周圍可見明顯光暈,誘導5h時細胞呈典型的神經(jīng)元形態(tài),細胞伸出較多的長突起,末端出現(xiàn)多級分支,且部分細胞的突起連接成網(wǎng)狀,誘導8h時,細胞形態(tài)與5h比無明顯變化,但此后細胞逐漸開始死亡,可見部分細胞脫離培養(yǎng)瓶瓶壁而浮起,至12 h左右大多數(shù)細胞已經(jīng)死亡。 2. MTT測定結果示成人脂肪基質(zhì)細胞在誘導分化過程中,細胞總數(shù)在預誘導組明顯低于誘導前組(P0.05),誘導1h組細胞數(shù)量明顯低于預誘導組(P0.05),誘導3h組、誘導5h組和誘導1h組相比,細胞數(shù)量無明顯差異(P0.05),此時細胞生長較平穩(wěn),但誘導8h組的細胞數(shù)量低于誘導5h組(P0.05)。 3.體外培養(yǎng)的成人脂肪基質(zhì)細胞誘導分化3h時神經(jīng)前體細胞標志物—神經(jīng)巢蛋白表達達高峰,為(86.25±4.82)%;誘導分化至5h時神經(jīng)巢蛋白表達明顯下降至誘導前以下水平;誘導5h時成熟神經(jīng)元標志物—神經(jīng)元特異性微管相關蛋白-2、神經(jīng)元特異性烯醇化酶的陽性細胞表達率均達高峰,分別為(77.69±1.53)%,(85.92±3.07)%;誘導5h和8h組間無明顯差異(P0.05),其余各組間差異均具有統(tǒng)計學意義(P0.05)。星形膠質(zhì)細胞的標志物—膠質(zhì)纖維酸性蛋白在誘導前后各時間點均未見表達。 4.透射電鏡觀察誘導分化3h時的神經(jīng)前體細胞的超微結構,可見細胞表面突起較多,胞質(zhì)中細胞器豐富,細胞核大,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;觀察誘導分化5h的神經(jīng)元細胞可見胞漿中特異性細胞器—尼氏體;同時電鏡下也觀察到誘導分化5h時具有典型凋亡超微結構特征的細胞。 5.膜電位檢測結果示誘導分化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.隨著誘導反應時間延長,存活細胞數(shù)量逐漸減少,在此過程中細胞凋亡率明顯上升(P0.05)。 結論 1.誘導分化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.誘導分化神經(jīng)元存活時間短,隨誘導反應時間延長細胞凋亡率呈上升趨勢。
[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.
【學位授予單位】：河北聯(lián)合大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329.28
本文編號：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.
【學位授予單位】：河北聯(lián)合大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329.28
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