發(fā)布時間：2018-01-15 17:20

  本文關鍵詞：MEF和hLEF對小鼠ES細胞支持作用的比較性研究　出處：《華中科技大學》2009年碩士論文　論文類型：學位論文

  更多相關文章： 絲裂霉素C MEF飼養(yǎng) 胚胎干細胞 MEF飼養(yǎng)層 胚胎干細胞 堿性磷酸酶 OCT-4 胚胎干細胞 絲裂霉素C濃度 絲裂霉素C 飼養(yǎng)層 胚胎干細胞 飼養(yǎng)層 胚胎干細胞 堿性磷酸酶 OCT4

【摘要】：第一部分MEF飼養(yǎng)層對小鼠胚胎干細胞（MES）的支持效果 實驗一小鼠成纖維細胞（MEF）的制備 目的制作原代小鼠成纖維細胞（MEF），為小鼠胚胎干細胞（MES）飼養(yǎng)層做準備。 方法將雌、雄昆明小鼠2：1合籠，取妊娠12~18d孕鼠，無菌取出胎鼠。去除胎鼠頭尾四肢內臟，PBS清洗數(shù)次后，剪碎組織，胰蛋白酶消化細胞，進行原代細胞培養(yǎng)，，并按常規(guī)方法傳代、凍存。 結果前三代小鼠成纖維細胞中雜細胞（非成纖維細胞）較多，細胞形態(tài)多樣。隨著傳代次數(shù)的增加，細胞形態(tài)逐漸一致,細胞連接成片，細胞之間連接緊密、界限不清。大部分細胞的胞質有2~3個突起，呈梭形，少數(shù)為圓形或不規(guī)則性。細胞胞體中間部分即細胞核所在的部位稍稍隆起，細胞輪廓清晰、立體感較強。細胞質內顆粒很少，細胞較為透明。 實驗二MEF飼養(yǎng)層的制備 目的制備MEF飼養(yǎng)層，為小鼠ES的培養(yǎng)做準備。 方法取對數(shù)期生長期的MEF，用終濃度10μg/ml絲裂霉素C處理2h。PBS充分洗滌五次。消化細胞調整濃度為0.5x106個/ml,均勻種植到經過0.1%明膠包被的6孔板里。 結果MEF飼養(yǎng)層細胞經過絲裂霉素C處理以后,種植到6孔板，2~3d后細胞融合成片，連接緊密單層生長。倒置顯微鏡下觀察，與藥物作用之前相比，MEF細胞數(shù)量略有減少，細胞形態(tài)沒有發(fā)生明顯的變化，細胞大多呈梭形，大小一致，排列規(guī)則、邊界清楚。 選擇適當?shù)慕z裂霉素C濃度和作用時間對飼養(yǎng)層的制作很關鍵。用濃度為10μg/ml的絲裂霉素C對MEF處理2h，飼養(yǎng)層細胞不再增殖同時保持良好的生存狀態(tài)。MEF細胞經過絲裂霉素C處理以后，1~2w左右會衰退死亡。 實驗三小鼠ES細胞在MEF飼養(yǎng)層上的生長特征 目的觀察小鼠胚胎干細胞（ES）在MEF飼養(yǎng)層上的生長情況。 方法選取的絲裂霉素C處理過的原代小鼠成纖維細胞（MEF）飼養(yǎng)層細胞，吸棄飼養(yǎng)層培養(yǎng)液，換用專用的小鼠胚胎干細胞（ES細胞）培養(yǎng)液，取ES細胞5x105個，均勻種植到飼養(yǎng)層細胞的表面。 結果胚胎干細胞（ES）接種到MEF飼養(yǎng)層兩天后細胞聚集生長形成ES克隆�？寺≈饾u增大，隆起生長，呈島嶼狀�？寺∵吘壡宄Y構致密，與周圍的飼養(yǎng)層細胞界限清楚。 實驗四MEF飼養(yǎng)層上的ES細胞分化情況的觀察 目的觀察胚胎干細胞在飼養(yǎng)層上分化情況。 方法 1.堿性磷酸酶(AKP)染色將兩種飼養(yǎng)層上的胚胎干細胞用PBS洗三次，用4%的多聚甲醛溶液室溫下固定10min，再用PBS洗三次，加入NBT/BCIP作用20-30min，倒置顯微鏡下觀察顯色情況，適時流水中止反應。 2. OCT-4檢測采用免疫熒光法染色技術。室溫下用正常驢血清（用PBS按1：10稀釋）封閉ES上的非特異特異性抗體。加入OCT-4抗體（用PBS按1：500稀釋）于4°°°C CC孵育過夜。PBS多次洗滌以后，加入熒光標記的驢抗兔二抗于室溫下孵育1h,PBS多次洗滌后，50%甘油封片。 結果免疫熒光法檢測ES細胞上OCT4的表達，MEF飼養(yǎng)層上的ES細胞克隆發(fā)出綠色熒光。MEF飼養(yǎng)層上的ES細胞克隆堿性磷酸酶(AKP)染色呈紫藍色。實驗結果表明MEF飼養(yǎng)層上的ES細胞仍處于未分化狀態(tài)，MEF飼養(yǎng)層可以維持胚胎干細胞的多分化潛能，保持其未分化狀態(tài)。 第二部分人胎肺成纖維細胞（hLEF）飼養(yǎng)層對小鼠胚胎干細胞（ES）的支持效果 實驗一hLEF的絲裂霉素最佳濃度的選擇 目的選擇合適的絲裂霉素濃度，為人胎肺成纖維細胞（hLEF）飼養(yǎng)層做準備。 方法消化培養(yǎng)細胞，調整細胞濃度，接種于96孔板中，37 oC、5%CO2培養(yǎng)培養(yǎng)24h。每孔加入MTT溶液（5mg/ml）100μL，繼續(xù)孵育4h，吸棄培養(yǎng)液，加入DMSO150μL，震蕩10min。利用酶標儀492nm波長下比色測定OD值。 結果不同濃度的絲裂霉素C，作用不同的時間，對人胎肺成纖維細胞（hLEF）產生的抑制細胞增殖的效果不同。隨著藥物作用時間的延長和藥物濃度的增加，藥物對hLEF的增殖抑制作用增強。用濃度為5μg /ml的絲裂霉素C作用1.5h就可以對hLEF產生一定的抑制作用但細胞仍然有明顯的增殖現(xiàn)象。用10μg/ml的絲裂霉素C處理細胞1.5~3.5h或者20μg /ml的絲裂霉素C處理細胞1.5h均可以保持hLEF細胞不再增殖也不會死亡。這是絲裂霉素C對人胎肺成纖維細胞（hLEF）的最佳處理濃度和作用時間范圍。當絲裂霉素C的濃度過低（10μg /ml）不能有效的抑制hLEF的增殖。而絲裂霉素C濃度過高（20μg/ml），同時作用的時間又過長（1.5h）則會引起hLEF細胞死亡。 實驗二hLEF飼養(yǎng)層的制備 目的制備hLEF飼養(yǎng)層,為小鼠ES的培養(yǎng)做準備。 方法取對數(shù)生長期hLEF，用終濃度10μg/ml絲裂霉素C處理2.5h。PBS充分洗滌五次。消化細胞調整濃度為0.5x106/ml,均勻種植到經過0.1%明膠包被的6孔板里。結果經絲裂霉素C處理過的hLEF細胞迅速融合成片，連接緊密單層生長。倒置顯微鏡下觀察細胞大多呈梭形，胞體透明、大小一致，排列規(guī)則、邊界清楚。選擇適當?shù)慕z裂霉素C的濃度和作用時間對飼養(yǎng)層的制備很關鍵。本實驗中選擇了濃度為10μg/ml的絲裂霉素C對hLEF的處理2.5h, hLEF飼養(yǎng)層細胞不再增殖同時保持良好的生存狀態(tài)。絲裂霉素C處理過的hLEF細胞大約可以存活2~3w。 實驗三小鼠ES細胞在hLEF飼養(yǎng)層上的生長特征 目的觀察小鼠胚胎干細胞（ES）在hLEF飼養(yǎng)層上的生長情況。 方法選取狀態(tài)良好的的胚胎干細胞種植到hLEF飼養(yǎng)層細胞，吸棄飼養(yǎng)層培養(yǎng)液，換用專用的小鼠胚胎干細胞（ES細胞）培養(yǎng)液，取ES細胞5x105個，均勻種植到飼養(yǎng)層細胞的表面。 結果胚胎干細胞（ES）接種到hLEF飼養(yǎng)層后第二天觀察細胞，ES克隆散在分布在飼養(yǎng)層細胞表面，ES克隆很小呈現(xiàn)一個個小圓點，凸起尚不明顯。2~3d后克隆逐漸增大，ES細胞克隆呈現(xiàn)集落狀（島嶼狀）隆起生長，邊緣清楚，結構致密，形成的克隆細胞之間的界限不明。同樣的培養(yǎng)條件下，培養(yǎng)同樣的天數(shù)，與MEF飼養(yǎng)層上的ES細胞克隆相比，hLEF飼養(yǎng)層上的ES細胞克隆生長較慢，克隆較小，不飽滿。 實驗四hLEF飼養(yǎng)層上的ES細胞分化情況的觀察 目的檢測hLEF飼養(yǎng)層是否可以維持ES細胞的未分化狀態(tài)。 方法 1.堿性磷酸酶(AKP)染色將兩種飼養(yǎng)層上的胚胎干細胞用PBS洗三次，用4%的多聚甲醛溶液室溫下固定10min，再用PBS洗三次，加入NBT/BCIP作用20-30min，倒置顯微鏡下觀察顯色情況，適時流水中止反應。 2. OCT4檢測采用免疫熒光法染色技術。室溫下用正常驢血清（用PBS按1：10稀釋）封閉ES上的非特異特異性抗體。加入OCT4抗體（用PBS按1：500稀釋）于4°°°C CC孵育過夜。PBS多次洗滌以后，加入熒光標記的驢抗兔二抗于室溫下孵育1h, PBS多次洗滌后，50%甘油封片。 結果hLEF飼養(yǎng)層上的胚胎干細胞OCT4檢測為陽性。熒光顯微鏡下觀察，ES細胞克隆發(fā)出綠色熒光。胚胎干細胞的AKP染色后，ES克隆被染成紫藍色。說明hLEF飼養(yǎng)層可以代替MEF飼養(yǎng)層維持ES細胞的生長狀態(tài)，保持胚胎干細胞處于未分化狀態(tài)。
[Abstract]:The support effect of MEF feeder layer on mouse embryonic stem cells (MES) in the first part
The preparation of experimental mouse fibroblast (MEF)
Objective to prepare the primary mouse fibroblast (MEF) and prepare the mouse embryonic stem cell (MES) feeder layer.
Methods the pregnant and 12~18d mice were taken from the male and female Kunming mice. The pregnant mice were taken out of the gestation. The fetuses were taken out of the fetal body. The fetal viscera and the viscera were removed. After PBS cleaning for several times, the tissues were cut, the cells were digested by trypsin, and the primary cells were cultured, and they were cryopreserved by routine methods. 2:1
The results of the first three generations of mice into fiber cells in complex cells (non fibroblast cells) more diverse forms. With the increase in the number of passages, the cells gradually, cells connected into cells connected closely, boundary is not clear. Most of the cells with cytoplasmic 2~3 bulges, spindle shaped, a few round or irregular. The cell body is the location of the middle part of the nucleus slightly uplift, clear cell outline, three-dimensional sense of strong granule in cytoplasm of cells. Rarely, more transparent.
Preparation of experiment two MEF feeder layer
Objective to prepare the MEF feeder layer for the cultivation of ES in mice.
Methods the logarithmic growth phase of the MEF, with a final concentration of 10 g/ml mitomycin C 2h.PBS fully washed five times. The digestive cell concentration was adjusted to 0.5x106 /ml, even after 0.1% planting to gelatin coated 6 well plates.
The results of MEF feeder cells after treatment with mitomycin C, implanted into 6 pore plate, 2~3d cell confluence, closely connected. Monolayer growth was observed under inverted microscope before action and drug compared to the number of MEF cells decreased slightly, cell morphology has not changed significantly, most of the cells were spindle shaped, uniform size, arrangement the rules, a clear boundary.
Select the appropriate time and concentration of mitomycin C on the function of feeder layer is critical. Mitomycin C was 10 g/ml with the concentration of MEF 2h, feeder cells no longer proliferation while maintaining good survival status of.MEF cells after mitomycin C treatment, 1~2w will decline to death.
The growth characteristics of experimental three mouse ES cells on the MEF feeder layer
Objective To observe the growth of mouse embryonic stem cells (ES) on the MEF feeder layer.
Methods the feeder cells from primary mouse fibroblast (MEF) treated by mitomycin C were removed, and the feeder layer culture fluid was removed, and the culture medium of mouse embryonic stem cells (ES cells) was replaced. 5x105 cells from ES cells were evenly distributed on the surface of feeder cells.
Results after embryonic stem cells (ES) were inoculated to MEF feeder layer two days later, cells grew and formed ES clones. The clones gradually increased, grew up and showed islands. The cloned edges were clear and dense, and the boundaries were clear with the surrounding feeder cells.
Observation on the differentiation of ES cells in Experiment four MEF feeder layer
Objective To observe the differentiation of embryonic stem cells on the feeder layer.
Method
1. alkaline phosphatase (AKP) staining was used to wash the embryonic stem cells from two feeder layers with PBS for three times, 4% 10min to immobilized 10min at room temperature, three times to wash with PBS, 20-30min was added to NBT/BCIP, the color was observed under inverted microscope, and the reaction was stopped at the right time.
2. OCT-4 were detected by immunofluorescence staining. At room temperature with normal Donkey Serum (PBS 1:10 dilution) blocking antibody specific non specific ES. Adding OCT-4 antibody (PBS 1:500 dilution) after 4 degrees CC degrees C degrees overnight incubation.PBS repeated washing, adding fluorescent labeled donkey anti two Rabbit anti 1H incubation at room temperature, PBS after repeated washing, 50% glycerol mounting.
Results the expression of ES on OCT4 cells was detected by immunofluorescence, ES cell clones MEF feeder layer on the green fluorescence on the.MEF feeder layer of ES cell clones of alkaline phosphatase (AKP) staining was purple blue. The experimental results show that the MEF feeder layer of ES cells is still in an undifferentiated state, MEF can maintain cell feeder layer pluripotent embryonic stem, maintaining their undifferentiated state.
The support effect of the second part of human fetal lung fibroblast (hLEF) feeder layer on mouse embryonic stem cells (ES)
The selection of the best concentration of mitomycin hLEF in Experiment 1
Objective to prepare the human fetal lung fibroblast (hLEF) feeder layer by selecting the proper mitomycin concentration.
Methods the digestive cells, adjust the cell concentration, inoculated in 96 well plates, 37 oC, 5%CO2 in cultured 24h. per hole adding MTT solution (5mg/ml) 100 L, incubate for 4h, suction abandon medium, adding DMSO150 L, 10min. 492nm wavelength calibration shock colorimetric determination of OD by enzyme.
Results different concentrations of mitomycin C, the effect of different time, of human fetal lung fibroblasts (hLEF) in different cell proliferation inhibition effect of the resulting increase with the prolongation of the time of drug action and drug concentration, enhance the inhibitory effect of drugs on the proliferation of hLEF. With the concentration of mitomycin C 1.5h 5 G /ml you can have a certain effect on hLEF cell proliferation but there are still obvious. Cells treated with 1.5~3.5h or 20 g /ml 10 g/ml mitomycin C mitomycin C treatment 1.5h cells can maintain hLEF cells no longer proliferation nor death. This is mitomycin C on human fetal lung fibroblasts (hLEF) the best treatment concentration and action time range. When the low concentration of mitomycin C (10 g /ml) can not effectively inhibit hLEF proliferation. But high concentration of mitomycin C (20 g/ml), while the role of the time too long (1.5h) causes death of hLEF cells.
Preparation of experiment two hLEF feeder layer
Objective to prepare the hLEF feeder layer for the cultivation of ES in mice.
Method of logarithmic growth phase hLEF, with a final concentration of 10 g/ml mitomycin C 2.5h.PBS fully washed five times. The digestive cell concentration was adjusted to 0.5x106/ml, even after planting to 0.1% gelatin coated 6 well plates. Results after mitomycin C treated hLEF cells rapidly confluent, closely connected monolayer growth. Most of the cells were spindle shape under inverted microscope, cell body transparent, uniform size, regular arrangement, clear boundary. Select the appropriate concentration and action time of mitomycin C feeder layer preparation is the key. This experiment chose 2.5h concentration of 10 g/ml mitomycin C on hLEF, hLEF feeder cells proliferation and no longer to maintain a good state of existence. Mitomycin C treated hLEF cells can survive for about 2~3w.
The growth characteristics of experimental three mouse ES cells on the hLEF feeder layer
Objective To observe the growth of mouse embryonic stem cells (ES) on the hLEF feeder layer.
Methods the well established embryonic stem cells were implanted into hLEF feeder layer cells, suck up the feeder layer culture medium, replace the culture medium of the mouse embryonic stem cells (ES cells), and take ES cells 5x105, evenly distributed on the surface of feeder cells.
The results of embryonic stem cells (ES) were inoculated into the hLEF feeder layer was observed second days after cells, ES clones were scattered in the feeder layer cell surface, ES cloning is very small a little dots, raised is not obvious after.2~3d clone gradually increased, ES cell clones showed colony like growth (Island) uplift, edge clearly, the compact structure, the boundaries between the formation of cloned cells is unknown. Under the same culture condition, the same culture days, compared with the MEF feeder layer on the ES cell clone, ES cell clone hLEF feeder layer on the cloning of slower growth, smaller, not full.
Observation on the differentiation of ES cells in Experiment four hLEF feeder layer
Objective to determine whether the hLEF feeder can maintain the undifferentiated state of ES cells.
Method
1. alkaline phosphatase (AKP) staining was used to wash the embryonic stem cells from two feeder layers with PBS for three times, 4% 10min to immobilized 10min at room temperature, three times to wash with PBS, 20-30min was added to NBT/BCIP, the color was observed under inverted microscope, and the reaction was stopped at the right time.
2. OCT4 were detected by immunofluorescence staining. At room temperature with normal Donkey Serum (PBS 1:10 dilution) blocking antibody specific non specific ES. Adding OCT4 antibody (PBS 1:500 dilution) after 4 degrees CC degrees C degrees overnight incubation.PBS repeated washing, adding fluorescent labeled donkey anti two Rabbit anti 1H incubation at room temperature, PBS after repeated washing, 50% glycerol mounting.
The hLEF feeder layer of embryonic stem cell OCT4 detection was positive. Fluorescence microscopy, ES cells emitted green fluorescence. Embryonic stem cells after AKP staining, ES clones were stained purple blue. The growth state of hLEF feeder layer can replace the MEF feeder layer to maintain ES cells, maintain embryonic stem cells in undifferentiated state.

【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

【參考文獻】

相關期刊論文 前7條

1 鄒雨汐;鄒飛;姜曉丹;;胚胎干細胞在神經移植治療中的研究進展[J];中華神經醫(yī)學雜志;2006年10期

2 曹娟,徐海偉,唐軍,范曉棠,吳旋;幾種培養(yǎng)方法對ES/GFP小鼠胚胎干細胞系增殖和分化能力作用比較[J];第三軍醫(yī)大學學報;2004年19期

3 呂魏,李堅;胚胎干細胞的培養(yǎng)特性[J];中國計劃生育學雜志;2002年02期

4 盛利琴;葉榮;陳學進;韓甫;張煥相;;飼養(yǎng)層體系的建立與胚胎干細胞的培養(yǎng)[J];解剖學雜志;2007年03期

5 熊吉信;劉小春;楊春江;劉兆軒;;小鼠胚胎干細胞飼養(yǎng)層的制備及兩種不同飼養(yǎng)層的比較[J];江西醫(yī)學院學報;2006年04期

6 高巍;劉福強;梅晰凡;劉暢;任甫;;昆明種小鼠胚胎干細胞的培養(yǎng)及鑒定[J];遼寧醫(yī)學院學報;2008年01期

7 王璐;王亞平;;小鼠胚胎干細胞飼養(yǎng)層制備條件的研究[J];重慶醫(yī)科大學學報;2008年01期

本文編號：1429318