MEF和hLEF对小鼠ES细胞支持作用的比较性研究

发布时间：2018-01-15 17:20

本文关键词：MEF和hLEF对小鼠ES细胞支持作用的比较性研究　出处：《华中科技大学》2009年硕士论文　论文类型：学位论文

【摘要】：第一部分MEF饲养层对小鼠胚胎干细胞（MES）的支持效果实验一小鼠成纤维细胞（MEF）的制备目的制作原代小鼠成纤维细胞（MEF），为小鼠胚胎干细胞（MES）饲养层做准备。方法将雌、雄昆明小鼠2：1合笼，取妊娠12~18d孕鼠，无菌取出胎鼠。去除胎鼠头尾四肢内脏，PBS清洗数次后，剪碎组织，胰蛋白酶消化细胞，进行原代细胞培养，，并按常规方法传代、冻存。结果前三代小鼠成纤维细胞中杂细胞（非成纤维细胞）较多，细胞形态多样。随着传代次数的增加，细胞形态逐渐一致,细胞连接成片，细胞之间连接紧密、界限不清。大部分细胞的胞质有2~3个突起，呈梭形，少数为圆形或不规则性。细胞胞体中间部分即细胞核所在的部位稍稍隆起，细胞轮廓清晰、立体感较强。细胞质内颗粒很少，细胞较为透明。实验二MEF饲养层的制备目的制备MEF饲养层，为小鼠ES的培养做准备。方法取对数期生长期的MEF，用终浓度10μg/ml丝裂霉素C处理2h。PBS充分洗涤五次。消化细胞调整浓度为0.5x106个/ml,均匀种植到经过0.1%明胶包被的6孔板里。结果MEF饲养层细胞经过丝裂霉素C处理以后,种植到6孔板，2~3d后细胞融合成片，连接紧密单层生长。倒置显微镜下观察，与药物作用之前相比，MEF细胞数量略有减少，细胞形态没有发生明显的变化，细胞大多呈梭形，大小一致，排列规则、边界清楚。选择适当的丝裂霉素C浓度和作用时间对饲养层的制作很关键。用浓度为10μg/ml的丝裂霉素C对MEF处理2h，饲养层细胞不再增殖同时保持良好的生存状态。MEF细胞经过丝裂霉素C处理以后，1~2w左右会衰退死亡。实验三小鼠ES细胞在MEF饲养层上的生长特征目的观察小鼠胚胎干细胞（ES）在MEF饲养层上的生长情况。方法选取的丝裂霉素C处理过的原代小鼠成纤维细胞（MEF）饲养层细胞，吸弃饲养层培养液，换用专用的小鼠胚胎干细胞（ES细胞）培养液，取ES细胞5x105个，均匀种植到饲养层细胞的表面。结果胚胎干细胞（ES）接种到MEF饲养层两天后细胞聚集生长形成ES克隆。克隆逐渐增大，隆起生长，呈岛屿状。克隆边缘清楚，结构致密，与周围的饲养层细胞界限清楚。实验四MEF饲养层上的ES细胞分化情况的观察目的观察胚胎干细胞在饲养层上分化情况。方法 1.碱性磷酸酶(AKP)染色将两种饲养层上的胚胎干细胞用PBS洗三次，用4%的多聚甲醛溶液室温下固定10min，再用PBS洗三次，加入NBT/BCIP作用20-30min，倒置显微镜下观察显色情况，适时流水中止反应。 2. OCT-4检测采用免疫荧光法染色技术。室温下用正常驴血清（用PBS按1：10稀释）封闭ES上的非特异特异性抗体。加入OCT-4抗体（用PBS按1：500稀释）于4°°°C CC孵育过夜。PBS多次洗涤以后，加入荧光标记的驴抗兔二抗于室温下孵育1h,PBS多次洗涤后，50%甘油封片。结果免疫荧光法检测ES细胞上OCT4的表达，MEF饲养层上的ES细胞克隆发出绿色荧光。MEF饲养层上的ES细胞克隆碱性磷酸酶(AKP)染色呈紫蓝色。实验结果表明MEF饲养层上的ES细胞仍处于未分化状态，MEF饲养层可以维持胚胎干细胞的多分化潜能，保持其未分化状态。第二部分人胎肺成纤维细胞（hLEF）饲养层对小鼠胚胎干细胞（ES）的支持效果实验一hLEF的丝裂霉素最佳浓度的选择目的选择合适的丝裂霉素浓度，为人胎肺成纤维细胞（hLEF）饲养层做准备。方法消化培养细胞，调整细胞浓度，接种于96孔板中，37 oC、5%CO2培养培养24h。每孔加入MTT溶液（5mg/ml）100μL，继续孵育4h，吸弃培养液，加入DMSO150μL，震荡10min。利用酶标仪492nm波长下比色测定OD值。结果不同浓度的丝裂霉素C，作用不同的时间，对人胎肺成纤维细胞（hLEF）产生的抑制细胞增殖的效果不同。随着药物作用时间的延长和药物浓度的增加，药物对hLEF的增殖抑制作用增强。用浓度为5μg /ml的丝裂霉素C作用1.5h就可以对hLEF产生一定的抑制作用但细胞仍然有明显的增殖现象。用10μg/ml的丝裂霉素C处理细胞1.5~3.5h或者20μg /ml的丝裂霉素C处理细胞1.5h均可以保持hLEF细胞不再增殖也不会死亡。这是丝裂霉素C对人胎肺成纤维细胞（hLEF）的最佳处理浓度和作用时间范围。当丝裂霉素C的浓度过低（10μg /ml）不能有效的抑制hLEF的增殖。而丝裂霉素C浓度过高（20μg/ml），同时作用的时间又过长（1.5h）则会引起hLEF细胞死亡。实验二hLEF饲养层的制备目的制备hLEF饲养层,为小鼠ES的培养做准备。方法取对数生长期hLEF，用终浓度10μg/ml丝裂霉素C处理2.5h。PBS充分洗涤五次。消化细胞调整浓度为0.5x106/ml,均匀种植到经过0.1%明胶包被的6孔板里。结果经丝裂霉素C处理过的hLEF细胞迅速融合成片，连接紧密单层生长。倒置显微镜下观察细胞大多呈梭形，胞体透明、大小一致，排列规则、边界清楚。选择适当的丝裂霉素C的浓度和作用时间对饲养层的制备很关键。本实验中选择了浓度为10μg/ml的丝裂霉素C对hLEF的处理2.5h, hLEF饲养层细胞不再增殖同时保持良好的生存状态。丝裂霉素C处理过的hLEF细胞大约可以存活2~3w。实验三小鼠ES细胞在hLEF饲养层上的生长特征目的观察小鼠胚胎干细胞（ES）在hLEF饲养层上的生长情况。方法选取状态良好的的胚胎干细胞种植到hLEF饲养层细胞，吸弃饲养层培养液，换用专用的小鼠胚胎干细胞（ES细胞）培养液，取ES细胞5x105个，均匀种植到饲养层细胞的表面。结果胚胎干细胞（ES）接种到hLEF饲养层后第二天观察细胞，ES克隆散在分布在饲养层细胞表面，ES克隆很小呈现一个个小圆点，凸起尚不明显。2~3d后克隆逐渐增大，ES细胞克隆呈现集落状（岛屿状）隆起生长，边缘清楚，结构致密，形成的克隆细胞之间的界限不明。同样的培养条件下，培养同样的天数，与MEF饲养层上的ES细胞克隆相比，hLEF饲养层上的ES细胞克隆生长较慢，克隆较小，不饱满。实验四hLEF饲养层上的ES细胞分化情况的观察目的检测hLEF饲养层是否可以维持ES细胞的未分化状态。方法 1.碱性磷酸酶(AKP)染色将两种饲养层上的胚胎干细胞用PBS洗三次，用4%的多聚甲醛溶液室温下固定10min，再用PBS洗三次，加入NBT/BCIP作用20-30min，倒置显微镜下观察显色情况，适时流水中止反应。 2. OCT4检测采用免疫荧光法染色技术。室温下用正常驴血清（用PBS按1：10稀释）封闭ES上的非特异特异性抗体。加入OCT4抗体（用PBS按1：500稀释）于4°°°C CC孵育过夜。PBS多次洗涤以后，加入荧光标记的驴抗兔二抗于室温下孵育1h, PBS多次洗涤后，50%甘油封片。结果hLEF饲养层上的胚胎干细胞OCT4检测为阳性。荧光显微镜下观察，ES细胞克隆发出绿色荧光。胚胎干细胞的AKP染色后，ES克隆被染成紫蓝色。说明hLEF饲养层可以代替MEF饲养层维持ES细胞的生长状态，保持胚胎干细胞处于未分化状态。
[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.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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