MEF和hLEF对小鼠ES细胞支持作用的比较性研究
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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。
part1 Support Eeffect of MEF as Feeder Layer toMouse Embryonic Stem Cells
Experiment one Preparation of Mouse Embryonic Fibroblasts
Objective Prepare Mouse Embryonic Fibroblast for culture of mouse embryonic stem cell.
Methods Primary mouse embryonic fibroblasts were isolated from 12~18days old fetusmouse. Cut the tissues into small pieces using dissecting scissors. Use trypsin to digestive thetissue. Incubate cells in a 37°C incubator supplied with 5% CO2.
Results Mouse embryonic fibroblast cells have many non-fibroblast cells before first 3generation. In vitro,It is adherent cell with good ability of proliferation affer passage 3.With theincreasing of the cell number, cell line gradually form. Most of cells look like a spindle, asmall number of cells are round or irregular, the cells are transparent. The middle part of cellsis a little uplift, a strong sense of three-dimensional. With the number of cells increasing, cellsis connected closely.
Experiment two Preparation of MEF feeder layer
Objective Preparation of MEF feeder layer for mouse embryonic stem cells.
Methods MitomycinC 10μg/ml treat MEF for 2h. PBS wash MEF for several times. plate thecells into wells of 6-well plates pre-coated with 0.1% Gelatin Solution.
Results Observed under inverted microscope ,the morphology of cells were no significantchanges. Most of the cells were spindle-shaped with clear boundaries.
Chose suitable concentration of mytomycin C and the time of treating cells is veryimportant..In our experiment we treat MEF using MitomycinC 10μg/ml for 2h.It is a good way to repress proliferation of ES cells. But the MEF treated by mytomycin C will die in 1~2weeks.
Experiment three The State of Es clones growing onMEF feeder layer
Objective Observe the state of Es clones growing on MEF of feeder layer.
Methods Chose suitable MEF feeder layer ,Change the medium into special mouseembryonic stem cells growth medium. Plate ES cell suspension (5x105)into MEF feeder layer.
Results ES is planted on MEF feeder layer ,2 days later, we can see the ES clones.The ESclones look like islands, the edge of ES clone is clear and smooth.There is clear boundarybetween ES clones and feeder layer.
Experiment four Observation the State of differentiation ofES cell
Objective Detect MEF feeder layers whether can maintain ES cells undiferentiation.
Methods (1) Detect Alkaline phosphatase (AKP) activity.
Put 4% Paraformalclehyde(PFA) into ES clones on two kinds of feeder layers for 10min. PBS wash several times. Then put NBT/BCIP into ES clones. Observe the Alkalinephosphatase (AKP) activity of ES cells .Use water to stop the reaction .
(2) Detect the expressioon of OCT-4 by Immunofluorescence.
After washing with phosphate buffered-saline PBS,the cells were permeabilized with 2%Triton X-100, blocked for 10 minutes with PBS containing 10% donkey serum albumin,thenincubated with primary rabbit monoclonal anti-mouse Oct-4 antibody (1:500) for a night at4°C. Negative control was performed with FITC-conjugated mouse anti-rabbit IgGsecondary antibodies.PBS wash three times.
Results ES clones on MEF feeder layers have strong AKP activity. It can express Oct4-greenfluorescent protein which can be observed by fluorescence microscopy. The experiment showed that MEF feeder layer can retaining the initial totipotency of ES cell.
part2 Support Effect of hLEF as Feeder Layer to MouseEmbryonic Stem Cells
Experiment one Chose Optional Concentration of Mytomycin C
Objective Chose optional concentration of mytomycin C for human embryonic lungfibroblats .
Methods Plate the MEF cell suspension into wells of 6-well plates pre-coated with GelatinSolution. After 24 h, put MTT solution（5mg/ml）100μL into every well of 6-wellplates.Incubate cells in a 37°C incubator for 4h. Put DMSO 150μL ,shake the 6-well plates for10 min.Than use ZS-3 Microplate Reader to obtain the optical density.
Results Using different concentration of mytomycin C and the time of treating hLEF,thestate of hLEF cells is different. Using MitomycinC 10~20μg/ml for 1.5~3.5h and MitomycinC20μg/ml for 1.5h can effeciently repress the proliferation of hLEF. Low concentration ofmytomycin C（<10μg /ml）can repress proliferation of hLEF effectively but high concentrationof mytomycin C（>20μg /ml）especially in long time（>1.5h）can kill the hLEF.
Experiment two Preparation of hLEF Feeder Layer
Objective Preparation of hLEF feeder layer for mouse embrynic stem cells
Methods MitomycinC 10μg/ml treat hLEF for 2.5h. PBS wash several times, plate the cellsinto wells of 6-well platespre-coated with 0.1% Gelatin Solution.
Results After treated by mytomycin C, hLEF were planted into wells of 6-wellplatespre-coated with 0.1% Gelatin Solution.The cells integrated into hLEF single-layer rapidly.Observed by Inverted microscope, the morphology of MEF were no significant changes. Mostof the cells were spindle-shaped with clear boundaries.
Select the appropriate concentration of mitomycin C and time is crucial for preparation ofhLEF feeder layer. In this experiment we used 10μg/ml of mitomycin C to treat hLEF for 2.5h, hLEF can keep alive for 2~3W. It is a good ways to repress proliferation.
Experiment three The State of Es Clones Growing on hLEFof Feeder Layer
Objective Observe the state of Es clones growing on hLEF of feeder layer.
Methods Using the same way as plating Es cell on MEF feeder layers.
Results ES is planted on two kinds of feeder layer ,the ES clone is round and small. 2~3dayslater, we can see the ES clones.There is no obviousely distinction of cell morphologycharacteristics and growth behaviors between ES cells on hLEF feeder layers and ES cells onMEF feeder layers .The ES clones look like islands, the edge of ES is clear and smooth.Thereis no clear boundary between ES cells.
Under the same culture condition ,ES clones on the MEF feeder layers is biger than ESclones on the hLEF feeder layers. ES cell on the hLEF feeder layers grow slower tnan ES cellon the MEF feeder layers.
Experiment four Observation of the State of Differentiationof ES cell
Objective Detect whether hLEF feeder layers and MEF feeder layers can maintain ES cellsundiferentiation state.
Methods (1) Detect Alkaline phosphatase (AKP) activity.As mentioned above.
(2) Detect the expressioon of OCT-4 by Immunofluorescence.As mentioned above.
Results ES clones on hLEF have the strong AKP activity. It can express Oct4-greenfluorescent protein which can be observed by fluorescence microscopy. The experimentshowed that hLEF feeder layer can retaining the initial totipotency of ES cell.
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。
part1 Support Eeffect of MEF as Feeder Layer toMouse Embryonic Stem Cells
Experiment one Preparation of Mouse Embryonic Fibroblasts
Objective Prepare Mouse Embryonic Fibroblast for culture of mouse embryonic stem cell.
Methods Primary mouse embryonic fibroblasts were isolated from 12~18days old fetusmouse. Cut the tissues into small pieces using dissecting scissors. Use trypsin to digestive thetissue. Incubate cells in a 37°C incubator supplied with 5% CO2.
Results Mouse embryonic fibroblast cells have many non-fibroblast cells before first 3generation. In vitro,It is adherent cell with good ability of proliferation affer passage 3.With theincreasing of the cell number, cell line gradually form. Most of cells look like a spindle, asmall number of cells are round or irregular, the cells are transparent. The middle part of cellsis a little uplift, a strong sense of three-dimensional. With the number of cells increasing, cellsis connected closely.
Experiment two Preparation of MEF feeder layer
Objective Preparation of MEF feeder layer for mouse embryonic stem cells.
Methods MitomycinC 10μg/ml treat MEF for 2h. PBS wash MEF for several times. plate thecells into wells of 6-well plates pre-coated with 0.1% Gelatin Solution.
Results Observed under inverted microscope ,the morphology of cells were no significantchanges. Most of the cells were spindle-shaped with clear boundaries.
Chose suitable concentration of mytomycin C and the time of treating cells is veryimportant..In our experiment we treat MEF using MitomycinC 10μg/ml for 2h.It is a good way to repress proliferation of ES cells. But the MEF treated by mytomycin C will die in 1~2weeks.
Experiment three The State of Es clones growing onMEF feeder layer
Objective Observe the state of Es clones growing on MEF of feeder layer.
Methods Chose suitable MEF feeder layer ,Change the medium into special mouseembryonic stem cells growth medium. Plate ES cell suspension (5x105)into MEF feeder layer.
Results ES is planted on MEF feeder layer ,2 days later, we can see the ES clones.The ESclones look like islands, the edge of ES clone is clear and smooth.There is clear boundarybetween ES clones and feeder layer.
Experiment four Observation the State of differentiation ofES cell
Objective Detect MEF feeder layers whether can maintain ES cells undiferentiation.
Methods (1) Detect Alkaline phosphatase (AKP) activity.
Put 4% Paraformalclehyde(PFA) into ES clones on two kinds of feeder layers for 10min. PBS wash several times. Then put NBT/BCIP into ES clones. Observe the Alkalinephosphatase (AKP) activity of ES cells .Use water to stop the reaction .
(2) Detect the expressioon of OCT-4 by Immunofluorescence.
After washing with phosphate buffered-saline PBS,the cells were permeabilized with 2%Triton X-100, blocked for 10 minutes with PBS containing 10% donkey serum albumin,thenincubated with primary rabbit monoclonal anti-mouse Oct-4 antibody (1:500) for a night at4°C. Negative control was performed with FITC-conjugated mouse anti-rabbit IgGsecondary antibodies.PBS wash three times.
Results ES clones on MEF feeder layers have strong AKP activity. It can express Oct4-greenfluorescent protein which can be observed by fluorescence microscopy. The experiment showed that MEF feeder layer can retaining the initial totipotency of ES cell.
part2 Support Effect of hLEF as Feeder Layer to MouseEmbryonic Stem Cells
Experiment one Chose Optional Concentration of Mytomycin C
Objective Chose optional concentration of mytomycin C for human embryonic lungfibroblats .
Methods Plate the MEF cell suspension into wells of 6-well plates pre-coated with GelatinSolution. After 24 h, put MTT solution（5mg/ml）100μL into every well of 6-wellplates.Incubate cells in a 37°C incubator for 4h. Put DMSO 150μL ,shake the 6-well plates for10 min.Than use ZS-3 Microplate Reader to obtain the optical density.
Results Using different concentration of mytomycin C and the time of treating hLEF,thestate of hLEF cells is different. Using MitomycinC 10~20μg/ml for 1.5~3.5h and MitomycinC20μg/ml for 1.5h can effeciently repress the proliferation of hLEF. Low concentration ofmytomycin C（<10μg /ml）can repress proliferation of hLEF effectively but high concentrationof mytomycin C（>20μg /ml）especially in long time（>1.5h）can kill the hLEF.
Experiment two Preparation of hLEF Feeder Layer
Objective Preparation of hLEF feeder layer for mouse embrynic stem cells
Methods MitomycinC 10μg/ml treat hLEF for 2.5h. PBS wash several times, plate the cellsinto wells of 6-well platespre-coated with 0.1% Gelatin Solution.
Results After treated by mytomycin C, hLEF were planted into wells of 6-wellplatespre-coated with 0.1% Gelatin Solution.The cells integrated into hLEF single-layer rapidly.Observed by Inverted microscope, the morphology of MEF were no significant changes. Mostof the cells were spindle-shaped with clear boundaries.
Select the appropriate concentration of mitomycin C and time is crucial for preparation ofhLEF feeder layer. In this experiment we used 10μg/ml of mitomycin C to treat hLEF for 2.5h, hLEF can keep alive for 2~3W. It is a good ways to repress proliferation.
Experiment three The State of Es Clones Growing on hLEFof Feeder Layer
Objective Observe the state of Es clones growing on hLEF of feeder layer.
Methods Using the same way as plating Es cell on MEF feeder layers.
Results ES is planted on two kinds of feeder layer ,the ES clone is round and small. 2~3dayslater, we can see the ES clones.There is no obviousely distinction of cell morphologycharacteristics and growth behaviors between ES cells on hLEF feeder layers and ES cells onMEF feeder layers .The ES clones look like islands, the edge of ES is clear and smooth.Thereis no clear boundary between ES cells.
Under the same culture condition ,ES clones on the MEF feeder layers is biger than ESclones on the hLEF feeder layers. ES cell on the hLEF feeder layers grow slower tnan ES cellon the MEF feeder layers.
Experiment four Observation of the State of Differentiationof ES cell
Objective Detect whether hLEF feeder layers and MEF feeder layers can maintain ES cellsundiferentiation state.
Methods (1) Detect Alkaline phosphatase (AKP) activity.As mentioned above.
(2) Detect the expressioon of OCT-4 by Immunofluorescence.As mentioned above.
Results ES clones on hLEF have the strong AKP activity. It can express Oct4-greenfluorescent protein which can be observed by fluorescence microscopy. The experimentshowed that hLEF feeder layer can retaining the initial totipotency of ES cell.
引文
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3. Joseph M. Metzger, Wan-In Lin, Ross A. Johnston, et at.Myosin Heavy Chain Expre- ssionin Contracting Myocytes Isolated During Embryonic Stem Cell Cardiogenesis. Circ. Res.,May 1995; 76: 710 - 719.
4. Liu xiong-ying ,jiang ning-yi ,et al Directed differentiation of embryonic stem cell towardsthyroid cell in vitro. Journal of Clinical Rahabilitative Tissue Engineering Research2008,12(17) 9389~9392.
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6.邹雨汐，邹飞，姜晓丹。胚胎干细胞在神经移植治疗中的研究进展。中华神经医学杂志，2006，5（10），1068~1070.
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8. Yusuke Nishimura, Tatsuo S. Hamazaki, Shinji Komazaki, et al.Ciliated CellsDifferentiated from Mouse Embryonic Stem Cells Stem Cells, May 2006; 24: 1381–1388
9.薛庆善肖渝平、林娟等体外培养的技术和原理2001，515~517.
10. Pease S.Isolation of Embryonic Stem Cell in Medium Supplemented with RecombinantLeukemia inhibitory Factor(LIF)[J].Dev Biol,1990,78(9):141-144.
11. ChoudhuryA ,J anesL , GajewskiJ C , et al. Use of leukemic dendritic cells for thegeneration of antileukemic cellular cytotoxicity against philadephia chromosome positivechronic myelogenous leukemia [J]. Blood ,1997 ,89 (4):1133.
12. NicholsJ,Evans EP,Smith AG. Establishment of germ-line-competent embryonic stem (ES)cells using differentiation inhibiting activity. Development, Dec 1990; 110: 1341 - 1348.
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14.曹娟，徐海尾，唐军等几种培养方法对ES/GFP胞系增殖和分化能力作用比较第三军医大学学报2004，26（19），1775.
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6.邹雨汐，邹飞，姜晓丹。胚胎干细胞在神经移植治疗中的研究进展。中华神经医学杂志，2006，5（10），1068~1070.
7. PaiaciosR,Golunski E，SamaridisJ. In vitro Generation of hematopoietic stem cells from anembryonic stem cell line.Pro Natl Acad Sci USA，1995，92(I6)7530—7534.
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9.薛庆善肖渝平、林娟等体外培养的技术和原理2001，515~517.
10. Pease S.Isolation of Embryonic Stem Cell in Medium Supplemented with RecombinantLeukemia inhibitory Factor(LIF)[J].Dev Biol,1990,78(9):141-144.
11. ChoudhuryA ,J anesL , GajewskiJ C , et al. Use of leukemic dendritic cells for thegeneration of antileukemic cellular cytotoxicity against philadephia chromosome positivechronic myelogenous leukemia [J]. Blood ,1997 ,89 (4):1133.
12. NicholsJ,Evans EP,Smith AG. Establishment of germ-line-competent embryonic stem (ES)cells using differentiation inhibiting activity. Development, Dec 1990; 110: 1341 - 1348.
13. Lim J W, Bodnar A Proteome 1analysis of conditioned medium from mouse embryonicfibroblast feeder layers which support the growth of human embryonic stem cells.Proteomics, 2002 , 2:1187
14.曹娟，徐海尾，唐军等几种培养方法对ES/GFP胞系增殖和分化能力作用比较第三军医大学学报2004，26（19），1775.
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