【作者】 卢晓；

【导师】 芮荣；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2007， 硕士

【摘要】 饲养层(feeder layer)是特定的细胞(如成纤维细胞、输卵管上皮细胞、子宫上皮细胞、胎儿睾丸细胞等)经丝裂霉素-C阻断有丝分裂处理后制成的单层细胞。目前，大多数实验室均采用小鼠成纤维细胞(MEF和STO细胞)作为饲养层分离培养胚胎干细胞(ES)。饲养层的存在不仅为ES细胞的生长繁殖提供了一个稳定的外环境，同时也维持了ES细胞无限增殖与分化的特性。然而衰老的饲养层会对ES细胞产生负面影响，死亡的饲养层细胞会释放一些DNA碎片和溶酶体的酶进入培养液，干扰了ES细胞的生长和正常核型的维持。本研究利用3种不同的冷冻方法对小鼠胚胎成纤维细胞进行冻存，筛选出最佳的冷冻方案。并对小鼠胚胎成纤维细胞在连续传代过程中细胞活力以及细胞骨架所发生的变化进行追踪观察，确定制作饲养层的最佳时机。本试验首先以12.5d的昆明小鼠胚胎和STO细胞为材料，对MEF进行了分离培养。并通过3种冷冻方法对STO细胞和MEF细胞分别进行冷冻，即方法A为将细胞在冰箱中平衡1h后放入-20℃冰箱中过夜，次日转移至液氮中；方法B为将细胞迅速放入-70℃冰箱过夜，次日转移至液氮中；方法C为将细胞置于液氮面上5-10cm处薰蒸20min后缓缓降入液氮中。细胞冻融后再以存活率(台盼蓝染色实验)为主，辅以细胞相对活力测定(MTT法检测)和冷冻前后的细胞形态比较，对这3种方法的冷冻效果作以比较。以其为小鼠成纤维细胞的保存提供依据。然后对STO细胞和MEF细胞进行了连续培养，观察其形态学变化，采用MTT法检测其细胞活力，并绘制了细胞生长曲线；对不同代次的小鼠胚胎成纤维细胞进行免疫荧光染色，观察其细胞骨架的变化。结果表明：1．采用方法B冻存的细胞存活率明显高于方法A和方法C，同时不同冷冻方法对细胞生长形态、相对活力的影响无明显差异，并推断出该实验中方法B为饲养层细胞冷冻最佳方案。2．STO细胞在解冻后，最初几代生长较快，胞体成典型的成纤维细胞形态，随传代次数的增加，特别是9代以后，细胞逐渐排列不规则，胞内出现大量颗粒和空泡，死细胞增加。14代以后，部分细胞出现胞质溶解，胞体破碎等现象。MEF细胞在分离培养后，在最初几代生长缓慢，贴壁细胞较少；4代后生长迅速，代谢旺盛，细胞形状规则，到6代以后，细胞急剧衰亡，细胞出现解体，有成片的细胞漂浮或悬浮在培养液中。3．解冻后的STO细胞在第5代细胞活力达到最大值，第9代出现下降，传至14代又有升高．MEF分离培养后，细胞活力先出现下降，随后上升，第5代细胞活力达最大值，6代以后细胞活力急剧下降。4．经激光共聚焦显微术观察发现，STO细胞解冻后，其前5代的细胞内，微丝与微管蛋白大多呈束装沿细胞延展的方向平行排列，细胞核居中，常染色质丰富，无异常核分裂相。9代以后的STO细胞，微丝变得模糊，呈散乱、无序分布，微管不均匀的分布于胞浆内，细胞核型不完整，14代以后的STO细胞微丝微管排列更为紊乱，染色体异常加剧。原代的MEF细胞，微丝微管最为规则，核型也最正常，随着传代次数的增加，到第5代以后，微丝微管逐渐变得紊乱，出现解聚、断裂甚至消失，核型变得异常。更多还原

【Abstract】 The feeder layer cells means those special types of cells used for feeder layers, such as fibroblast, oviduct epithelia, uterus epithelia, embryonic testicle cells etc., they were treated with mitomycin C and then seeded for monolayer, which is called feeder layer. Currently, murine embryonic fibroblasts (including MEF and STO cells) were adopted for feeder layer cells to isolate embryonic stem (ES) cells in most related laboratories all over the world. Feeder cells could not only provide steady condition for ES cells, but also stimulate ES cell proliferation in vitro and keep its pluripotency. However, aged feeder cells would yield negative effects to ES cells. Dead feeder cells could release DNA fragments and enzymes into culture system, disturb the growth of ES cells and maintaining the normal type nucleus. In this experiment, three kinds of freezing method were compared for the cryopreservation of mouse fibroblasts. And changes of the post-thaw viability of the cells and cytoskeleton during continuous passages were observed and analysed.In this experiment, we isolate and culture MEF cells, then freeze STO cells and MEF cells by three kinds of method, including Method A as balancing cells in the refrigerator of 4℃for 1h before transferring cells into the refrigerator of -20℃overnight and then put them into the Liquid Nitrogen the next day. Method B as having cells in Nalgene Cryo-freezer cold down in a -70℃refrigerator overnight and transferring them into the Liquid Nitrogen the next day and Method C as steaming cells on the level of 5-10cm above the surface of Liquid Nitrogen for 20min and put them into the liquid slowly. After cells’ revivification, the freezing effects of three different approaches are compared mainly with livability, which is measured in Trypan Blue dyeing experiment, and the comparative cell viability, which is measured by MTT assay, and the compares between the cells’ morphological observations before freezing and after revivification are considered as additional targets. Then we culture mouse embyonic fibroblast continuously, Observing the change of morphology, measuring cell viability by MTT assay, drawing the cell growth curve, and using immunostainin to observe the change of cytoskeleton. The results shown as follows.1. The livability of cells freezed by Method B is remarkably higher than that freezed by Method A and Method C, meanwhile, there is no noticeable differences of cells’ morphological development and of the comparative cell viability among the three freezing approaches. And the further conclude is that the freezing approach B is confirmed to be the best scheme of freezing on the feeder layer cells in this experiment.2. STO cells grow rapidly after resuscitation, and present typical fibroblast morphology. As the times of passageing increasing, especially after the ninth passage, cells arrange is out of order, lots of grains and vacuoles are appeared, dead cells is increasing. After fourteenth passage, cytoplasm is dissolving and cell is crashing in part of the cells. MEF cells grow slowly in the first three passsages; after the forth passage, MEF cells grow rapidly, metabolism is vigorous, the shape of the cells is regular, after the sixth passage, the cells are decrepit rapidly, cells are broken, flaky cells are floating or suspending in cultural system.3. The cell viability of STO cells reach the most value in the fifth passage, then decline after the ninth passage, ascend in fourteenth passage. The cell viability of MEF cells reach the most value in the fifth passage, the decline after the sixth passage.4. With technology of CLSM, we observe the change of embryonic fibroblast cytoskeleton. in the first five passages after resuscitation, the tubulin and actin of STO ceils arranged regularly, nucleolus locate in the middle of the cells, the ordinary karyotin is abundant, and abnormal nucleus is absent, after the ninth passage, actin become in disorder, tubulin distribute asymmetrily in cytoplasm, the type of nucleolus is unintegrity, after the fourteenth passage, the actin and tubulin of STO cells are even more disorder, chromosome is even more abnormal. The arrangement of actin and tubulin of primary MEF cells are the most regularly, the type of nucleolus is normal, but after continuous passaging, especially after the fifth passage the actin and tubulin of MEF cells become turbulent, dissolved, ruptured, even disappeared, and the type of nucleus is abnormal.更多还原