小鼠成纤维细胞制备保存及和胚体心肌细胞共培养研究
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摘要
小鼠胚体成纤维细胞的培养是制备饲养层的重要途径，其制备、传代及冷冻保存均有不同的研究报道，饲养层的质量受许多因素的影响，如动物的品种、培养液、所传代数及实验条件等，关于饲养层制备过程中的胚体细胞培养、传代、冷冻后的细胞形态、组织学等方面的研究报道很少，故本实验以昆明小白鼠为实验动物，研究其胚体培养过程中细胞的形态学、组织学等方面的变化，以期为本实验室建立饲养层细胞库提供理论依据，同时探讨心肌细胞和成纤维细胞层共培养的可行性，以期为心脏生物学特性的体外研究奠定基础。
选取妊娠11～16天的昆明系小白鼠胚胎为实验材料，通过对不同胚龄的小鼠胚胎成纤维细胞进行培养，以确定分离原代胚胎成纤维细胞的最适胚龄，并且通过对相同条件的小鼠胚胎成纤维细胞用几种不同的方法进行冷冻保存，从而确定冷冻保存小鼠胚胎成纤维细胞的最适方法，此外，将小鼠胚胎成纤维细胞与小鼠胚胎心肌组织及乳鼠心肌组织进行共培养，以期心肌组织能够与成纤维细胞层共培养且发生自律性收缩，并且通过电镜观察对小鼠胚胎成纤维细胞、小鼠胚胎心肌细胞以及与集团性心肌细胞相似但未收缩的细胞团进行结构比较。
研究结果表明：(1)用不同日龄小鼠胚胎(11～16d)分离培养成纤维细胞，其生长状况、铺层时间及寿命以13d、14d小鼠胚胎效果最佳。(2)体外培养的原代小鼠胚体成纤维细胞随培养时间的增加，核质比逐渐增加。(3)胚胎冷冻法可以使成纤维细胞取得较好的冷冻保存效果，解冻活率最高可达0.86。降温速率可以加快到0.60～1.0℃／min，平衡时间以2～2.5h为宜。(4)小鼠胚体成纤维细胞可以与心肌细胞团共培养，并出现自律性收缩，细胞团逐渐增长，其收缩情况随温度变化而变化，随时间变化其形态学发生变化，而且并非所有心肌细胞团都能收缩。
The culture of marine embryonic body fibroblast is an important approach to produced feeder layer. There are different report for its production, in vitro and cry preservation. The quality of feeder layer is affected by a lot of factors, such as animal breed, culture medium, passages in vitro and experiment condition, etc. As to the production of feeder layer, there are a few reports about morphological and histologic change when of embryonic body fibroblast when culturing in vitro and cryopreservation, so Kunming mouse were chosen as experimental animals and morphological and histologic changes were studied in course of its embryonic body culturing. We expect to offer theoretical foundation to our laboratory for setting up feeder layer storehouse. At the same time, the feasibility of myocardium tissue culturing with fibroblast layer altogether was studied so that established foundation for studied the biological characteristic of heart outside body.
In this experiment, we chose 11~16d pregnancy Kunming mouse as experimental animals, in order to verify the optimum pregnant period for producing embryonic body fibroblast cells. The several methods were used to refrigerate murine embryonic body fibroblast cells to make sure the optimum cryopreservation method. Also mouse embryonic body fibroblast cells were cultured with embryonic body myocardium tissue altogether in order to culture myocardium tissue outside body successfully. The histological structure were researched and compared on murine embryonic body fibroblast cells , murine embryonic body myocardium tissue and uncontracting myocardium cell group which was quite indentical to contracting one in morphology under electron microscope.
The results are showed as followed: (1) The embryo from mouse pregnant 13d, 14d could provide better results in growth states, time of spread layer and lifespan for making and production of murine embryonic body fibroblast cells in our laboratory conditions. (2) The ratio of nucleus to cytoplasm about fibroblast increased gradually with culturing time in the same generation. (3) Murine embryonic body fibroblast cells could be refrigerated well by method of embryonic freezing. The cells motility thawed was 0.86. The speed of decreasing temperature was 0.60~1.0 /min and the proper equilibrium duration was 2~2.5. (4) The myocardium cell group on embryonic body fibroblast feeder was cultured successfully. The myocardium tissue grew gradually, its contraction changed with temperature and morphology changed with time, and not all myocardium tissue could contract.
选取妊娠11～16天的昆明系小白鼠胚胎为实验材料，通过对不同胚龄的小鼠胚胎成纤维细胞进行培养，以确定分离原代胚胎成纤维细胞的最适胚龄，并且通过对相同条件的小鼠胚胎成纤维细胞用几种不同的方法进行冷冻保存，从而确定冷冻保存小鼠胚胎成纤维细胞的最适方法，此外，将小鼠胚胎成纤维细胞与小鼠胚胎心肌组织及乳鼠心肌组织进行共培养，以期心肌组织能够与成纤维细胞层共培养且发生自律性收缩，并且通过电镜观察对小鼠胚胎成纤维细胞、小鼠胚胎心肌细胞以及与集团性心肌细胞相似但未收缩的细胞团进行结构比较。
研究结果表明：(1)用不同日龄小鼠胚胎(11～16d)分离培养成纤维细胞，其生长状况、铺层时间及寿命以13d、14d小鼠胚胎效果最佳。(2)体外培养的原代小鼠胚体成纤维细胞随培养时间的增加，核质比逐渐增加。(3)胚胎冷冻法可以使成纤维细胞取得较好的冷冻保存效果，解冻活率最高可达0.86。降温速率可以加快到0.60～1.0℃／min，平衡时间以2～2.5h为宜。(4)小鼠胚体成纤维细胞可以与心肌细胞团共培养，并出现自律性收缩，细胞团逐渐增长，其收缩情况随温度变化而变化，随时间变化其形态学发生变化，而且并非所有心肌细胞团都能收缩。
The culture of marine embryonic body fibroblast is an important approach to produced feeder layer. There are different report for its production, in vitro and cry preservation. The quality of feeder layer is affected by a lot of factors, such as animal breed, culture medium, passages in vitro and experiment condition, etc. As to the production of feeder layer, there are a few reports about morphological and histologic change when of embryonic body fibroblast when culturing in vitro and cryopreservation, so Kunming mouse were chosen as experimental animals and morphological and histologic changes were studied in course of its embryonic body culturing. We expect to offer theoretical foundation to our laboratory for setting up feeder layer storehouse. At the same time, the feasibility of myocardium tissue culturing with fibroblast layer altogether was studied so that established foundation for studied the biological characteristic of heart outside body.
In this experiment, we chose 11~16d pregnancy Kunming mouse as experimental animals, in order to verify the optimum pregnant period for producing embryonic body fibroblast cells. The several methods were used to refrigerate murine embryonic body fibroblast cells to make sure the optimum cryopreservation method. Also mouse embryonic body fibroblast cells were cultured with embryonic body myocardium tissue altogether in order to culture myocardium tissue outside body successfully. The histological structure were researched and compared on murine embryonic body fibroblast cells , murine embryonic body myocardium tissue and uncontracting myocardium cell group which was quite indentical to contracting one in morphology under electron microscope.
The results are showed as followed: (1) The embryo from mouse pregnant 13d, 14d could provide better results in growth states, time of spread layer and lifespan for making and production of murine embryonic body fibroblast cells in our laboratory conditions. (2) The ratio of nucleus to cytoplasm about fibroblast increased gradually with culturing time in the same generation. (3) Murine embryonic body fibroblast cells could be refrigerated well by method of embryonic freezing. The cells motility thawed was 0.86. The speed of decreasing temperature was 0.60~1.0 /min and the proper equilibrium duration was 2~2.5. (4) The myocardium cell group on embryonic body fibroblast feeder was cultured successfully. The myocardium tissue grew gradually, its contraction changed with temperature and morphology changed with time, and not all myocardium tissue could contract.
引文
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