小鼠胎盘双核细胞的分离与培养

发布时间：2018-08-19 11:31

【摘要】：胎盘具有供给、代谢、排泄、分泌和免疫等功能，胎盘异常则会引起胎儿缺氧、窘迫、营养不良、发育迟缓、智力受损、甚至胎死腹中等现象。滋养层细胞中的双核细胞在胎盘形成与功能维持中的变化与胎盘异常的发生有密切联系。本研究以建立小鼠双核细胞的体外细胞培养模型为目的，为研究胎盘功能及妊娠相关疾病奠定基础。 1双核细胞的分离：本实验分别用序贯消化法、多次胰蛋白酶法、胰蛋白酶+胶原蛋白酶法、单次胰蛋白酶法和单次胶原蛋白酶法对胎盘组织进行消化，发现序贯消化法获得的单细胞活性最高为73.3%±1.8%，获得的细胞数为（0.65±0.10）×106个，胰蛋白酶＋胶原蛋白酶法获得的细胞活性次之，为69.8%±1.3%，二者间无显著性差异（P＞0.05)，但二者细胞活性均显著高于其他三组，分别为54.4%±0.7%、49.4%±1.4%、51.5%±1.5%，三者之间无显著性差异（P＞0.05)；将用序贯消化法获得的细胞悬液通过Percoll密度梯度离心法离心，获得的细胞用HE及台盼蓝染色法进行评估，双核细胞的纯度为45.5%±0.2%，细胞活性约94.3%±0.3%。结果表明，本实验所建立的方法可以用于小鼠胎盘双核细胞的分离。 2双核细胞的培养与纯化：本实验将Percoll密度梯度离心法获得的双核细胞随机分为2组，分别采用反复贴壁法和多次差别消化法进行纯化，，结果发现，传至第3代时，反复贴壁法获得的细胞纯度(74.5%±1.2%）略低于多次差别消化法（76.5%±2.3%），且反复贴壁法纯化双核细胞的时间为10d，显著少于多次差别消化法，需要12d。将纯化后的细胞分3组培养，发现添加谷胱甘肽组、50%成纤维细胞条件培养基+β巯基乙醇组和用胶原处理培养瓶组的双核细胞的活性分别为90.3%±0.5%、95.1%±0.2%和92.5%±0.4%，三者之间无显著性差异（P㧐0.05）。这些结果表明，原代培养物中的成纤维样细胞可以通过2或3次的反复贴壁法和多次差别消化法除去，本研究的培养条件可以用于双核细胞的体外培养。
[Abstract]:Placenta has the functions of supply, metabolism, excretion, secretion and immunity. Placental abnormalities can cause fetal hypoxia, distress, malnutrition, growth retardation, mental impairment, and even fetal death. The changes of the binuclear cells in trophoblastic cells during placental formation and maintenance are closely related to the occurrence of placental abnormalities. Objective To establish a cell culture model of mouse binuclear cells in vitro and lay a foundation for the study of placental function and pregnancy-related diseases.
1. Dinuclear cell isolation: In this experiment, the placenta tissues were digested by sequential digestion, multiple trypsin, trypsin + collagenase, single trypsin and single collagenase, respectively. It was found that the highest single cell activity was 73.3% + 1.8% and the number of cells obtained by sequential digestion was (0.65 +0.10)*106. The cell viability obtained by trypsin + collagenase method was 69.8% + 1.3%, and there was no significant difference between the two groups (P > 0.05), but the cell viability of the two groups was significantly higher than that of the other three groups, 54.4% + 0.7%, 49.4% + 1.4%, 51.5% + 1.5%, respectively. There was no significant difference between the three groups (P > 0.05); the cell suspension obtained by sequential digestion method was passed through the cell suspension. The purity of the binuclear cells was 45.5%+0.2% and the activity of the cells was 94.3%+0.3%. The results showed that the method could be used to isolate the binuclear cells from mouse placenta.
2. Culture and purification of Binuclear cells: Dinuclear cells obtained by Percoll density gradient centrifugation were randomly divided into two groups and purified by repeated adherence method and multiple differential digestion method. The results showed that the purity of cells obtained by repeated adherence method (74.5% + 1.2%) was slightly lower than that by multiple differential digestion method (76.5% + 2.3%). The purified cells were cultured in three groups. The results showed that the activities of the three groups were 90.3% + 0.5%, 95.1% + 0.2% and 90.3% + 0.5% respectively. The results showed that fibroblast-like cells in primary culture could be removed by two or three times of repeated adherence and multiple differential digestion. The culture conditions in this study could be used for the in vitro culture of Binuclear cells.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R321

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