【摘要】：胎盤具有供給、代謝、排泄、分泌和免疫等功能，胎盤異常則會(huì)引起胎兒缺氧、窘迫、營(yíng)養(yǎng)不良、發(fā)育遲緩、智力受損、甚至胎死腹中等現(xiàn)象。滋養(yǎng)層細(xì)胞中的雙核細(xì)胞在胎盤形成與功能維持中的變化與胎盤異常的發(fā)生有密切聯(lián)系。本研究以建立小鼠雙核細(xì)胞的體外細(xì)胞培養(yǎng)模型為目的，為研究胎盤功能及妊娠相關(guān)疾病奠定基礎(chǔ)。 1雙核細(xì)胞的分離：本實(shí)驗(yàn)分別用序貫消化法、多次胰蛋白酶法、胰蛋白酶+膠原蛋白酶法、單次胰蛋白酶法和單次膠原蛋白酶法對(duì)胎盤組織進(jìn)行消化，發(fā)現(xiàn)序貫消化法獲得的單細(xì)胞活性最高為73.3%±1.8%，獲得的細(xì)胞數(shù)為（0.65±0.10）×106個(gè)，胰蛋白酶＋膠原蛋白酶法獲得的細(xì)胞活性次之，為69.8%±1.3%，二者間無(wú)顯著性差異（P＞0.05)，但二者細(xì)胞活性均顯著高于其他三組，分別為54.4%±0.7%、49.4%±1.4%、51.5%±1.5%，三者之間無(wú)顯著性差異（P＞0.05)；將用序貫消化法獲得的細(xì)胞懸液通過Percoll密度梯度離心法離心，獲得的細(xì)胞用HE及臺(tái)盼藍(lán)染色法進(jìn)行評(píng)估，雙核細(xì)胞的純度為45.5%±0.2%，細(xì)胞活性約94.3%±0.3%。結(jié)果表明，本實(shí)驗(yàn)所建立的方法可以用于小鼠胎盤雙核細(xì)胞的分離。 2雙核細(xì)胞的培養(yǎng)與純化：本實(shí)驗(yàn)將Percoll密度梯度離心法獲得的雙核細(xì)胞隨機(jī)分為2組，分別采用反復(fù)貼壁法和多次差別消化法進(jìn)行純化，，結(jié)果發(fā)現(xiàn)，傳至第3代時(shí)，反復(fù)貼壁法獲得的細(xì)胞純度(74.5%±1.2%）略低于多次差別消化法（76.5%±2.3%），且反復(fù)貼壁法純化雙核細(xì)胞的時(shí)間為10d，顯著少于多次差別消化法，需要12d。將純化后的細(xì)胞分3組培養(yǎng)，發(fā)現(xiàn)添加谷胱甘肽組、50%成纖維細(xì)胞條件培養(yǎng)基+β巰基乙醇組和用膠原處理培養(yǎng)瓶組的雙核細(xì)胞的活性分別為90.3%±0.5%、95.1%±0.2%和92.5%±0.4%，三者之間無(wú)顯著性差異（P㧐0.05）。這些結(jié)果表明，原代培養(yǎng)物中的成纖維樣細(xì)胞可以通過2或3次的反復(fù)貼壁法和多次差別消化法除去，本研究的培養(yǎng)條件可以用于雙核細(xì)胞的體外培養(yǎng)。
[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.
【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321

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