缺氧对体外滋养细胞及脐静脉内皮细胞中巢蛋白表达的影响
发布时间：2019-06-16 12:23
【摘要】：目的： 重度子痫前期（Severe preeclampsia, SPE）被认为是一种继发于胎盘缺氧的严重妊娠合并症，表现为妊娠20周后出现高血压、蛋白尿及多组织功能损伤，严重危胁着母儿的生命与健康。巢蛋白（Nestin）是一种中间丝细胞骨架蛋白，参与神经细胞发育，是传统观念的神经干细胞标记物。近年来发现Nestin也参与血管的发生与维护，是新生血管内皮细胞标志物之一，其表达状况与缺氧环境有关。 本研究利用缺氧诱导剂建立体外细胞缺氧环境，观察人绒毛膜上皮JEG-3细胞系和人脐静脉内皮细胞（Human umbilical veinendothelial cell, HUVEC）-两种胚胎干细胞在不同缺氧时间和缺氧程度中Nestin的表达状况，为胎盘缺氧在SPE发生、发展中的作用提供实验依据。 材料与方法： 利用二氯化钴（CoCl2）模拟体外缺氧环境，分别诱导JEG-3细胞系及HUVEC化学缺氧。提取不同缺氧条件下各组细胞mRNA,在核酸水平上检测细胞Nestin基因的表达水平；同时收集各组细胞培养基，利用Nestin ELISA试剂盒检测细胞培养基中Nestin蛋白水平并进行统计学分析。 结果 1、缺氧对人绒毛膜上皮JEG-3细胞系中Nestin mRNA及蛋白表达的影响 1）、JEG-3细胞系在CoCl2浓度为300umol/L化学缺氧条件下分别培养6h、12h、24h及48h,后三者Nestin mRNA表达量均明显高于6h者（P0.01）。JEG-3细胞系在CoCl2浓度300umol/L的缺氧环境中培养24h,与未加入CoCl2的培基中培养24h者比较，Nestin mRNA表达量增加（P0.05）。随着缺氧时间的延长，培养基中Nestin蛋白水平逐渐降低，其中6h与48h之间Nestin蛋白水平差异有统计学意义（P0.05）。JEG-3细胞系在CoCl2浓度300umol/L培养基中培养24h,与未加入CoCl2的培基中培养24h者比较，培养基中Nestin蛋白水平下降，但无统计学意义（P0.05）。 2）、JEG-3细胞系在CoCl2培养基中培养24h，随着CoCl2浓度增加，Nestin mRNA表达水平变化不大（P0.05）；培养基中Nestin蛋白水平也无统计学差异（P0.05）。 2、缺氧对人脐静脉内皮细胞-HUVEC中Nestin mRNA及蛋白表达的影响 1）、HUVEC在CoCl2浓度300umol/L化学缺氧条件下，，随着缺氧时间延长，12h、24h及48h者Nestin mRNA表达量均显著高于6h（P0.01）。CoCl2浓度300umol/L化学缺氧24h，与未加入CoCl2化学缺氧情况者比较，Nestin mRNA表达量增加（P0.05）。随着缺氧时间延长，HUVEC培养基中Nestin蛋白水平无明显变化（P0.05）。CoCl2浓度300umol/L化学缺氧24h，与未加入CoCl2化学缺氧情况者比较，培养基中Nestin蛋白水平下降（P0.05）。 2）、HUVEC在CoCl2化学缺氧条件下培养24h，其浓度在400umol/L时Nestin mRNA表达量高于200umol/L及600umol/L（P0.05；P0.01），CoCl2浓度在600umol/L时Nestin mRNA表达量低于200umol/L者（P0.01）。随着CoCl2浓度增加，Nestin蛋白浓度无明显改变（P0.05）。 结论 1、短时间慢性缺氧可以促进Nestin mRNA在滋养细胞和胎盘血管内皮细胞中表达，可能与子痫前期发病过程中机体应对缺氧的保护性机制有关。 2、慢性缺氧时间（一定范围内）延长是影响Nestin在滋养细胞中表达的不利因素，可能与子痫前期患者胎盘浅着床及血管重塑功能障碍有关。 3、慢性缺氧时间（一定范围内）延长对Nestin在胎盘血管内皮细胞中长时间表达及其细胞保护功能起到支持作用。 4、慢性缺氧程度（一定范围内）对Nestin在滋养细胞中表达影响较弱。慢性缺氧程度稍加重可促进Nestin mRNA在胎盘血管内皮细胞中表达，但缺氧程度明显加重可能是限制Nestin mRNA在胎盘血管内皮细胞中表达的不利因素。
[Abstract]:Purpose: Severe pre-eclampsia (SPE) is considered to be a serious pregnancy complication that is secondary to the hypoxia of the placenta, showing the presence of high blood pressure, proteinuria and multi-tissue functional damage after 20 weeks of pregnancy, with a serious risk of life and health of the mother Concortin (Nestin) is an intermediate wire cytoskeleton protein, which is involved in the development of nerve cells. It is a neural stem cell marker of the traditional concept. In recent years, Nestin is also involved in the development and maintenance of blood vessels. It is one of the markers of the new vascular endothelial cells. The expression of Nestin in human chorioepitheliocyte (JEG-3 cell line and human umbilical vein endothelial cell (HUVEC)-two embryonic stem cells was observed under different hypoxia time and hypoxia, and the hypoxia of the placenta was found in the SP. E. The role of development in development Basis for inspection. Materials and Methods: The JEG-3 cell line and HU were induced by using cobalt dichloride (CoCl2) to simulate the in vitro hypoxia environment. the expression level of the nestin gene in the cell culture medium is detected at the same time, the cell culture medium of each group is collected, the nestin protein level in the cell culture medium is detected by using the nestin ELISA kit, carry out Statistical analysis. Results 1, hypoxia on human chorioepithelial JEG-3 cell line, Nestin The expression of mRNA and protein 1), the JEG-3 cell line was cultured under the condition of CoCl2 at a concentration of 300 umol/ L under the condition of chemical oxygen deficiency. After h,12 h,24 h and 48 h, the expression of Nestin mRNA was significantly higher than that of 6 h (P0.01). The JEG-3 cell line was cultured in an anoxic environment with a CoCl2 concentration of 300 umol/ L for 24 h, and the Nestin mRN was compared with the culture of 24 h in the culture medium without CoCl2. The expression of protein A was increased (P0.05). With the increase of the time of hypoxia, the level of the Nestin protein in the culture medium was gradually decreased, and the level of the Nestin protein between 6 h and 48 h was statistically significant (P0.05). The JEG-3 cell line was cultured in a CoCl2 concentration of 300 umol/ L medium for 24 h, and the culture medium was compared with the culture of 24 h in the culture medium which was not added with CoCl2. The level of the mestin protein is decreased, but There was no statistical significance (P0.05). The JEG-3 cell line was cultured in CoCl2 medium for 24 h, and the expression level of Nestin mRNA in the medium increased with the increase of the concentration of CoCl2 (P0.05); the Nestin protein water in the culture medium There was no significant difference in the level of human umbilical vein endothelial cells (HUVEC) in human umbilical vein endothelial cells (HUVEC). The expression of Nestin mRNA and protein was 1), and the expression of Nestin mRNA in 12 h,24 h and 48 h was significantly higher than that of 6 h (P0.01). 2 concentration of 300 umol/ L for 24 h, compared with those without CoCl2 chemical oxygen deficiency, Nes The expression of tin mRNA was increased (P0.05). As the time of hypoxia prolonged, the level of Nestin in HUVEC culture medium did not change significantly (P0.05). The expression of Nestin mRNA was higher than 200 umol/ L and 600 umol/ L at 400 uml/ L (P0.05; P0.01), and the concentration of CoCl2 was at 600 uml/ L. As the concentration of CoCl2 increased, N was lower than that of 200 uml/ L (P0.01). es Conclusion 1. Short-term chronic hypoxia can promote the expression of Nestin mRNA in trophoblast and placental vascular endothelial cells. Can be related to the protective mechanism of the body to cope with the hypoxia in the course of pre-eclampsia.2. The prolongation of chronic hypoxia time (in certain range) is the negative factor that affects the expression of Nestin in the trophoblastic cells. In particular, that duration of chronic hypoxia (in a range) may be extended to Nestin. Long-term expression and cell protection in placental vascular endothelial cells support role.4, Slow The degree of sexual hypoxia (in a certain range) has a weak effect on the expression of Nestin in the trophoblastic cells. The slight increase in the degree of chronic hypoxia can promote the expression of the Nestin mRNA in the vascular endothelial cells of the placenta, but the degree of hypoxia is significantly increased.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714.244
[Abstract]:Purpose: Severe pre-eclampsia (SPE) is considered to be a serious pregnancy complication that is secondary to the hypoxia of the placenta, showing the presence of high blood pressure, proteinuria and multi-tissue functional damage after 20 weeks of pregnancy, with a serious risk of life and health of the mother Concortin (Nestin) is an intermediate wire cytoskeleton protein, which is involved in the development of nerve cells. It is a neural stem cell marker of the traditional concept. In recent years, Nestin is also involved in the development and maintenance of blood vessels. It is one of the markers of the new vascular endothelial cells. The expression of Nestin in human chorioepitheliocyte (JEG-3 cell line and human umbilical vein endothelial cell (HUVEC)-two embryonic stem cells was observed under different hypoxia time and hypoxia, and the hypoxia of the placenta was found in the SP. E. The role of development in development Basis for inspection. Materials and Methods: The JEG-3 cell line and HU were induced by using cobalt dichloride (CoCl2) to simulate the in vitro hypoxia environment. the expression level of the nestin gene in the cell culture medium is detected at the same time, the cell culture medium of each group is collected, the nestin protein level in the cell culture medium is detected by using the nestin ELISA kit, carry out Statistical analysis. Results 1, hypoxia on human chorioepithelial JEG-3 cell line, Nestin The expression of mRNA and protein 1), the JEG-3 cell line was cultured under the condition of CoCl2 at a concentration of 300 umol/ L under the condition of chemical oxygen deficiency. After h,12 h,24 h and 48 h, the expression of Nestin mRNA was significantly higher than that of 6 h (P0.01). The JEG-3 cell line was cultured in an anoxic environment with a CoCl2 concentration of 300 umol/ L for 24 h, and the Nestin mRN was compared with the culture of 24 h in the culture medium without CoCl2. The expression of protein A was increased (P0.05). With the increase of the time of hypoxia, the level of the Nestin protein in the culture medium was gradually decreased, and the level of the Nestin protein between 6 h and 48 h was statistically significant (P0.05). The JEG-3 cell line was cultured in a CoCl2 concentration of 300 umol/ L medium for 24 h, and the culture medium was compared with the culture of 24 h in the culture medium which was not added with CoCl2. The level of the mestin protein is decreased, but There was no statistical significance (P0.05). The JEG-3 cell line was cultured in CoCl2 medium for 24 h, and the expression level of Nestin mRNA in the medium increased with the increase of the concentration of CoCl2 (P0.05); the Nestin protein water in the culture medium There was no significant difference in the level of human umbilical vein endothelial cells (HUVEC) in human umbilical vein endothelial cells (HUVEC). The expression of Nestin mRNA and protein was 1), and the expression of Nestin mRNA in 12 h,24 h and 48 h was significantly higher than that of 6 h (P0.01). 2 concentration of 300 umol/ L for 24 h, compared with those without CoCl2 chemical oxygen deficiency, Nes The expression of tin mRNA was increased (P0.05). As the time of hypoxia prolonged, the level of Nestin in HUVEC culture medium did not change significantly (P0.05). The expression of Nestin mRNA was higher than 200 umol/ L and 600 umol/ L at 400 uml/ L (P0.05; P0.01), and the concentration of CoCl2 was at 600 uml/ L. As the concentration of CoCl2 increased, N was lower than that of 200 uml/ L (P0.01). es Conclusion 1. Short-term chronic hypoxia can promote the expression of Nestin mRNA in trophoblast and placental vascular endothelial cells. Can be related to the protective mechanism of the body to cope with the hypoxia in the course of pre-eclampsia.2. The prolongation of chronic hypoxia time (in certain range) is the negative factor that affects the expression of Nestin in the trophoblastic cells. In particular, that duration of chronic hypoxia (in a range) may be extended to Nestin. Long-term expression and cell protection in placental vascular endothelial cells support role.4, Slow The degree of sexual hypoxia (in a certain range) has a weak effect on the expression of Nestin in the trophoblastic cells. The slight increase in the degree of chronic hypoxia can promote the expression of the Nestin mRNA in the vascular endothelial cells of the placenta, but the degree of hypoxia is significantly increased.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R714.244
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相关期刊论文 前5条
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