缺氧對體外滋養(yǎng)細(xì)胞及臍靜脈內(nèi)皮細(xì)胞中巢蛋白表達(dá)的影響
發(fā)布時(shí)間：2019-06-16 12:23
【摘要】：目的： 重度子癇前期（Severe preeclampsia, SPE）被認(rèn)為是一種繼發(fā)于胎盤缺氧的嚴(yán)重妊娠合并癥，表現(xiàn)為妊娠20周后出現(xiàn)高血壓、蛋白尿及多組織功能損傷，嚴(yán)重危脅著母兒的生命與健康。巢蛋白（Nestin）是一種中間絲細(xì)胞骨架蛋白，參與神經(jīng)細(xì)胞發(fā)育，是傳統(tǒng)觀念的神經(jīng)干細(xì)胞標(biāo)記物。近年來發(fā)現(xiàn)Nestin也參與血管的發(fā)生與維護(hù)，是新生血管內(nèi)皮細(xì)胞標(biāo)志物之一，其表達(dá)狀況與缺氧環(huán)境有關(guān)。 本研究利用缺氧誘導(dǎo)劑建立體外細(xì)胞缺氧環(huán)境，觀察人絨毛膜上皮JEG-3細(xì)胞系和人臍靜脈內(nèi)皮細(xì)胞（Human umbilical veinendothelial cell, HUVEC）-兩種胚胎干細(xì)胞在不同缺氧時(shí)間和缺氧程度中Nestin的表達(dá)狀況，為胎盤缺氧在SPE發(fā)生、發(fā)展中的作用提供實(shí)驗(yàn)依據(jù)。 材料與方法： 利用二氯化鈷（CoCl2）模擬體外缺氧環(huán)境，分別誘導(dǎo)JEG-3細(xì)胞系及HUVEC化學(xué)缺氧。提取不同缺氧條件下各組細(xì)胞mRNA,在核酸水平上檢測細(xì)胞Nestin基因的表達(dá)水平；同時(shí)收集各組細(xì)胞培養(yǎng)基，利用Nestin ELISA試劑盒檢測細(xì)胞培養(yǎng)基中Nestin蛋白水平并進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果 1、缺氧對人絨毛膜上皮JEG-3細(xì)胞系中Nestin mRNA及蛋白表達(dá)的影響 1）、JEG-3細(xì)胞系在CoCl2濃度為300umol/L化學(xué)缺氧條件下分別培養(yǎng)6h、12h、24h及48h,后三者Nestin mRNA表達(dá)量均明顯高于6h者（P0.01）。JEG-3細(xì)胞系在CoCl2濃度300umol/L的缺氧環(huán)境中培養(yǎng)24h,與未加入CoCl2的培基中培養(yǎng)24h者比較，Nestin mRNA表達(dá)量增加（P0.05）。隨著缺氧時(shí)間的延長，培養(yǎng)基中Nestin蛋白水平逐漸降低，其中6h與48h之間Nestin蛋白水平差異有統(tǒng)計(jì)學(xué)意義（P0.05）。JEG-3細(xì)胞系在CoCl2濃度300umol/L培養(yǎng)基中培養(yǎng)24h,與未加入CoCl2的培基中培養(yǎng)24h者比較，培養(yǎng)基中Nestin蛋白水平下降，但無統(tǒng)計(jì)學(xué)意義（P0.05）。 2）、JEG-3細(xì)胞系在CoCl2培養(yǎng)基中培養(yǎng)24h，隨著CoCl2濃度增加，Nestin mRNA表達(dá)水平變化不大（P0.05）；培養(yǎng)基中Nestin蛋白水平也無統(tǒng)計(jì)學(xué)差異（P0.05）。 2、缺氧對人臍靜脈內(nèi)皮細(xì)胞-HUVEC中Nestin mRNA及蛋白表達(dá)的影響 1）、HUVEC在CoCl2濃度300umol/L化學(xué)缺氧條件下，，隨著缺氧時(shí)間延長，12h、24h及48h者Nestin mRNA表達(dá)量均顯著高于6h（P0.01）。CoCl2濃度300umol/L化學(xué)缺氧24h，與未加入CoCl2化學(xué)缺氧情況者比較，Nestin mRNA表達(dá)量增加（P0.05）。隨著缺氧時(shí)間延長，HUVEC培養(yǎng)基中Nestin蛋白水平無明顯變化（P0.05）。CoCl2濃度300umol/L化學(xué)缺氧24h，與未加入CoCl2化學(xué)缺氧情況者比較，培養(yǎng)基中Nestin蛋白水平下降（P0.05）。 2）、HUVEC在CoCl2化學(xué)缺氧條件下培養(yǎng)24h，其濃度在400umol/L時(shí)Nestin mRNA表達(dá)量高于200umol/L及600umol/L（P0.05；P0.01），CoCl2濃度在600umol/L時(shí)Nestin mRNA表達(dá)量低于200umol/L者（P0.01）。隨著CoCl2濃度增加，Nestin蛋白濃度無明顯改變（P0.05）。 結(jié)論 1、短時(shí)間慢性缺氧可以促進(jìn)Nestin mRNA在滋養(yǎng)細(xì)胞和胎盤血管內(nèi)皮細(xì)胞中表達(dá)，可能與子癇前期發(fā)病過程中機(jī)體應(yīng)對缺氧的保護(hù)性機(jī)制有關(guān)。 2、慢性缺氧時(shí)間（一定范圍內(nèi)）延長是影響Nestin在滋養(yǎng)細(xì)胞中表達(dá)的不利因素，可能與子癇前期患者胎盤淺著床及血管重塑功能障礙有關(guān)。 3、慢性缺氧時(shí)間（一定范圍內(nèi)）延長對Nestin在胎盤血管內(nèi)皮細(xì)胞中長時(shí)間表達(dá)及其細(xì)胞保護(hù)功能起到支持作用。 4、慢性缺氧程度（一定范圍內(nèi)）對Nestin在滋養(yǎng)細(xì)胞中表達(dá)影響較弱。慢性缺氧程度稍加重可促進(jìn)Nestin mRNA在胎盤血管內(nèi)皮細(xì)胞中表達(dá)，但缺氧程度明顯加重可能是限制Nestin mRNA在胎盤血管內(nèi)皮細(xì)胞中表達(dá)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R714.244
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