子痫前期外周血及胎盘组织中HNP3、PAPPA2的研究
发布时间：2019-06-03 02:07
【摘要】：目的:筛选子痫前期患者与正常妊娠孕妇血清中的差异蛋白,对相关差异蛋白进行检测并初步探讨其在子痫前期发病过程中可能的作用机制,以期筛选出能够早期预测子痫前期的潜在血清生物标志物。方法:收集2015年6月至2016年6月在青岛大学附属医院产科住院治疗的重度子痫前期患者30例,设为病例组;同期收集30例正常妊娠,因社会因素、产道异常、胎位不正等原因行择期剖宫产的孕妇,设为对照组。懫血分离血清提取总蛋白,经iTRAQ标记后,用质谱仪进行鉴定,获得差异表达的蛋白质。差异蛋白的研究采用定量蛋白质组学技术,差异蛋白功能分析采用生物信息学的方法。同期收集另外30例重度子痫前期患者,作为重度组;30例轻度子痫前期患者,作为轻度组;30例正常妊娠孕妇,作为正常组。采血分离血清、收集胎盘组织。选取有统计学意义的差异蛋白:中性粒细胞防御素3(HNP3)、冠毛素2(PAPPA2),应用酶联免疫吸附试验以及实时荧光定量PCR技术分别检测其在三组研究对象外周血及胎盘组织中的表达。结果:(1)共筛选出血清差异蛋白234个,病例组与对照组相比表达丰度相差1.6倍以上或0.625倍以下且经t检验确认,差异有统计学意义的差异蛋白点共25个,其中表达上调的蛋白点有16个,另9个蛋白点表达下调;(2)重度组及轻度组子痫前期患者血清中HNP3水平分别为(144.0±22.35)ng/m L、(98.9±11.70)ng/m L均高于正常组(83.8±11.71)ng/m L(P0.0001、P=0.045,P0.05),差异有统计学意义;且重度组高于轻度组(P=0.0074,P0.01),差异具有统计学意义;(3)重度组及轻度组子痫前期患者胎盘组织中HNP3m RNA的相对表达量分别为(4.856±0.077)、(1.978±0.156)均高于正常组(1.001±0.219)(P=0.001、P=0.032,P0.05),差异有统计学意义;重度组与轻度组相比(P=0.015,P0.05),差异亦具有统计学意义;(4)子痫前期患者血清中HNP3水平与胎盘组织中HNP3m RNA相对表达量无相关性,两者相关系数r=0.554,P=0.626,P0.05,无统计学意义;(5)重度组及轻度组子痫前期患者血清中PAPPA2水平分别为(549.1±36.68)pg/m L、(166.1±16.41)pg/m L均高于正常组(138.1±9.05)pg/m L(P0.0001、P=0.014,P0.05),差异有统计学意义;且重度组高于轻度组(P=0.0015,P0.01),差异有统计学意义;(6)重度组及轻度组子痫前期患者胎盘组织中PAPPA2m RNA的相对表达量分别为(13.136±0.230)、(4.523±0.282)均高于正常组(1.000±0.169)(P0.0001、P=0.015,P0.05),差异有统计学意义;比较重度组与轻度组(P=0.001,P0.01),差异具有统计学意义;(7)子痫前期患者血清中PAPPA2水平与胎盘组织中PAPPA2m RNA相对表达量呈正相关,两者相关系数r=0.944,P=0.005,P0.05,相关有统计学意义;(8)子痫前期患者血清中HNP3与PAPPA2水平呈正相关,两者相关系数r=0.852,P0.0001,相关有统计学意义。结论:(1)iTRAQ联合LC-MS/MS技术为子痫前期早期血清标志物的筛选与鉴定提供了有效的技术支持;(2)中性粒细胞防御素3(HNP3)和冠毛素2(PAPPA2)可能参与了子痫前期的发生发展;(3)中性粒细胞防御素3(HNP3)和冠毛素2(PAPPA2)在子痫前期的发病过程中可能存在相互作用;(4)冠毛素2(PAPPA2)有可能成为早期预测子痫前期的潜在血清生物标志物。
[Abstract]:Objective: To screen the differential protein in the serum of pre-eclampsia and the normal pregnant women, to test the related differential protein and to explore the possible mechanism of the early preeclampsia in the early stage of preeclampsia, with a view to screening the potential serum biomarkers in the early preeclampsia. Methods:30 cases of severe preeclampsia were collected from June,2015 to June,2016 in the hospital of the Affiliated Hospital of the University of Wisconsin, and 30 cases of normal pregnancy were collected in the same period. Set as the control group. The total protein of the serum was isolated from the serum. After the iTRAQ marker, the protein with differential expression was obtained by mass spectrometer. The research of differential protein is based on the quantitative proteomics technology, and the functional analysis of the differential protein is a bioinformatics method. In the same period,30 cases of severe preeclampsia were collected as the severe group,30 of the 30 patients with mild preeclampsia were treated as mild group, and 30 normal pregnant women were used as the normal group. Blood was collected to separate the serum and the placental tissue was collected. The differentially expressed proteins were selected: Neutrophil Defensin 3 (HNP3), PAPP-2 (PAPPA2), Enzyme-linked Immunosorbent Assay (ELISA) and real-time fluorescence quantitative PCR (RT-PCR). Results: (1) A total of 234 serum differential proteins were selected, and the expression abundance was more than 1.6 times or 0.625 times or 0.625 times as compared with the control group. The levels of HNP3 in serum of patients with severe and mild preeclampsia were (144.0-22.35) ng/ mL, (98.9-11.70) ng/ mL higher than that of normal group (83.8-11.71) ng/ mL (P0.01, P = 0.045, P0.05). The relative expression of HNP3m in the placenta was higher than that in the normal group (1.001 and 0.219) (P = 0.001, P = 0.032, P0.05). (4) There was no correlation between the level of HNP3 in serum of preeclampsia and the relative expression of HNP3m in placenta, and the correlation coefficient was r = 0.554, P = 0.626, P <0.05, and there was no statistical significance. (5) The levels of PAPPA2 in serum of severe and mild group of preeclampsia were (549.1, 36.68) pg/ mL, (166.1-16.41) pg/ m L were higher than that of normal group (138.1-9.05) pg/ m L (P = 0.014, P0.05), and the difference was statistically significant; and the severe group was higher than that of mild group (P = 0.0015, P0.01). (6) The relative expression of PAPPA2 mRNA in the placenta of the patients with severe and mild preeclampsia were (13.136, 0.230) and (4.523, 0.282) higher than that in the normal group (1.000-0.169) (P = 0.015, P <0.05). (7) The level of PAPPA2 in the serum of preeclampsia was positively correlated with the relative expression of PAPPA2 mRNA in the placenta tissue, the correlation coefficient r = 0.944, P = 0.005, P 0.05, and the correlation was statistical significance; (8) The level of HNP3 in the serum of preeclampsia was positively correlated with the level of PAPPA2, and the correlation coefficient was r = 0.852, P <0001, and the correlation was of statistical significance. Conclusion: (1) The combination of iTRAQ and LC-MS/ MS provides effective technical support for screening and identification of early-stage serum markers in pre-eclampsia. (2) Neutrophil-Defensin 3 (HNP3) and Seaponin 2 (PAPPA2) may be involved in the development of pre-eclampsia. (3) Neutrophil-3 (HNP3) and hypertrichostatin 2 (PAPPA2) may interact in the pathogenesis of pre-eclampsia; (4) the preeclampsia-2 (PAPPA2) is likely to be a potential serum biomarker for early preeclampsia.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.244
本文编号：2491589
[Abstract]:Objective: To screen the differential protein in the serum of pre-eclampsia and the normal pregnant women, to test the related differential protein and to explore the possible mechanism of the early preeclampsia in the early stage of preeclampsia, with a view to screening the potential serum biomarkers in the early preeclampsia. Methods:30 cases of severe preeclampsia were collected from June,2015 to June,2016 in the hospital of the Affiliated Hospital of the University of Wisconsin, and 30 cases of normal pregnancy were collected in the same period. Set as the control group. The total protein of the serum was isolated from the serum. After the iTRAQ marker, the protein with differential expression was obtained by mass spectrometer. The research of differential protein is based on the quantitative proteomics technology, and the functional analysis of the differential protein is a bioinformatics method. In the same period,30 cases of severe preeclampsia were collected as the severe group,30 of the 30 patients with mild preeclampsia were treated as mild group, and 30 normal pregnant women were used as the normal group. Blood was collected to separate the serum and the placental tissue was collected. The differentially expressed proteins were selected: Neutrophil Defensin 3 (HNP3), PAPP-2 (PAPPA2), Enzyme-linked Immunosorbent Assay (ELISA) and real-time fluorescence quantitative PCR (RT-PCR). Results: (1) A total of 234 serum differential proteins were selected, and the expression abundance was more than 1.6 times or 0.625 times or 0.625 times as compared with the control group. The levels of HNP3 in serum of patients with severe and mild preeclampsia were (144.0-22.35) ng/ mL, (98.9-11.70) ng/ mL higher than that of normal group (83.8-11.71) ng/ mL (P0.01, P = 0.045, P0.05). The relative expression of HNP3m in the placenta was higher than that in the normal group (1.001 and 0.219) (P = 0.001, P = 0.032, P0.05). (4) There was no correlation between the level of HNP3 in serum of preeclampsia and the relative expression of HNP3m in placenta, and the correlation coefficient was r = 0.554, P = 0.626, P <0.05, and there was no statistical significance. (5) The levels of PAPPA2 in serum of severe and mild group of preeclampsia were (549.1, 36.68) pg/ mL, (166.1-16.41) pg/ m L were higher than that of normal group (138.1-9.05) pg/ m L (P = 0.014, P0.05), and the difference was statistically significant; and the severe group was higher than that of mild group (P = 0.0015, P0.01). (6) The relative expression of PAPPA2 mRNA in the placenta of the patients with severe and mild preeclampsia were (13.136, 0.230) and (4.523, 0.282) higher than that in the normal group (1.000-0.169) (P = 0.015, P <0.05). (7) The level of PAPPA2 in the serum of preeclampsia was positively correlated with the relative expression of PAPPA2 mRNA in the placenta tissue, the correlation coefficient r = 0.944, P = 0.005, P 0.05, and the correlation was statistical significance; (8) The level of HNP3 in the serum of preeclampsia was positively correlated with the level of PAPPA2, and the correlation coefficient was r = 0.852, P <0001, and the correlation was of statistical significance. Conclusion: (1) The combination of iTRAQ and LC-MS/ MS provides effective technical support for screening and identification of early-stage serum markers in pre-eclampsia. (2) Neutrophil-Defensin 3 (HNP3) and Seaponin 2 (PAPPA2) may be involved in the development of pre-eclampsia. (3) Neutrophil-3 (HNP3) and hypertrichostatin 2 (PAPPA2) may interact in the pathogenesis of pre-eclampsia; (4) the preeclampsia-2 (PAPPA2) is likely to be a potential serum biomarker for early preeclampsia.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.244
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