子痫前期外周血及胎盘组织相关蛋白的研究
发布时间：2019-01-28 19:24
【摘要】：目的应用同位素标记相对和绝对定量iTRAQ)技术联合液相色谱—串联质谱(LC-MS/MS)技术检测子痫前期及正常妊娠孕妇血浆中的差异蛋白,从而筛选出潜在的子痫前期血浆生物标志物,并初步探讨其在子痫前期中的可能作用机制。 方法临床收集2012年6月～2013年6月在青岛大学附属医院产科住院期间子痫前期患者60例,其中子痫前期重度患者30例(重度组),子痫前期轻度患者30例(轻度组),同期正常妊娠孕妇30例(对照组)。采血分离血浆提取总蛋白,随后对重度组和对照组血浆总蛋白进行变性、还原、酶解,iTRAQ标记后用质谱仪进行鉴定,得到差异表达蛋白。选取差异蛋白Gelsolin、S100A8应用ELIAS、实时荧光定量PCR技术进行进一步验证。 结果①iTRAQ联合LC-MS/MS技术共鉴定到血浆差异蛋白234个,重度组较对照组表达丰度相差1.5倍以上(上调比值1.50或下调比值0.67)且经t检验证实差异有统计学意义的蛋白质点共有24个,其中14个蛋白点较对照组表达上调,另10个点较之表达下调。②轻度组及重度组子痫前期患者血浆中Gelsolin水平分别为(0.288±0.013)ng/L、(0.363±0.152)ng/L,明显高于对照组(0.204±0.101)ng/L,分别比较,差异均有统计学意(P0.05)；③轻度组及重度组子痫前期患者血浆中S100A8水平分别为(0.687±0.064)ng/L、(0.928±0.101)ng/L,明显高于对照组(0.550±0.073)ng/L,分别比较,差异均有统计学意(P0.05)；④单因素方差分析显示子痫前期患者胎盘组织中Gelsolin mRNA水平明显高于对照组(0.90±0.037),再比较轻度组(2.44±0.614)及重度组(4.42±1.059),差异有统计学意义(P0.05)；⑤单因素方差分析显示子痫前期患者胎盘组织中S100A8mRNA水平明显高于对照组(0.98±0.04),再比较轻度组(3.83±0.879)及重度组(7.59±1.803),差异有统计学意义(P0.05)；⑥子痫前期患者血浆中Gelsolin水平与胎盘组织中Gelsolin mRNA的表达水平无相关性(r=0.163,P0.05)；⑦子痫前期患者血浆中S100A8水平与胎盘组织中S100A8mRNA的表达水平呈正相关(r=0.701,P0.01)。 结论iTRAQ联合LC-MS/MS技术能够有效地筛选出子痫前期血浆差异蛋白。Gelsolin和S100A8的差异表达与子痫前期的发生发展存在一定关系,S100A8或许可以作为子痫前期特异性血浆蛋白标记物,为子痫前期的早期诊断打下一定的理论基础。
[Abstract]:Objective to detect the differential proteins in plasma of preeclampsia and normal pregnant women by isotope labeled relative and absolute quantitative iTRAQ and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential biomarkers of preeclampsia plasma were screened and the possible mechanism of preeclampsia was discussed. Methods from June 2012 to June 2013, 60 cases of preeclampsia were collected in the obstetrical department of Qingdao University affiliated Hospital, including 30 cases of severe preeclampsia (severe group) and 30 cases of mild preeclampsia (mild group). 30 normal pregnant women (control group). The total protein was extracted from the blood samples, then denatured, reduced, hydrolyzed by enzyme and identified by mass spectrometer. The differentially expressed proteins were obtained after the denaturation, reduction, enzymatic hydrolysis and iTRAQ labeling of the plasma total proteins in the severe group and the control group. The differential protein Gelsolin,S100A8 was selected for further verification by ELIAS, real-time fluorescence quantitative PCR. Results 234 plasma differentially expressed proteins were identified by 1iTRAQ and LC-MS/MS. The expression abundance of protein in severe group was more than 1.5 times higher than that in control group (up-regulation ratio 1.50 or down-regulation ratio 0.67) and there were 24 protein spots with statistical significance confirmed by t test, of which 14 protein spots were up-regulated than that of control group. 2 the plasma Gelsolin levels in mild and severe preeclampsia patients were (0.288 卤0.013) ng/L, (0.363 卤0.152) ng/L, higher than those in control group (0.204 卤0.101) ng/L, respectively. The differences were statistically significant (P0.05). 3The plasma S100A8 levels in mild and severe preeclampsia patients were (0.687 卤0.064) ng/L, (0.928 卤0.101) ng/L, higher than those in control group (0.550 卤0.073) ng/L, respectively. The difference was statistically significant (P0.05). 4 the level of Gelsolin mRNA in placenta of preeclampsia patients was significantly higher than that of control group (0.90 卤0.037), mild group (2.44 卤0.614) and severe group (4.42 卤1.059). The difference was statistically significant (P0.05). 5 the level of S100A8mRNA in placenta of preeclampsia patients was significantly higher than that of control group (0.98 卤0.04), mild group (3.83 卤0.879) and severe group (7.59 卤1.803). The difference was statistically significant (P0.05). 6There was no correlation between the level of Gelsolin in plasma and the expression of Gelsolin mRNA in placenta (r = 0.163 P 0.05). 7There was a positive correlation between plasma S100A8 level and placental S100A8mRNA expression in preeclampsia (r = 0.701 / P0.01). Conclusion iTRAQ combined with LC-MS/MS can effectively screen plasma differential proteins of preeclampsia. The differential expression of Gelsolin and S100A8 may be related to the occurrence and development of preeclampsia. S100A8 may serve as a specific plasma protein marker for preeclampsia and provide a theoretical basis for early diagnosis of preeclampsia.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.244
本文编号：2417236
[Abstract]:Objective to detect the differential proteins in plasma of preeclampsia and normal pregnant women by isotope labeled relative and absolute quantitative iTRAQ and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential biomarkers of preeclampsia plasma were screened and the possible mechanism of preeclampsia was discussed. Methods from June 2012 to June 2013, 60 cases of preeclampsia were collected in the obstetrical department of Qingdao University affiliated Hospital, including 30 cases of severe preeclampsia (severe group) and 30 cases of mild preeclampsia (mild group). 30 normal pregnant women (control group). The total protein was extracted from the blood samples, then denatured, reduced, hydrolyzed by enzyme and identified by mass spectrometer. The differentially expressed proteins were obtained after the denaturation, reduction, enzymatic hydrolysis and iTRAQ labeling of the plasma total proteins in the severe group and the control group. The differential protein Gelsolin,S100A8 was selected for further verification by ELIAS, real-time fluorescence quantitative PCR. Results 234 plasma differentially expressed proteins were identified by 1iTRAQ and LC-MS/MS. The expression abundance of protein in severe group was more than 1.5 times higher than that in control group (up-regulation ratio 1.50 or down-regulation ratio 0.67) and there were 24 protein spots with statistical significance confirmed by t test, of which 14 protein spots were up-regulated than that of control group. 2 the plasma Gelsolin levels in mild and severe preeclampsia patients were (0.288 卤0.013) ng/L, (0.363 卤0.152) ng/L, higher than those in control group (0.204 卤0.101) ng/L, respectively. The differences were statistically significant (P0.05). 3The plasma S100A8 levels in mild and severe preeclampsia patients were (0.687 卤0.064) ng/L, (0.928 卤0.101) ng/L, higher than those in control group (0.550 卤0.073) ng/L, respectively. The difference was statistically significant (P0.05). 4 the level of Gelsolin mRNA in placenta of preeclampsia patients was significantly higher than that of control group (0.90 卤0.037), mild group (2.44 卤0.614) and severe group (4.42 卤1.059). The difference was statistically significant (P0.05). 5 the level of S100A8mRNA in placenta of preeclampsia patients was significantly higher than that of control group (0.98 卤0.04), mild group (3.83 卤0.879) and severe group (7.59 卤1.803). The difference was statistically significant (P0.05). 6There was no correlation between the level of Gelsolin in plasma and the expression of Gelsolin mRNA in placenta (r = 0.163 P 0.05). 7There was a positive correlation between plasma S100A8 level and placental S100A8mRNA expression in preeclampsia (r = 0.701 / P0.01). Conclusion iTRAQ combined with LC-MS/MS can effectively screen plasma differential proteins of preeclampsia. The differential expression of Gelsolin and S100A8 may be related to the occurrence and development of preeclampsia. S100A8 may serve as a specific plasma protein marker for preeclampsia and provide a theoretical basis for early diagnosis of preeclampsia.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.244
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