【摘要】：目的應(yīng)用同位素標(biāo)記相對(duì)和絕對(duì)定量iTRAQ)技術(shù)聯(lián)合液相色譜—串聯(lián)質(zhì)譜(LC-MS/MS)技術(shù)檢測(cè)子癇前期及正常妊娠孕婦血漿中的差異蛋白,從而篩選出潛在的子癇前期血漿生物標(biāo)志物,并初步探討其在子癇前期中的可能作用機(jī)制。 方法臨床收集2012年6月～2013年6月在青島大學(xué)附屬醫(yī)院產(chǎn)科住院期間子癇前期患者60例,其中子癇前期重度患者30例(重度組),子癇前期輕度患者30例(輕度組),同期正常妊娠孕婦30例(對(duì)照組)。采血分離血漿提取總蛋白,隨后對(duì)重度組和對(duì)照組血漿總蛋白進(jìn)行變性、還原、酶解,iTRAQ標(biāo)記后用質(zhì)譜儀進(jìn)行鑒定,得到差異表達(dá)蛋白。選取差異蛋白Gelsolin、S100A8應(yīng)用ELIAS、實(shí)時(shí)熒光定量PCR技術(shù)進(jìn)行進(jìn)一步驗(yàn)證。 結(jié)果①iTRAQ聯(lián)合LC-MS/MS技術(shù)共鑒定到血漿差異蛋白234個(gè),重度組較對(duì)照組表達(dá)豐度相差1.5倍以上(上調(diào)比值1.50或下調(diào)比值0.67)且經(jīng)t檢驗(yàn)證實(shí)差異有統(tǒng)計(jì)學(xué)意義的蛋白質(zhì)點(diǎn)共有24個(gè),其中14個(gè)蛋白點(diǎn)較對(duì)照組表達(dá)上調(diào),另10個(gè)點(diǎn)較之表達(dá)下調(diào)。②輕度組及重度組子癇前期患者血漿中Gelsolin水平分別為(0.288±0.013)ng/L、(0.363±0.152)ng/L,明顯高于對(duì)照組(0.204±0.101)ng/L,分別比較,差異均有統(tǒng)計(jì)學(xué)意(P0.05)；③輕度組及重度組子癇前期患者血漿中S100A8水平分別為(0.687±0.064)ng/L、(0.928±0.101)ng/L,明顯高于對(duì)照組(0.550±0.073)ng/L,分別比較,差異均有統(tǒng)計(jì)學(xué)意(P0.05)；④單因素方差分析顯示子癇前期患者胎盤組織中Gelsolin mRNA水平明顯高于對(duì)照組(0.90±0.037),再比較輕度組(2.44±0.614)及重度組(4.42±1.059),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；⑤單因素方差分析顯示子癇前期患者胎盤組織中S100A8mRNA水平明顯高于對(duì)照組(0.98±0.04),再比較輕度組(3.83±0.879)及重度組(7.59±1.803),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；⑥子癇前期患者血漿中Gelsolin水平與胎盤組織中Gelsolin mRNA的表達(dá)水平無(wú)相關(guān)性(r=0.163,P0.05)；⑦子癇前期患者血漿中S100A8水平與胎盤組織中S100A8mRNA的表達(dá)水平呈正相關(guān)(r=0.701,P0.01)。 結(jié)論iTRAQ聯(lián)合LC-MS/MS技術(shù)能夠有效地篩選出子癇前期血漿差異蛋白。Gelsolin和S100A8的差異表達(dá)與子癇前期的發(fā)生發(fā)展存在一定關(guān)系,S100A8或許可以作為子癇前期特異性血漿蛋白標(biāo)記物,為子癇前期的早期診斷打下一定的理論基礎(chǔ)。
[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 鹵0.013) ng/L, (0.363 鹵0.152) ng/L, higher than those in control group (0.204 鹵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 鹵0.064) ng/L, (0.928 鹵0.101) ng/L, higher than those in control group (0.550 鹵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 鹵0.037), mild group (2.44 鹵0.614) and severe group (4.42 鹵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 鹵0.04), mild group (3.83 鹵0.879) and severe group (7.59 鹵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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.244

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