本文選題：HtrA4 + GCM1��； 參考：《山東大學(xué)》2014年碩士論文

【摘要】：研究目的 HtrA4(high temperature reguirement A4, HtrA4)是HtrA家族蛋白中不典型一員,HtrA家族蛋白是絲氨酸蛋白酶,對(duì)折疊錯(cuò)誤和位置錯(cuò)誤的蛋白起質(zhì)控作用。最近幾年,對(duì)HtrA1等已經(jīng)有所報(bào)道,主要在調(diào)控滋養(yǎng)細(xì)胞分化及侵襲中起一定作用,但對(duì)HtrA4研究不多。研究表明子癇前期病人的HtrA4在胎盤及血清中表達(dá)水平均較正常血壓的孕婦水平要低；相較正常絨毛,子癇前期的病人的HtrA4在合體滋養(yǎng)細(xì)胞層表達(dá)顯著增多。已知GCM1(Glial cells missing1)是胎盤形成過程中的一個(gè)重要轉(zhuǎn)化因子,調(diào)節(jié)作用于合體滋養(yǎng)細(xì)胞層分化的syncytin融合蛋白的表達(dá),也調(diào)節(jié)促進(jìn)胎盤血管生成的胎盤生長(zhǎng)因子(PGF)的表達(dá)。Liang-Jie Wang[3]等通過染色質(zhì)免疫沉淀芯片分析,確認(rèn)HtrA4基因是GCM1的靶基因。本研究就HtrA4和GCM1在妊娠期高血壓患者胎盤組織中的表達(dá),探討兩者參與調(diào)節(jié)滋養(yǎng)細(xì)胞侵襲機(jī)制的關(guān)系,為子癇前期等病理妊娠的發(fā)病機(jī)制提供理論基礎(chǔ)。 實(shí)驗(yàn)方法 1.研究對(duì)象隨機(jī)收集山東大學(xué)齊魯醫(yī)院2013年4月～2013年7月產(chǎn)科住院的產(chǎn)婦胎盤60例,其中妊高癥患者40例作為實(shí)驗(yàn)組(早發(fā)型子癇前期23例,晚發(fā)型子癇子癇前期17例),另選同期在本院正常晚期妊娠孕婦20例為對(duì)照組。子癇前期的診斷標(biāo)準(zhǔn)參照《婦產(chǎn)科學(xué)》(第8版)。兩組的孕周、年齡均無統(tǒng)計(jì)學(xué)差異。所選對(duì)象無慢性高血壓、糖尿病、心臟病、腎臟病等病史。 2. RT-PCR提取總RNA后,選取合格的mRNA作為模板進(jìn)行反轉(zhuǎn)錄反應(yīng),反轉(zhuǎn)錄條件嚴(yán)格按照說明書進(jìn)行操作。PCR引物由北京博愛森生物技術(shù)公司合成,GCMl上游：5'-GTTAGAAGCTGAGGCAAGAA-3',下游：5'-TAAAGATTACCCGCGGAT-3'; HtrA4上游：5'-GTCAGCACCAAACAGCG-3',下游：5'-GGAGATTCCATCAGTCACCC-3'; β-actin上游：5'-AACTCCATCATGAAGTGAC-3'下游：5’-GATCCACATCTGCTGGAAGG-3',均按說明操作進(jìn)行PCR,擴(kuò)增產(chǎn)物經(jīng)凝膠電泳后,在D-140圖像記錄分析系統(tǒng)上進(jìn)行分析。HtrA4和GCM1的mRNA表達(dá)量分別以HtrA4或GCM1擴(kuò)增DNA條帶的灰度值計(jì)算。 Western blotting:取100mg組織剪碎進(jìn)行蛋白提取,凝膠電泳,轉(zhuǎn)膜,雜交,顯色,成像儀成像,分析結(jié)果 3.統(tǒng)計(jì)學(xué)方法：應(yīng)用SPSS17.0軟件包進(jìn)行統(tǒng)計(jì)分析,以均數(shù)±標(biāo)準(zhǔn)差表示,組間比較采用單因素方差分析,采用t檢驗(yàn),組間相關(guān)性分析用spearman檢驗(yàn)。 結(jié)果 在mRNA水平,HtrA4與GCM1在組中均有表達(dá),HtrA4與GCM1mRNA表達(dá)量在研究組中低于健康對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(P0.01)；子癇前期組胎盤組織中HtrA4與GCM1的表達(dá)均呈正相關(guān)(mRNA:r-0.426,蛋白：r=0.686、P0.01)。 討論 GCM1與子癇前期發(fā)病的關(guān)系GCM1即GCMa, GCM蛋白是一種具有保守DNA結(jié)合區(qū)域的轉(zhuǎn)錄因子。人類的GCM1主要在胎盤組織,是胎盤發(fā)育過程中一個(gè)必要的轉(zhuǎn)錄因子。在低氧情況下GCM1泛素化降解而活性被抑制,GCM1主要通過促進(jìn)金合胞素和PGF進(jìn)而促進(jìn)滋養(yǎng)細(xì)胞融合分化,而HtrA4可以通過PDZ結(jié)構(gòu)域與合胞素的表面亞結(jié)構(gòu)結(jié)合,已有報(bào)道HtrA4和GCM1交叉表達(dá)于母胎界面的間質(zhì)EVTs中。我們實(shí)驗(yàn)證明了在子癇前期的胎盤中GCM1和HtrA4在基因和蛋白水平均降低,且具有相關(guān)性,因此,GCM1有可能通過激活HtrA4的表達(dá)來調(diào)節(jié)EVTs的分化,進(jìn)而促進(jìn)EVTs侵襲,并同時(shí)抵消合胞素的融合活性,但HtrA家族其他蛋白主要通過PDZ結(jié)構(gòu)進(jìn)行活化作用,所以,HtrA4也有可能促進(jìn)合胞素表達(dá),還需要進(jìn)一步實(shí)驗(yàn)研究。 結(jié)論 HtrA4與GCM1在子癇前期胎盤中均表達(dá)異常,而且具有相關(guān)性,兩者在子癇前期發(fā)展過程中可能相互作用,有助于進(jìn)一步了解子癇前期發(fā)病機(jī)制。
[Abstract]:research objective
HtrA4 (high temperature reguirement A4, HtrA4) is an untypical member of the HtrA family protein. The HtrA family protein is serine protease, which plays a quality control role in the error of folding and position error. In recent years, some reports have been made on HtrA1 and so on, which mainly play a role in regulating the differentiation and invasion of trophoblastic cells, but for HtrA4 studies. Not much. The expression level of HtrA4 in preeclampsia patients in preeclampsia was lower than that of normal blood pressure pregnant women; compared with normal villi, the expression of HtrA4 in the syncytio cell layer was significantly increased in the patients with preeclampsia. The known GCM1 (Glial cells missing1) is an important transformation factor in the process of fetal disc formation. The expression of syncytin fusion protein and the expression of placental growth factor (PGF), the expression of placental growth factor (PGF), which promote the formation of placenta, and the expression of.Liang-Jie Wang[3] by chromatin immunoprecipitation chip, confirm that the HtrA4 gene is the target gene of GCM1. This study was on HtrA4 and GCM1 in placental group of hypertensive patients with pregnancy. The relationship between the two involved in regulating the invasion mechanism of trophoblastic cells was explored, providing a theoretical basis for the pathogenesis of preeclampsia and other pathological pregnancies.
Experimental method
1. the subjects randomly collected 60 cases of maternal placenta in the Qilu Hospital of Shandong University from April 2013 to July 2013, of which 40 cases of pregnancy induced hypertension were used as the experimental group (23 cases of early onset preeclampsia and 17 cases of late eclampsia), and 20 cases of pregnant women in the same period were selected as the control group. There was no statistical difference in gestational age between the two groups (Eighth Edition). There was no significant difference in age, gestational age, diabetes, heart disease and kidney disease.
2. RT-PCR extract total RNA, select the qualified mRNA as a template for reverse transcription reaction, reverse transcription conditions strictly according to the instructions to operate.PCR primers by Beijing Bo arson Biotech Corp, GCMl upstream: 5'-GTTAGAAGCTGAGGCAAGAA-3', downstream: 5'-TAAAGATTACCCGCGGAT-3'; HtrA4 upstream: 5'-GTCAGCACCAAACAGCG-3', downstream. : 5'-GGAGATTCCATCAGTCACCC-3'; upstream of beta -actin: downstream of 5'-AACTCCATCATGAAGTGAC-3': 5 '-GATCCACATCTGCTGGAAGG-3', all PCR is carried out according to instructions. After gel electrophoresis, the amplified products are analyzed on the D-140 image recording analysis system for the mRNA expression of.HtrA4 and GCM1 in HtrA4 or GCM1 amplification of the DNA strip, respectively. Count.
Western blotting: was taken from 100mg tissue to cut and break for protein extraction, gel electrophoresis, transmembrane, hybridization, color rendering, imaging instrument analysis, and results.
3. statistical method: the statistical analysis was carried out by SPSS17.0 software package, the average number of standard deviation was expressed. The single factor variance analysis was used in the group. The t test was used, and the correlation analysis between groups was tested by Spearman.
Result
At mRNA level, both HtrA4 and GCM1 were expressed in the group, and the expression of HtrA4 and GCM1mRNA in the study group was lower than that in the healthy control group. The difference was statistically significant (P0.01). The expression of HtrA4 and GCM1 in the placental tissue of the preeclampsia group was positively correlated (mRNA:r-0.426, protein: r= 0.686, P0.01).
discuss
The relationship between GCM1 and preeclampsia is GCM1, GCMa, and GCM protein is a transcription factor with conservative DNA binding region. Human GCM1 is mainly in placental tissue. It is a necessary transcription factor in the development of placenta. In hypoxic conditions, the activity of GCM1 is degraded and the activity of GCM1 is inhibited, and GCM1 is mainly through the promotion of syncytin and PGF. Promoting the fusion and differentiation of trophoblastic cells, while HtrA4 can bind to the surface substructure of the PDZ domain with the surface substructure of syncytin, it has been reported that HtrA4 and GCM1 are intersecting in the interstitial EVTs of the maternal fetal interface. Our experiment proved that GCM1 and HtrA4 in the placenta of preeclampsia have an average decrease in gene and protein water and are related, therefore, GCM1 has a possible effect. The expression of HtrA4 can regulate the differentiation of EVTs, and then promote the invasion of EVTs and counteract the fusion activity of the syncytin, but the other proteins of the HtrA family are activated by the PDZ structure. So, HtrA4 may also promote the expression of syncytin, and a further experimental study is needed.
conclusion
HtrA4 and GCM1 are abnormal in the placenta of preeclampsia and are related, and they may interact in the development of preeclampsia, which can help to further understand the pathogenesis of preeclampsia.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.244

【共引文獻(xiàn)】

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