摘要
妊娠高血压综合征是一种复杂的疾病,主要临床表现为妊娠20周后出现高血压和蛋白尿。目前疾病的病因学仍然不清楚,推测妊娠高血压患者胎盘绒毛组织中金属蛋白酶的表达异常,造成细胞外基质水解发生障碍,影响胎盘滋养层正常侵袭,可能是造成疾病发生重要原因。
研究目的:
研究金属蛋白酶MMP-9和ADAMTS家族(ADAMTS-1、-4、-5)表达与妊娠高血压的相关性,以及启动子区DNA甲基化状态调控的作用。
研究方法:
选取末期妊娠的妊娠高血压患者(20例)和正常孕产妇(18例)的胎盘组织,利用石蜡包埋切片.免疫组化染色、荧光实时定量PCR技术,研究病例组和对照的金属蛋白酶在蛋白水平和mRNA水平的表达差异。同时,利用限制性内切酶-PCR检测技术,检测基因启动子区的甲基化状态,利用生物信息学分析特异位点结合的转录因子,分析DNA甲基化状态调控金属蛋白酶的异常表达。
研究结果:
1)免疫组化结果:
病例组的MMP-9在滋养层细胞和绒毛间质细胞高表达,而对照组只有少量表达;病例组的ADAMTS-4在合体滋养层细胞高表达,在细胞滋养层细胞也有少量表达,而对照组表达水平很低;病例组的ADAMTS-5在滋养层细胞中高表达,明显高于对照组。
2)荧光实时定量PCR结果:
MMP-9、ADAMTS-1、-4、-5在病例组中的表达高于在对照组的表达。
3)启动子区甲基化状态检测:
病例组和对照组MMP-9的整体甲基化水平没有差异,但是在-712位点差异显著,去甲基化的比例分别为90%对44%(p＜0.05);病例组中ADAMTS-1的整体甲基化水平显著降低,去甲基化的比例为48.8%对30.6%(p＜0.05),并且-944位病例组与对照组去甲基化的比例为55%对22.2%(p＜0.05);ADAMTS-4的整体甲基化水平在病例组中也有显著性降低,去甲基化的比例分别20%对4.4%(p＜0.05),并且-323位病例组与对照组去甲基化的比例为25%对0(p＜0.05);病例组ADAMTS-5的整体甲基化水平相对对照组降低了一半,去甲基化的比例为40%对20.8%(p＜0.05),并且+72位病例组与对照组去甲基化的比例为30%对0(p＜0.05)。
结论:
1)首次证实了ADAMTS家族(ADAMTS-1、-4、-5)与妊娠高血压疾病发生相关;
2)首次检测金属蛋白酶MMP-9和ADAMTS家族(ADAMTS-1、-4、-5)在妊娠高血压患者胎盘中的甲基化状态,证实了表观遗传甲基化状态的改变造成基因表达量升高,可能是疾病发生的原因。
3)筛选出基因启动子区的特异位点,推测可能单个位点的甲基化状态起着关键作用,为产前妊娠高血压的早期诊断与药物研发提供了特异的分子标记。
Pregnancy-induced hypertension syndrome(PIH) is a multisystem disease classically defined on the basis of hypertension and proteinuria after 20 weeks gestation.The etiology of the disease remains poorly understood but it is known that altered expression of some metalloproteinases resulted extracellular matrix degrading disorder may be responsible for the abnormal trophoblast invasion.
Objective:To perform a systematic expression analysis of MMP-9 and ADAMTS family (ADAAMTS-1,-4,-5) genes in normal and pathological placentas and to pinpoint epigenetic alterations inside promoter regions of genes.
Methods:Placentas were obtained from 20 patients with PIH and 18 normal pregnancies in the third trimester.Immunohistochemistry of paraffin wax-embedded slices and Quantitative Real-Time PCR methods were used to detect the protein and mRNA expression level of different genes.The methylation status of the promoter regions was analyzed with methylation-sensitive restriction enzymes,followed by PCR amplification in order to study the association between demethylation of specific CpG sitesand abnormal expression.
Results:
1) Immunohistochemistry:MMP-9 was highly expressed in trophoblast and Villous stromal cells in PIH samples,while much lower in normal controls;ADAMTS-4 was highly expressed syncytiotrophoblast cells and lower in cytotrophoblast cells,while there was almost no diction in normal controls;ADAMTS-5 expressed highly in trophoblast cells,apparently higher than normal controls.There was no suitable antibody for ADAMTS-1.
2) Quantitative Real-Time PCR results showed MMP-9,ADAMTS-1,-4,-5 were expressed higher in PIH samples than normal controls.
3) The methylation status of the promoter regions:There was no significant higher level of MMP-9 demethylation between PIH samples and normal controls.But the percentage of unmethylated-712 site was higher in preeclampsia patients(90%) comparing with controls 44%(p＜0.05);ADAMTS-1 was significant higher demethylation in PIH samples than normal controls,the percentage was 48.8%vs. 30.6%(p＜0.05),with the specific site-944 which the unmethylation percentage was 55%vs.22.2%(p＜0.05);ADAMTS-4 was significant higher demethylation in PIH samples than normal controls,the percentage was 20%vs.4.4%(p＜0.05),with the specific site -323 which the unmethylation percentage was 25%vs.0(p＜0.05); ADAMTS-5 was significant higher demethylation in PIH samples than normal controls,the percentage was 40%vs.20.8%(p＜0.05).with the specific site +72 which the unmethylation percentage was 33%vs.0(p＜0.05).
Conclusion:
1) Demonstrated the association between the altered synthesis of ADAMTS-1,-4,-5 and PIH etiology for the first time;
2) For the first time to study the expression of MMP-9,ADAMTS-1,-4,-5 may result from epigenetic changes of the methylation status of CpG sites in the promoter region and was responsible for PIH happens.
3) Screened out specific sites of promoter regions may play key roles in gene regulation. This study provided new biomarkers for early diagnose and drug investigations.
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