【作者】 王利平；

【导师】 李筱梅；

【作者基本信息】 郑州大学 ， 妇产科学， 2012， 硕士

【摘要】 背景与目的子痫前期(preeclampsia,PE)是妊娠期高血压疾病的一种,是妊娠期常见而特有的疾病。该病严重影响母婴健康,是孕产妇和围生儿死亡的重要原因。20世纪80年代,国外学者首次提出,根据本病的发病时间可分为早发型子痫前期(early onset preeclampsia)和晚发型子痫前期(late onset preeclampsia)。目前,国外报导早发型子痫前期占妊娠高血压疾病的20.4%,而在我国流行病学研究发现为11.09%。且在城市,孕产妇的死亡率达18.9/10万。关于子痫前期发病机制的假说很多,近年来,测定孕妇外周血中游离核酸也成为探讨其病因及发病机理的热点之一。Michael等人首先在恶性黑色素瘤患者外周血中检测到游离mRNA,随后Poon等人首次在孕育男胎的孕妇血浆中发现Y染色体特异性锌指蛋白mRNA,并证实了孕妇外周血中存在胎儿游离mRNA。Ng等人在孕妇外周血中发现了促肾上腺激素释放激素(CRH) mRNA,肿瘤转移抑制基因(KISS-1) mRNA和选择素P(selectin-P)mRNA等,并证明它们是胎盘特异性表达的。KISS-1与妊娠滋养细胞在子宫内膜的浸润有关,是一种新的肿瘤转移抑制基因。CRH可通过激活NO/cGMP(一氧化氮/环磷酸鸟苷)通路控制胎儿-胎盘血循环,从而影响胎盘血管的生物学效应。selectin-P属于细胞粘附分子家族,介导白细胞滚动过程中与内皮细胞的粘附,致内皮细胞功能紊乱。本研究通过实时荧光定量PCR方法,分别对晚发型重度子痫前期、早发型重度子痫前期、正常妊娠孕4周>34周、正常妊娠孕周≤34周孕妇外周血中的胎儿游离KISS-1mRNA, CRHmRNA和selectin-PmRNA的表达进行相对定量,分析它们与子痫前期的相关性。探讨胎儿游离：RNA在子痫前期早期筛查中的价值。材料与方法1.研究对象：选2010年7月——2011年9月在郑州大学第二附属医院和郑州大学第三附属医院妇产科门诊及住院部的患者共105例,分为正常对照组和重度子痫前期组,具体如下：(1)重度子痫前期组：共65例,分为两组。早发型重度子痫前期组患者30例；晚发型重度子痫前期组患者35例。(2)正常对照组：共40例,分为两组,早发型对照组孕周≤34周的正常孕妇20例；晚发型对照组孕周>34周的正常孕妇20例。研究对象的筛选标准：既往体健,单胎妊娠,无其他内外科合并症及并发症,未临产,符合重度子痫前期的诊断标准,均以剖宫产终止妊娠。诊断标准参考乐杰主编人民卫生出版社出版《妇产科学》第7版。分组标准参考文献[’][2]。2.研究方法(1)提取血浆游离mRNA,反转录为cDNA,采用实时荧光定量PCR方法检测各组标本中目的基因的表达量。(2)用相对定量公式法对目标基因进行相对定量。RQ=2-ΔΔCt(3)实验数据使用SPSS17.0统计软件系统分析,a=0.05为显著性检验水准,P<0.05具有统计学意义。结果1.临床一般资料分析：重度子痫前期组和正常对照组中,年龄与生育次数之间,差异无统计学意义(P>0.05)。早发型重度子痫前期和早发型对照组孕周比较,晚发型重度子痫前期和晚发型对照组孕周比较,差异均无统计学意义(P>0.05)。2. KISS-1mRNA的表达：与正常对照组比较,重度子痫前期组明显升高(P<0.05),其中早发型重度子痫前期组高于早发型对照组(P<0.05),晚发型重度子痫前期组表达量高于晚发型对照组,差异有统计学意义(P<0.05)；早发型重度子痫前期组表达量明显高于晚发型重度子痫前期组(P<0.01)；在正常对照组中,KISS-1mRNA在早发型对照组的表达量低于晚发型对照组(P<0.05)。3CRHmRNA的表达：与正常对照组比较,重度子痫前期组明显升高(P<0.05),其中早发型重度子痫前期组明显高于早发型对照组(P<0.05),晚发型重度子痫前期组表达量明显高于晚发型对照组,差异有统计学意义(P<0.01)；早发型重度子痫前期组表达量明显高于晚发型重度子痫前期组(P<0.01)；在正常对照组中,CRHmRNA早发型对照组的表达量低于晚发型对照组(P<0.01)。4. selectin-PmRNA的表达：与正常对照组比较,重度子痫前期组明显升高(P<0.05),其中早发型重度子痫前期组高于早发型对照组(P<0.01),晚发型重度子痫前期组表达量高于晚发型对照组,差异有统计学意义(P<0.01)；且早发型重度子痫前期组表达量明显高于晚发型重度子痫前期组(P<0.01)；在正常对照组中,selectin-PmRNA早发型对照组的表达量低于晚发型对照组(P<0.05)。5. KISS-1mRNA和CRHmRNA呈正相关(r=0.696,P<0.05),CRHmRNA和selectin-PmRNA呈正相关(r=0.897,P<0.05),KISS-1mRNA和selectin-PmRNA呈正相关(r=0.712,P<0.05)。结论1.CRHmRNA、KISS-1mRNA和selectin-PmRNA可能参与了子痫前期的发生发展。2.CRHmRNA、KISS-1mRNA和selectin-PmRN A在子痫前期的发生发展中可能起协同作用。3. CRHmRNA、KISS-1mRNA和selectin-PmRNA可能成为诊断子痫前期的指标。更多还原

【Abstract】 Background and Objective:Preeclampsia(PE) is a kind of hypertensive disorder complicating pregnancy(HDCP),and it is a commom and specific disease in pregnancy.PE influences the health of mother and her baby seriously,and it is also an important reason of mortality. In1980s,some scholar divided it into two class according to the time:early onset preeclampsia and late onset preeclampsia. For now,the morbidity of early onset severe preeclampsia in HDCP is20.4%overseas,but it is11.09%at present in China and also the mortality is18.9/100000in city.There are many hypothesizes about nosogenesis of PE, the detection of free nucleic acid is becoming a hotspot in maternal plasma recently.Michael et al detected free mRNA in peripheral blood of malignant melanoma patients first. Poon et al detected fetal free ZFYmRNA in maternal plasma who conceived a boy and confirmed fetal free mRNA was existing.Ng and Tsui detected KISS-1mRNA, CRHmRNA and selectin-PmRNA were espcially expressed on plactenal.KISS-1is a new tumor metastasis suppressor gene,which is concerned with gestational trophoblastic cells the uterine endometrium invasion and placental development.CRH control the fetal placental circulation by activating NO/cGMP pathways, thus affecting placental vasculature. Selectin-P is a member of cell adhesion molecule family,which mediates the adhesion reaction between leukocyte and endothelial, inducing dysfunction of endothelial cell.The experiment detected the expression of fetal free KISS-1mRNA、 CRHmRNA and selectin-PmRNA in maternal plasma,which was divided into early onset preeclampsia,late onset preeclampsia and the control to early onset preeclampsia,the control to late onset preeclampsia,by realtime fluorescence quantitative polymerase chain reaction(PCR),to analyse the correlation between fetal free mRN A and PE.To explore the value of fetal free mRNA in PE.Materials and Methods:1. object of reseach:105cases were selected from July2010to September2011,who admitted to the Second Affiliated Hospital of Zhengzhou University and the Third Affiliated Hospital of Zhengzhou University. All pregnant were divided into two groups,as follows:(1)severe preeclampsia group:65cases,divided into two groups:30early onset preeclampsia;35cases of late onset preeclampsia.(2)control group:40cases,divided into two groups:control to early onset preeclampsia:20cases of who concepted baby≤34weeks;control to late onset preeclampsia:20cases of who concepted baby>34weeks.The selected standard of subject investigated should obey these requirement: previously healthy,singleton pregnancy,non-complication on pregnancy,not in labour,in accord with the diagnostic criteria of severe preeclampsia, select the way of termination of pregnancy is uterine-incision delivery.The standard of diagnosis should refer to the textbook<Obstetrics and Gynecology>,which punlished in renmin Medical Publishing House,the7edition, lejie chief editor.The standard of classification should refer to references.2. The study method:(1)Extracting free mRNA from maternal plasma,reverse mRNA to cDNA,detecting the content of fetal free KISS-1mRNA、CRHmRNAand selectin-PmRNA in maternal plasma in all groups.(2)Relative Quantification of target gene by RQ formula,RQ=2-ΔΔCt(3)The result of experiment were analyzed by SPSS17.0and P<0.05were defined as statistical significance.Result:1. Analysis of clinical data:comparing severe preeclampsia group and control group:we found that the age and birth number within these groups were non-statistical significance (P>0.05);gestational weeks between early onset preeclampsia group and the control to it were non-statistical(p>0.05),and it also were non-statistical between late onset preeclampsia and the control to it (P>0.05)2.The expression of KISS-1mRNA:severe preeclampsia group were obviously rised comparing to control group, and we found that early onset preeclampsia group were higher than the control to early onset preeclampsia group obviously (P<0.01), also late onset preeclampsia were higher than the control to late onset preeclampsia group obviously (P<0.05), the difference was regarded as statistical significance;the expression of kiss-1mRNA was ralated to severity of diease, and early onset severe preeclampsia were higher obviously than later onset severe preeclampsia (P<0.01); the control to early onset preeclampsia group expression of kiss-1mRNA were lower than control to late onset preeclampsia group (P<0.05)3.The expression of CRHmRNA:severe preeclampsia group were obviously rised comparing to control group, and we found that early onset preeclampsia group were higher than the control to early onset preeclampsia group obviously (P<0.01), also late onset preeclampsia were higher than the control to late onset preeclampsia group obviously (P<0.01), the difference was regarded as statistical significance;the expression of CRHmRNA was ralated to severity of diease, and early onset severe preeclampsia were higher obviously than later onset severe preeclampsia (P<0.01);the control to early onset preeclampsia group expression of CRHmRNA were lower than control to late onset preeclampsia group (P<0.01)4. The expression of selectin-PmRNA:severe preeclampsia group were obviously rised comparing to control group, and we found that early onset preeclampsia group were higher than the control to early onset preeclampsia group obviously (P<0.01),also late onset preeclampsia were higher than the control to late onset preeclampsia group obviously (P<0.01),the difference was regarded as statistical significance;the expression of selectin-PmRNA was ralated to severity of diease, and early onset severe preeclampsia were higher obviously than later onset severe preeclampsia (P<0.01);the control to early onset preeclampsia group expression of selectin-PmRNA were lower than control to late onset preeclampsia group (P<0.05)5. Positive correlation between the KISS-1mRNA and CRHmRNA(r=0.696, P<0.05); positive correlation between the CRHmRNA and selectin-PmRNA (r=0.897, P<0.05) positive correlation between the KISS-1mRNA and selectin-PmRNA (r=0.712, P<0.05)Conclusion:1.KISS-1mRNA、CRHmRNA and selectin-PmRNA might play important role in the occurrence and development of preeclampsia.2.KISS-1mRNA、CRHmRNA and selectin-PmRNA play synergistic effect role in the development of preeclampsia.3.KISS-1mRNA、CRHmRNA and selectin-PmRNA might be the diagnostic criteria of preeclampsia.更多还原