摘要
稽留流产是指在妊娠20周前,在没有任何外界干预因素的情况下,胚胎或胎儿在宫内死亡后未及时排出。造成稽留流产的原因很多,如染色体的数量、结构异常,免疫功能不全,内分泌功能紊乱,子宫内环境异常,遗传性血栓形成倾向,全身感染性疾病,环境不良因素,这些因素大约占稽留流产发病原因的50%。稽留流产的发生可能是多因素共同作用的结果,但是确切机制尚不明确。
有研究发现成功的妊娠依赖于必要的滋养层细胞凋亡和适当足量的血管生成。稽留流产可能与过度的细胞凋亡和较少的血管生成有关。细胞凋亡是个体发育过程中基因调控下的细胞自杀活动,是多细胞有机体为调控机体发育、维护内环境稳定,由基因编码的细胞主动程序性死亡过程。正常妊娠存在一定程度的滋养层细胞凋亡,在胚胎发育过程中,适度的细胞凋亡有利于绒毛内血管腔分支形成及早孕绒毛的发育。成功的妊娠依赖于绒毛足够的血管生成,只有这样才能给胎儿供应足够的氧和营养物质。而研究证明,血管的生成过程受局部氧浓度的调节。在早期妊娠中,滋养细胞的早期发育(妊娠12周前)是在低氧环境中进行的,滋养细胞适应缺氧环境,是妊娠能成功的关键。但在异常情况下,绒毛发育不良或出现不同程度的退行性改变,增殖与退变并存,细胞滋养层细胞、合体滋养层细胞明显减少甚至消失,绒毛滋养层细胞基底膜及毛细血管基底膜轻度增厚,导致孕卵发育受阻,妊娠失败,发生稽留流产。
p53蛋白作为转录因子,通过调节多个基因的表达,在细胞周期的调控,维持细胞基因组的完整性,诱导细胞分化和凋亡过程中起着重要的作用。在正常细胞,p53基因的功能保持在较低水平,从而使细胞周期不中断或细胞不会遭受最终的死亡。当细胞的DNA受到损伤,适当的p53基因介导的细胞修复途径被激活;如果过多的损害使修复不能顺利进行,则p53介导的凋亡途径开始起作用,从而导致细胞凋亡,细胞周期停滞在G1期。有研究表明滋养层细胞凋亡相关因素p53蛋白的表达与稽留流产的发生有关。p53蛋白是一种短寿命蛋白,其细胞内浓度受其负调控因子鼠双微体基因2(MDM2)基因的严格调控。MDM2是p53调控网络中的下游基因,作为p53的重要调节因子参与细胞的生长、凋亡、细胞周期调控等过程。通过对包含p53和MDM2的负反馈回路的研究发现,野生型p53蛋白的高表达能刺激MDM2蛋白的表达升高;然而,增加的MDM2蛋白又能够抑制野生型p53蛋白的功能,从而增加了负反馈循环的作用。能够影响到p53、MDM2或p53-MDM2相互作用的因素都会对细胞命运(凋亡、细胞周期阻滞等)产生重要影响。
当基因序列的变化发生极为稀有时称为突变,当其发生较为常见时即称为多态。基因多态位点的单核苷酸改变(SNP)会明显改变基因的功能,现在越来越多的目光转向了对等位基因功能的研究。p53基因的第4外显子调控着细胞的生长抑制和凋亡,并且其第72位密码子具CCC/CGC的单核苷酸多态,使编码的精氨酸(Arg)被脯氨酸(Pro)取代。含Arg和Pro的两种p53蛋白均为野生型p53蛋白,虽然在大多数细胞中两者的稳定性相同,但两种蛋白的转录激活作用、抑制转化细胞生长和诱导细胞调亡的能力有所不同。Pro型p53似乎能更有效的激活转录,从而使许多下游基因表达上调;然而Pro型p53抑制转化细胞生长和诱导细胞调亡的能力较Arg型弱。
MDM2基因启动子的序列多态改变可以使MDM2蛋白的表达发生改变。最近,在MDM2基因启动子的内在多态位点SNP309(第一内含子的T→G核苷酸位点改变)被发现。研究结果显示,携带SNP309 G/G基因型的MDM2蛋白比携带SNP309 T/T基因型的MDM2蛋白更能够通过增加对转录子Sp1的结合,提高MDM2蛋白的表达,使p53稳定性降低,从而改变p53介导的对于DNA损伤的凋亡反应。
基于p53、MDM2基因多态及p53、MDM2基因在调控细胞凋亡、改变细胞周期和血管生成中起着重要的作用,我们将研究分为两部分,第一部分主要探讨早孕患者血液及绒毛组织p53 codon72和MDM2 SNP309的基因多态分型及绒毛组织中p53 codon72和MDM2 SNP309的基因多态与p53、MDM2基因表达的关系;第二部分检测绒毛组织中p53、MDM2、VEGF和HIF-1α基因的表达,进而通过将重组质粒pcDNA3-MDM2转染入绒毛滋养细胞株JEG-3、Bewo,研究p53、MDM2基因在绒毛滋养细胞生长过程中的作用机制,阐明稽留流产的发病原因,从而为稽留流产的早期诊治提供新的策略。
第一部分p53、MDM2基因多态与稽留流产的关系研究
【研究目的】
(1)探讨p53 codon 72和MDM2 SNP309基因多态在稽留流产及人工流产患者血液和绒毛组织中的分型,试图发现与稽留流产发生有关的高危基因型。
(2)探讨绒毛组织中p53 codon72和MDM2 SNP309基因多态与p53、MDM2基因表达的关系,进而明确多态等位基因的作用原理。
【研究方法】
(1)DNA提取试剂盒提取稽留流产和人工流产患者血液和绒毛组织中的DNA。
(2)序列特异性引物多聚酶链反应(PCR-SSP)分析稽留流产和人工流产患者血液及绒毛组织中p53 codon72的多态基因分型。
(3)限制性片段长度多态性多聚酶链反应(PCR-RFLP)分析稽留流产和人工流产患者血液及绒毛组织中MDM2 SNP309的多态基因分型。
(4)提取稽留流产和人工流产患者绒毛组织mRNA,通过实时荧光定量PCR(qReal-time PCR)检测绒毛组织中p53与MDM2基因mRNA的表达。
(5)免疫组织化学方法检测稽留流产和人工流产患者绒毛组织中p53、MDM2蛋白的定位及表达。
【结果】
(1)血液及绒毛组织中p53 codon72多态基因分型:p53 codon72 Pro/Pro基因型在稽留流产组中的发生比例明显高于人工流产组(9.47%vs.3.66%;20.00%vs.12.5%),但是两组比较,差别没有显著性(P=0.094;P=0.330)。p53 codon72 Aag/Arg基因型在稽留流产组中的发生比例低于人工流产组(34.74%vs.37.80%;20.00%vs.25.00%),但是两组比较,差别没有显著性(P=0.689;P=0.528)。
(2)血液及绒毛组织中MDM2 SNP309 G/G基因多态分型:MDM2 SNP309 G/G基因型在稽留流产组中的发生比例明显高于人工流产组(32.63%vs.18.29%:28.33%VS.12.50%),并且两组比较,差别有显著性(P=0.010;P=0.043)。MDM2 SNP309 T/T基因型在稽留流产组中的发生比例明显低于人工流产组(21.05%vs.28.05%;31.67%vs.40.625%),但是两组比较,差别没有显著性(P=0.239;P=0.352)。
(3)绒毛组织中p53 mRNA表达情况比较:p53 codon72 Arg/Arg基因型比Pro/Pro基因型更能诱导p53 mRNA水平的高表达,但是两者比较,差异没有显著性(19=0.278);MDM2 SNP309 T/T基因型比G/G基因型更能诱导p53 mRNA水平的高表达,但是两者比较,差异没有显著性(P=0.367)。
(4) p53蛋白在绒毛组织中的定位及表达:p53蛋白主要表达于早孕绒毛细胞滋养细胞、合体滋养细胞和绒毛外细胞滋养细胞柱的细胞核中;p53codon72 Arg/Arg基因型与其他基因型比较,不能诱导p53蛋白的高表达(P=0.563)。MDM2 SNP309 T/T基因型与其他基因型比较,不能诱导p53蛋白的高表达(P=0.289)。
(5)绒毛组织中MDM2 mRNA表达情况比较:p53 codon72 Pro/Pro基因型比Arg/Arg基因型更能诱导MDM2 mRNA水平的高表达,且两者比较,差异有显著性(P=0.013);MDM2 SNP309 G/G基因型比T/T基因型更能诱导MDM2mRNA水平的高表达,且两者比较,差异有显著性(P=0.04)。
(6) MDM2蛋白在绒毛组织中的定位及表达:MDM2蛋白主要表达于早孕绒毛细胞滋养细胞、合体滋养细胞和绒毛外细胞滋养细胞柱的细胞核中;p53codon72 Pro/Pro基因型可诱导MDM2蛋白的高表达,且与其他基因型比较,差别有显著性(P=0.037),MDM2 SNP309 G/G基因型可诱导MDM2蛋白的高表达,且与其他基因型比较,差别有显著性(P=0.001)。
【结论】
(1) MDM2 SNP309 G/G基因型是稽留流产发生的高危基因型。
(2) p53 codon72 Pro/Pr0基因型和MDM2 SNP309 G/G基因型能够诱导MDM2基因的高表达,最终导致稽留流产的发生。
第二部分p53、MDM2基因在稽留流产中的作用机制研究
【研究目的】
(1)探讨p53、MDM2、血管内皮生长因子(VEGF)和缺氧诱导因子-1α(HIF-1α)在稽留流产和人工流产患者绒毛组织中mRNA的表达与蛋白的表达及定位。
(2)探讨p53、MDM2基因在绒毛滋养细胞生长过程中的作用机制。
【研究方法】
(1) qReal-time PCR检测稽留流产和人工流产患者绒毛组织中p53、MDM2、VEGF和HIF-1α基因mRNA的表达。
(2)免疫组织化学方法检测稽留流产和人工流产患者绒毛组织中p53、MDM2、VEGF和HIF-1α蛋白的表达及定位。
(3)提取JEG-3、Bewo细胞的mRNA,扩增MDM2基因,鉴定两种细胞株的MDM2 mRNA的表达情况。
(4)将质粒pcDNA3和重组质粒pcDNA3-MDM2分别转染入绒毛滋养细胞株JEG-3和Bewo中,经G418筛选,用反转录-PCR(RT-PCR)法检测MDM2mRNA的表达,筛选出高表达的细胞株。
(5)将筛选成功的细胞株给予MDM2的拮抗剂(Nutlin3)处理,置于乏氧(1%O_2;5%CO_2;94%N_2)条件下培养。JEG-3细胞株分为五组:A组:JEG-3组;B组:JEG-3-pcDNA3-DMSO组;C组:JEG-3-pcDNA3-Nutlin3组;
D组:JEG-3-pcDNA3-MDM2-DMSO组;E组:JEG-3-pcDNA3-MDM2-Nutlin3组(Bewo细胞株的分组方法同JEG-3细胞株)。培养24小时后收集以上各组细胞:
1)收集5×10~5细胞加入1ml 4℃预冷的70%的乙醇-PBS溶液吹打成单细胞悬液,置于4度冰箱过夜。经PI处理后,流式细胞仪检测各组细胞的细胞周期改变;
2)剩余细胞分为两部分,分别用qReal-time PCR和免疫印迹(Westernblotting)法检测各组细胞p53、MDM2、VEGF和HIF-1α的mRNA和蛋白质表达。
【结果】
(1)稽留流产患者绒毛组织中p53、MDM2、VEGF和HIF-1αmRNA表达的相关性结果:稽留流产患者绒毛组织中,p53的表达与MDM2、HIF-1α的表达呈正相关(r=0.35;r=0.63),且相关性有显著意义(P=0.01;P＜0.001);但与VEGF的表达呈负相关(r=-0.30),且相关性有显著意义(P=0.03)。MDM2的表达与HIF-1α的表达呈正相关(r=0.28),且相关性有显著意义(P=0.04);与VEGF的表达呈负相关(r=-0.08),但是相关性没有显著意义(P=0.57)。HIF-1α的表达与VEGF的表达呈负相关(r=-0.37),且相关性有显著意义(P=0.007)。
(2)人工流产患者绒毛组织中p53、MDM2、VEGF和HIF-1αmRNA表达的相关性:在正常早孕绒毛组织中,p53的表达与MDM2、VEGF和HIF-1α的表达呈正相关(r=0.31:r=0.48;r=0.67),且相关性有显著意义(P=0.03;P=0.003;P＜0.001)。MDM2的表达与VEGF的表达呈正相关(r=0.23),但是相关性没有显著意义(P=0.11);与HIF-1α的表达呈负相关(r=-0.03),但是相关性没有显著意义(P=0.84)。HIF-1α表达与VEGF的表达呈正相关(r=0.35),且相关性有显著意义(P=0.01)。
(3)稽留流产和人工流产患者绒毛组织中p53、MDM2、VEGF和HIF-1α蛋白的定位:
p53和MDM2蛋白主要表达于两组患者绒毛组织细胞滋养细胞、合体滋养细胞和绒毛外细胞滋养细胞柱的细胞核中。VEGF蛋白主要表达于两组患者绒毛组织细胞滋养细胞、合体滋养细胞、绒毛外细胞滋养细胞柱和绒毛间质细胞的细胞浆中。HIF-1α蛋白主要表达于两组患者绒毛组织细胞滋养细胞、合体滋养细胞和绒毛外细胞滋养细胞柱的细胞浆中,偶可表达于绒毛组织合体滋养细胞和绒毛外细胞滋养细胞柱的细胞核中。
(4)稽留流产和人工流产患者绒毛组织中p53、MDM2、VEGF和HIF-1α蛋白表达情况的比较:
p53、MDM2和HIF-1α在稽留流产患者绒毛组织中的表达升高,与人工流产患者比较,差异有显著性(P＜0.001;P＜0.001;P=0.035);VEGF在稽留流产的表达明显低于人工流产患者,两组比较,差别有显著性(P=0.026)。
(5) JEG-3细胞株有MDM2 mRNA的弱表达,Bewo细胞株无MDM2 mRNA的表达。
(6)流式细胞仪检测细胞周期发现:JEG-3细胞株各组细胞加入Nutlin3处理后,细胞周期明显停滞在G1期,S期的比例减少。Bewo细胞株转染pcDNA3-MDM2的细胞株加入Nutlin3处理后,细胞周期明显停滞在G1期,S期的比例减少,而转染pcDNA3的细胞株加入Nutlin3处理后,细胞周期没有明显改变。
(7)乏氧条件下培养的JEG-3各组细胞p53、MDM2、VEGF和HIF-1αmRNA表达水平的检测:
1) p53 mRNA表达水平的比较:A组与B组比较,p53 mRNA的表达没有差别(P＞0.05);p53 mRNA的表达在C组明显高于B组,D组明显高于B组,E组明显高于D组,差别有显著性(P＜0.05)。
2) MDM2 mRNA表达水平的比较:A组与B组比较,MDM2mRNA的表达没有差别(P＞0.05);MDM2 mRNA的表达在C组明显高于B组,D组明显高于C组,E组明显高于D组,差别有显著性(P＜0.05)。
3) VEGF mRNA表达水平的比较:A组与B组比较,VEGF mRNA的表达没有差别(P＞0.05);VEGF mRNA的表达在C组明显低于B组,D组明显高于B组和E组,差别有显著性(P＜0.05)。
4) HIF-1αmRNA表达水平的比较:A组与B组比较,HIF-1αmRNA的表达没有差别(P＞0.05);HIF-1αmRNA的表达在B组明显高于C组,D组高于E组,差别有显著性(P＜0.05)。
(8)乏氧条件下培养的Bewo各组细胞p53、MDM2、VEGF和HIF-1αmRNA表达水平的检测:
1) p53 mRNA表达水平的比较:A组与B组,B组与C组,p53 mRNA的表达没有明显差别(P＞0.05);p53 mRNA的表达在D组明显高于B组,E组明显高于D组,差别有显著性(P＜0.05)。
2) MDM2 mRNA表达水平的比较:MDM2 mRNA在A组、B组和C组没有表达;MDM2 mRNA在E组的表达明显高于D组,差别有显著性(P＜0.05)。
3) VEGF mRNA表达水平的比较:A组、B组和C组比较,VEGF mRNA的表达没有差别(P＞0.05);VEGF mRNA的表达在D组明显高于B组和E组,差别有显著性(P＜0.05)。
4) HIF-1αmRNA表达水平的比较:A组和B组比较,HIF-1αmRNA的表达没有差别(P＞0.05);HIF-1αmRNA的表达在D组明显高于E组,差别有显著性(P＜0.05)。
(9)乏氧条件下培养的JEG-3各组细胞p53、MDM2、VEGF和HIF-1α蛋白表达水平的检测:
1) p53蛋白表达水平的比较:B组的表达高于A组,但低于C组和D组,并且D组的表达高于C组。
2) MDM2蛋白表达水平的比较:B组的表达高于A组,但低于C组和D组,并且D组的表达高于C组。
3) VEGF蛋白表达水平的比较:B组的表达低于A组,C组的表达明显升高,D组的表达低于C组。
4) HIF-1α蛋白表达水平的比较:A组和B组有弱表达,C组的表达略有升高,D组的表达低于C组。
(10)乏氧条件下培养的Bewo各组细胞p53、MDM2、VEGF和HIF-1α蛋白表达水平的检测:
1) p53蛋白表达水平的比较:A组和B组比较,p53蛋白的表达差别不大,C组的表达高于A组和B组,D组的表达明显高于C组。
2) MDM2蛋白表达水平的比较:A组和B组比较,MDM2蛋白的表达差别不大,但低于C组,但D组的表达高于C组。
3) VEGF蛋白表达水平的比较:A组和B组的表达差别不大,D组的表达低于C组。
4) HIF-1α蛋白表达水平的比较:A组和B组的表达差别不大,C组的表达升高,D组的表达低于C组。
【结论】
(1)稽留流产患者绒毛的血管生成明显减少。
(2) p53、MDM2参与早孕绒毛的血管生成,不同的是p53、MDM2对正常早孕绒毛血管生成起促进作用,而当细胞DNA遭到损伤或缺氧加剧,则会诱导p53、MDM2的高表达,抑制早孕绒毛的血管生成。
(3) p53、MDM2基因通过调节HIF-1α和VEGF基因的表达调控绒毛滋养细胞的生长,最终影响妊娠结局。
本研究创新点
1.首次发现MDM2 SNP309 G/G基因型是稽留流产发生的高危基因型,从而有利于筛选高危人群,早期发现及预防稽留流产。
2.首次发现p53 codon72 Pro/Pro基因型和MDM2 SNP309 G/G基因型可以诱导MDM2基因的高表达,最终导致稽留流产的发生。
3.首次发现早孕绒毛生长受p53、MDM2基因调控。
本研究发现了稽留流产发病的高危因素,为稽留流产的预测、诊断和治疗提供了新思路。
Missed abortion refers to pregnancy at 20 weeks ago,in the absence of any outside interference factors,embryonic or fetal death in utero is not timely discharge. Missed abortion caused by many reasons such as the number of chromosomes, structural abnormalities,immune dysfunction,endocrine dysfunction,abnormal intrauterine environment,genetic tendency to thrombosis,systemic infection, environmental adverse factors,these factors are about the 50%incidence of missed abortion.The occurrence of missed abortion is probably more the result of factors, but the exact mechanism is not clear.
Study found that a successful pregnancy depended on the necessary trophoblast apoptosis and appropriate sufficient angiogenesis.Apoptosis is the course under the control of gene activity of cell suicide,is a multi-cell organisms to control the body development,the maintenance of environmental stability,the initative procedure by the gene encoding the cell death process.Normal pregnancy has certain degree of trophoblast apoptosis,in embryonic development process,there is an appropriate degree of apoptosis within the lumen conducive to villi and chorionic villi branch formation and development.The success of pregnancy depends on enough villous angiogenesis,only with this can it supply adequate oxygen and nutrients.In early pregnancy,the early development of trophoblast cells(before 12 weeks of pregnancy) is a hypoxic environment,and the adaption of trophoblast cells to hypoxic environment is the key to successful pregnancy.And studies have proved that the process of angiogenesis was regulated by local oxygen concentration.However,in exceptional cases,villi dysplasia or degree of degenerative changes in both proliferation and degeneration,cytotrophoblast cells,syncytiotrophoblast cells significantly reduced or even disappear,villous trophoblast basement membrane and capillary basement membrane light degree of thickening,cytula cause developmental delay,failure of pregnancy,the occurrence of missed abortion.
P53 protein as a transcription factor,by regulating the expression of multiple genes in cell cycle regulation and control,plays an important role in maintaining the integrity of cell genome,inducing cell differentiation and apoptosis.In normal cells, p53 gene remained at a relatively low level,so that does not interrupt the cell cycle or eventual cell death.When the cells were under DNA damage,p53 appropriate cell-mediated gene repair is activated;if too much to repair the damage,the p53-mediated apoptosis began to work,resulting in apoptosis,cell cycle arrest at G1 phase.Research had shown that the expression of trophoblast apoptosis-related factor p53 protein related to the occurrence of missed abortion.P53 is a short-lived protein in cells,the concentration of it is strict regulated and controlled by its negative regulator murine double minutes2(MDM2) gene.MDM2 is of the p53 regulation network and and as an important regulator of p53 involved in cell growth inhibition,apoptosis,cell cycle regulation and control processes.Through the study of the p53 and MDM2 negative feedback loop,they found that wild-type p53 protein can stimulate the high expression of MDM2 protein;however,the increase in the MDM2 protein can inhibit the function of wild-type p53 protein,thus increasing the role of the negative feedback cycle.Those can affect p53,MDM2 gene,or the factors of the p53-MDM2 interaction can have an important impact on cell fate (apoptosis,cell cycle arrest,etc.).
When the change of the gene sequence was rare,it was called mutation;when it changed commonly,it was called gene polymorphism.Many gene single nucleotide change(SNP) will significant change the gene function,now,more and more eyelight are turning to the function of the polymorphism alleles,p53 exon 4 regulates the cell growth inhibition and apoptosis,and the 72 codons has the CCC/ CGC single nucleotide polymorphisms,then replaced arginine(Arg) by proline(Pro). Those included Arg and Pro p53 protein are all wild-type p53 protein,although in most cells the stability are same,but the ability of the two proteins of activating transcription,inhibiting cell growth and inducing apoptosis capacity are different.P ro-p53 seems to be more effective to activate transcription,so that upregulate the expression of the downstream gene;However the ability of Pro-p53 inhibiting cell growth and inducing apoptosis is weaker than Arg-p53.
MDM2 gene promoter polymorphisms of the sequence can change the expression of MDM2 protein.Recently,inherent SNP309 in the MDM2 promoter polymorphism(first intron of the T→G change in nucleotide sites) was found.The research showed that the MDM2 protein carrying SNP309 G/G genotype of MDM2 protein also can enhance their effectiveness in stimulating the adoption of the combination of Sp1 protein,then enhancing the expression of MDM2 protein to reduce the stability of p53 than the MDM2 protein carrying the T/T genotype,thus changing the p53 mediated apoptosis in the response to DNA damage.
Based on p53,MDM2 polymorphisms and p53、MDM2 play an important role in the regulation of apoptosis、cell cycle changes and angiogenesis,we divided our study into two parts,the first part mainly did the research of the polymorphism genotypes of p53 codon72 and MDM2 SNP309 in early pregnancy blood and villous samples and the relationship of the polymorphism of p53 codon72 and MDM2 SNP309 polymorphism with the expression of p53 and MDM2;The second part examined the expression of p53、MDM2,VEGF and HIF-1α,then transfected the recombined pcDNA3-MDM2 plasmid into trophoblast cell lines JEG-3、Bewo, investigated the mechanism of p53、MDM2 gene in the growing precess of villous trophoblast cells,further clarified the causes of missed abortion and to provide early diagnosis and treatment of the new strategy.
PARTⅠTHE RELATIONSHIP OF P53、MDM2 POLYMORPHISMS WITH MISSED ABORTION
Objective:
(1) To investigate the polymorphisms of p53 codon72 and MDM2 SNP309 in the blood and villous samples of missed abortion and induced abortion patients, which attempt to discover the high risk genotype of missed abortion.
(2) To investigate the relationship of p53 codon72 and MDM2 SNP309 polymorphisms with the mRNA and protein expression levels of p53 and MDM2, then to identify the mechanism of the effect of polymorphisms.
Methods:
(1) Extract the DNA of the blood and villous samples of missed abortion and induced abortion with DNA extract kit.
(2) PCR with sequence specific primers(PCR-SSP) method to analysis the genotype of p53 codon72 in blood and villous samples of missed abortion and induced abortion groups.
(3) PCR restriction fragment length polymorphism(PCR-RFLP) method to analysis the genotype of MDM2 SNP309 in blood and villous samples of missed abortion and induced abortion groups.
(4)Extract mRNA of the villous samples of missed abortion and induced abortion, using qReal-time PCR method to examine the mRNA expression level of p53 and MDM2 in villous samples.
(5) Immunohistochemistry method to examine the location and expression levels of p53、MDM2 protein in villous samples of missed abortion and induced abortion group.
Results:
(1) The genotypes of p53 codon72 in blood and villous samples:the distribution of p53 codon72 Pro/Pro genotype in missed abortion group was higher than induced abortion group(9.47%vs.3.66%;20.00%vs.12.50%),but in the comparison of the two group,the difference was not significant(P=0.094; P=0.330).The distribution of p53 codon72 Arg/Arg genotype in missed abortion group was lower than induced abortion group(34.74%vs.37.80%;20.00% vs.25.00%),but in the comparison of the two group,the difference was not significant(P=0.689;P=0.528).
(2) The genotypes of MDM2 SNP309 in blood and villous samples:the distribution of MDM2 SNP309 G/G genotype in missed abortion group was higher than induced abortion group(32.63%vs.18.29%;28.33%vs.12.50%),and in the comparison of the two group,difference was significant(P=0.010;P=0.043). The distribution of MDM2 SNP309 T/T genotype in missed abortion group was lower than induced abortion group(21.05%vs.28.05%;31.67%vs.40.625%), and in the comparison oft he two group,difference was not significant(P=0.239; P=0.352).
(3) The comparison of p53 mRNA expression in villous samples:p53 codon 72 Arg/Arg genotype induced higher p53 mRNA expression than Pro/Pro genotype, but in the comparison of the two genotypes,the difference was not significant (P=0.278);MDM2 SNP309 T/T genotype induced higher p53 mRNA expression than G/G genotype,but in the comparison of the two genotypes,the difference was not significant(P=0.367).
(4) The location and expression of p53 protein in villous samples:p53 protein was mainly expressed in the nucleuses of villous cytotrophoblast、syneytiotrophoblast and extravillous trophoblastic cell column;p53 codon 72 Arg/Arg genotype can not induce higher expression of p53 protein with compared with other genotypes (P=0.563).MDM2 SNP309 T/T genotype can not induce higher expression of p53 protein with compared with other genotypes(P=0.289).
(5) The comparison of MDM2 mRNA expression in villous samples:p53 codon 72 Pro/Pro genotype can induce higher expression of MDM2 mRNA than Arg/Arg genotype,and in the comparison of the two genotypes,the difference was significant(P=0.013);MDM2 SNP309 G/G genotype can induce higher expression of MDM2 mRNA than T/T genotype,and in the comparison of the two genotypes,the difference was significant(P=0.04).
(6)The location and expression of MDM2 protein in villous tissues:MDM2 protein was mainly expressed in the nucleuses of villous cytotrophoblast、syneytiotrophoblast and extravillous trophoblastic cell column;p53 codon 72 Pro/Pro genotype can induce higher expression of MDM2 protein,and compared with other genotypes,the difference was significant(P=0.037),MDM2 SNP309 G/G genotype can induce higher expression of MDM2 protein,and compared with other genotypes,the difference was significant(P=0.001).
Conclusions:
(1)MDM2 SNP309 G/G genotype is the high risk genotype of missed abortion.
(2) p53 codon72 Pro/Pro genotype and MDM2 SNP309 G/G genotype could induce high expression level of MDM2,then induced the occurrence of missed abortion.
PARTⅡTHE EFFECT OF P53、MDM2 ON MISSED ABORTION
Objective:
(1)To explore the mRNA expression and protein expression and location of the related genes of p53、MDM2、vascular endothelial growth factor(VEGF) and hypoxia inducible transcription factors-1α(HIF-1α) in villous samples of missed abortion and induced abortion patients.
(2)To explore the effect of p53、MDM2 on the growth of villous trophoblast cells.
Methods:
(1) qReal-time PCR method to detect the mRNA expression levels of p53、MDM2、VEGF and HIF-1αin villous samples of missed abortion and induced abortion groups.
(2) Immunohistochemistry method to detect the protein expression levels and location of p53、MDM2、VEGF and HIF-1αin villous samples of missed abortion and induced abortion groups.
(3) Extract the total mRNA of JEG-3 and Bewo,and amplying the MDM2 gene,to characterizate the expression of MDM2 mRNA in the two trophoblast cell lines.
(4) The plasmid pcDNA3 and the recombinant plasmid pcDNA3-MDM2 were transfected into trophoblast cell lines JEG-3 and Bewo,separately;after the screening of G418,using reverse transcription-PCR(RT-PCR) method to detecte the expression of MDM2 mRNA,screening out the high expression cell.
(5) With screening out high expression of MDM2 mRNA transfection cells given MDM2 antagonist(Nutlin3) treatment,under hypoxia(1%O_2;5%CO_2;94%N_2) conditions,the JEG-3 cell line was divided into five groups:A:JEG-3 group; B:JEG-3- pcDNA3-DMSO group;C,JEG-3-pcDNA3-Nutlin3 group;D, JEG-3-pcDNA3-MDM2-DMSO group;E,JEG-3-pcDNA3-MDM2-Nutlin3 group(the group of Bewo cell line was same to JEG-3 cell line).Above cells were collected after being cultured for 24 hours:
1) Collecting 5×10~5 cells added 1 ml 70%precooling alcohol-PBS stored at 4 degree,wind and percussion blending refrigerator overnight at before the treatment adopted by the PI flow cytometry to examing the changes of cell cycles in each group;
2) The remaining cells were divided into two parts,the mRNA and protein expression levels of p53、MDM2、VEGF and HIF-1αwere detected by qReal-time PCR and Western blotting,separately.
Results:
(1) The relationship of the mRNA expression level of p53、MDM2、VEGF and HIF-1αin villous samples of missed abortion patients:In the villous smples of missed abortion patients,the expression of p53 was positive correlated with the expression of MDM2、HIF-1α(r=0.35;r=0.63),and the relationship was significant(P=0.01;P＜0.001);but negatively correlated to the expression of VEGF(r=-0.30),and the relationship was significant(P=0.03).The expression of MDM2 was positive correlated with the expression of HIF-1α(r=0.28),and the relationship was significant(P=0.04);and negatively correlated with the expression of VEGF(r=-0.08),but the relationship was not significant(P=0.57). The expression of HIF-1αwas negatively correlated with the expression of VEGF(r=-0.37),and the relationship was significant(P=0.007).
(2) The relationship of the mRNA expression level of p53、MDM2、VEGF and HIF-1αin villous samples of induced abortion patients:In the villous samples of induced abortion patients,the expression of p53 was positive correlated with the expression of MDM2、VEGF and HIF-1α(r=0.31;r=0.48;r=0.67),and the relationship was significant(P=0.03;P=0.003;P＜0.001).The expression of MDM2 was positively correlated with the expression of VEGF(r=0.23),but the relationship was not significant(P=0.11);and negatively correlated with the expression of HIF-1α(r=-0.03),but the relationship was not significant (P=0.84).The expression of HIF-1αwas positively correlated with the expression of VEGF(r=0.35),and the relationship was significant(P=0.01).
(3) The location of p53、MDM2、VEGF and HIF-1αprotein in early pregnancy villous samples:
p53 and MDM2 protein was mainly expressed in the nucleuses of villous cytotrophoblast、syneytiotrophoblast and extravillous trophoblastic cell column. VEGF protein mainly expressed in the cytoplasm of syneytiotrophoblasts, cytotrophoblasts,extravillous trophoblastic cell columns of villouses and the villous mesenchymocyte of the two groups.HIF-1αprotein mainly expressed in the cytoplasm of syneytiotrophoblasts,cytotrophoblasts,extravillous trophoblastic cell columns of villouses and the villous mesenchymocyte of the two groups,occasionally expressed in the nucleuses of villous syneytiotrophoblast and extravillous trophoblastic cell column.
(4) The comparison of the expression of p53、MDM2、VEGF and HIF-1αprotein in early pregnancy villous samples:
Higher expression of p53、MDM2 and HIF-1αin villous samples of missed abortion group can be seen,and in the comparison of induced abortion group,the difference was significant(P＜0.001;P＜0.001;P=0.035);The expression of VEGF in villous samples of missed abortion group was less than induced abortion group,and in the comparison of the two group,the difference was significant(P=0.026).
(5) The JEG-3 cell line had faint MDM2 mRNA expression level,Bewo cell line had little MDM2 mRNA expression.
(6) Flow cytometry in examing the changes of cell cycle was found:After treatmented with Nutlin3,the cell cycles apparent stagnated at G1 phase,and the ratio of S phase decreased in all the groups of JEG-3 cell line.The cell cycles apparent stagnated at G1 phase,and the ratio of S phase decreased in the transfected of pcDNA3-MDM2 Bewo cell line.
(7) Under hypoxic conditions,the detection of the p53、MDM2、VEGF and HIF-1αmRNA expression levels of JEG-3 cells
1) Comparison of p53 mRNA expression level:comparing A group and B group, p53 mRNA expression level was not different(P＞0.05);p53 mRNA expression in C group was significantly higher than B group,D group was significantly higher than the B group,E group was significantly higher than D group,and the difference was significant(P＜0.05).
2) Comparison of MDM2 mRNA expression level:comparing A group and B group, MDM2 mRNA expression level was not different(P＞0.05);MDM2 mRNA expression in C group was significantly higher than B group,D group was significantly higher than C group,E group was significantly higher than D group, and the difference was significant(P＜0.05).
3) Comparison of VEGF mRNA expression level:comparing A and B group,VEGF mRNA expression level was not different(P＞0.05);VEGF mRNA expression in C group was significantly lower than B group,D group was significant higher than B and E group,the difference was significant(P＜0.05).
4) Comparison of HIF-1αmRNA expression level:comparing A and B group, HIF-1αmRNA expression level was not different(P＞0.05);HIF-1αmRNA expression in B group was significant higher than C group,D group was higher than E group,the difference was significant(P＜0.05).
(8) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αmRNA expression levels of Bewo cells
1) Comparison of p53 mRNA expression level:comparing A group and B group,B group and C group,p53 mRNA expression level was not different(P＞0.05);p53 mRNA expression in the D group was significantly higher than B group,E group was significantly higher than D group,the difference was significant(P＜0.05).
2) Comparison of MDM2 mRNA expression level:the MDM2 mRNA expression was not detected in the A group,B group and C group;MDM2 mRNA expression level in the E group was significantly higher than D group,the difference was significant(P＜0.05).
3) Comparison of VEGF mRNA expression level:comparing A、B and C group, VEGF mRNA expression level was not different(P＞0.05);VEGF mRNA expression in D group was significantly higher than B and E group,the differences were significant(P＜0.05).
4) Comparison of HIF-1αmRNA expression level:comparing A group and B group, HIF-1αmRNA expression level was not different(P＞0.05);HIF-1αmRNA expression in D group was significantly higher than E group,the difference was significant(P＜0.05).
(9) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αprotein expression levels of JEG-3 cells
1) Comparison of p53 protein expression:B group was higher than A group,but lower than C group and D group,and D group was higher than C group.
2) Comparison of MDM2 protein expression:B group was higher than A group,but lower than C group and D group,and D group was higher than C group.
3) Comparison of VEGF protein expression:B group was lower than A group,and the expression of C group was significant high,D group was lower than C group.
4) Comparison of HIF-1αprotein expression:A group and B group had faint expression,C group increased the expression slightly,D group was lower than C group.
(10) Under hypoxic conditions,the detection of the p53,MDM2,VEGF and HIF-1αprotein expression levels of Bewo cells
1) Comparison of p53 protein expression:Comparing A and B group,the expression of p53 protein was not different,C group was higher than A and B group,D group was higher than C group.
2) Comparison of MDM2 protein expression:Comparing A and B group,the expression of MDM2 protein was not different,but lower than C group,D group was higher than C group.
3) Comparison of VEGF protein expression:the expression in A group and B group was not different,D group was lower than C group.
4) Comparison of HIF-1αprotein expression:the expression in A group and B group was not different,,D group was lower than C group.
Conclusions;
(1) Agiogenesis in villous of missed abortion patients decreased obviously.
(2) p53、MDM2 participated in the angiogenesis of early pregnancy villous,but it is disparate that p53、MDM2 played a role of facilitating villous angiogenesis in normal early pregnancy,nevertheless when the DNA damaged or hypoxia intensified,p53、MDM2 would overexpress and could decrease the angiogenesis of early pregnancy villous.
(3)p53、MDM2 gene regulated the growth of villous trophoblast cells through the regulated HIF-1αand VEGF,ultimately influenced the pregnancy outcome.
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