Abstract
目的
研究辅助生殖中体外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)技术对早期胎盘滋养层细胞黏着斑激酶(focal adhension kinase,FAK)信号通路基因表达的影响,探讨IVF-ET技术对早期胎盘发育和功能的影响。
方法
收集IVF-ET来源的于7~8周经超声引导下减胎获得的胎盘绒毛组织作为研究组,对照组采用自然妊娠双胎7~8周人工流产术中获得的胎盘绒毛组织。利用美国Affymetrix HG-U133 Plus 2.0基因芯片对两组胎盘绒毛组织进行芯片杂交分析,实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,qRT-PCR)验证其中8个差异表达基因,选取差异表达基因进行无监督聚类分析和生物信息学分析。
结果
获得4例IVF-ET减胎绒毛组织和4例自然妊娠人工流产绒毛组织进行基因芯片检测。与自然妊娠组相比,IVF-ET组FAK信号通路中有32个基因差异表达,差异表达倍数≥2,其中12个基因上调,20个基因下调。经qRT-PCR验证,IVF-ET与自然妊娠早期胎盘绒毛中的8个FAK信号通路基因表达确实存在差异,与基因芯片检测结果一致。FAK信号通路基因定位显示,IVF-ET来源胎盘绒毛组织FAK信号通路上游基因表达受到影响,胎盘滋养层细胞通过基因表达代偿维持FAK信号通路功能基本正常。
结论
IVF-ET来源和自然妊娠来源的早期胎盘FAK信号通路存在基因表达差异,差异表达基因涉及多种FAK信号通路关键功能,影响IVF-ET胎盘绒毛早期发育和功能,同时胎盘滋养层细胞通过改变相关基因表达来代偿IVF-ET技术本身的干扰,以维持FAK信号通路正常功能,满足胎盘绒毛和胎儿发育需要。
Keywords: 受精, 体外, 胚胎移植, 滋养层, 黏着斑激酶, 基因表达
Abstract
Objective
To study the effects of in vitro fertilization-embryo transfer (IVF-ET) technique on gene expression of focal adhension kinase (FAK) signaling pathway in early placental trophoblast cells, and to explore the effects of IVF-ET technology on the development and function of early placenta.
Methods
We collected 7-8 weeks of gestation placenta tissue as a study group by ultrasound guided reduction of fetal from double embryo transfer under IVF-ET technology. In the control group, placenta tissues were obtained from the spontaneous abortion of natural pregnancy twin 7-8 weeks. Microarray hybridization analysis was performed on the placenta tissue of the two groups using the Affymetrix HG-U133 Plus 2.0 gene chip. Eight differentially expressed genes were identified by real-time quantitative polyme-rase chain reaction (qRT-PCR), and unsupervised clustering analysis and functional bioinformatics analy-sis were performed for the differentially expressed genes.
Results
Twenty-eight cases of IVF-ET reduced fetal villi and 8 cases of spontaneous abortion villi were collected. A total of 8 placental villi were detected by the gene chip. Compared with the natural pregnancy control group, 32 differentially expressed genes in the placental FAK signaling pathway were expressed in IVF-ET. The differential expression was greater than or equal to 2 times, of which 12 genes were up-regulated and 20 were down-regulated. The qRT-PCR showed that the expression of the 8 genes in FAK signaling pathways of IVF-ET was significantly different from that in the placenta of natural pregnancy, which was consistent with the result of the gene chip detection. The FAK signal pathway gene localization showed that the FAK gene was mainly located in the upstream of the signal pathway in the placenta of IVF-ET. The placental trophoblast cells maintained the FAK signaling pathway function through gene expression compensation.
Conclusion
There are gene expression differences in the FAK signaling pathway between the IVF-ET derived early placenta and the natural pregnancy placenta. The differentially expressed genes are involved in many key functions of the FAK signaling pathway and affect the early development and function of the IVF-ET placenta, while the placental trophoblast cells change gene expression for interference to compensate for IVF-ET technology itself, maintain normal function of the FAK signaling pathway, and satisfy the need for placental and fetal development.
Keywords: Fertilization in vitro, Embryo transfer, Trophoblasts, Focal adhension kinase, Gene expression
辅助生殖技术(assisted reproductive technology,ART)的安全性一直广受关注,流行病学调查研究显示,即使经过混杂因素的调整,辅助生殖的围产期合并症发病率仍高于普通人群,其病理生理机制与胎盘发育不良有关,表现为胎盘滋养层细胞侵袭能力下降,对子宫螺旋动脉重塑不足[1]。动物模型研究证实,与自然受孕相比,体外受精-胚胎移植(in vitro fertilization-embryo transfer,IVF-ET)会增加胎盘滋养层细胞功能异常和流产的发病率[2]。
黏着斑激酶(focal adhension kinase,FAK)是一种非受体型络氨酸蛋白激酶,能够依赖整合素将细胞外信号向细胞内转导,其磷酸化激活以及由此产生的下游蛋白质磷酸化,是细胞外基质与细胞相互作用并产生一系列生物学效应的关键环节,参与了细胞增殖、迁移与凋亡的调节过程,是细胞内多条信号通路的交汇点,对胎盘滋养层细胞的粘附、侵袭功能起关键作用,FAK信号转导机制已成为生物学研究热点和治疗的新靶点[3]。
本研究将自然妊娠与IVF-ET来源的早期胎盘绒毛组织进行比较,探讨IVF-ET对早期胎盘FAK信号通路功能的影响,寻找差异表达基因和调控分子机制,从胎盘早期发育视角初步探讨ART的安全性和可能的病理生理机制。
1. 资料与方法
1.1. 资料来源与分组
选取2014—2017年在北京大学第三医院生殖中心接受IVF-ET并双胎移植的4例孕妇作为研究组,同期计划生育手术室行人工流产4例双胎妊娠孕妇作为对照组。IVF-ET组临床资料通过生殖中心临床资料数据库收集,入选标准为年龄30~35岁,因为输卵管因素接受IVF-ET治疗后双胚胎移植,为双绒毛膜双胎,妊娠7~8周经超声引导下减为单胎,剩余胚胎妊娠经过正常,无妊娠并发症及出生缺陷。其双胎减为单胎的原因有:高血压等慢性疾病、瘢痕子宫、子宫平滑肌瘤、宫颈机能不全或患者要求。对照组采用同年龄、同孕龄自然妊娠双绒毛膜双胎,人工流产前超声确定为双绒毛膜双胎,采用物理方法扩张宫颈管。纳入本研究的孕妇基本临床资料见表1,两组孕妇均身体健康,无妊娠合并症,减胎操作和人工流产过程中均未使用前列腺素制剂。两组孕妇均签署知情同意书,本研究方案和标本获取经北京大学第三医院医学科学研究伦理委员会批准(医伦审:2014075)。
1.
基因芯片检测病例一般资料
General information on gene chip detection cases
| Case number | Age/years | Number of pregnancies | Number of births | Pregnancy time |
| IVF-ET, in vitro fertilization-embryo transfer. | ||||
| Control 1 | 34 | 3 | 0 | 7 weeks+1 |
| Control 2 | 34 | 3 | 0 | 7 weeks+5 |
| Control 3 | 31 | 1 | 0 | 7 weeks+2 |
| Control 4 | 33 | 2 | 0 | 7 weeks+6 |
| IVF-ET 1 | 34 | 3 | 1 | 7 weeks+2 |
| IVF-ET 2 | 30 | 2 | 0 | 7 weeks+6 |
| IVF-ET 3 | 33 | 3 | 1 | 7 weeks+1 |
| IVF-ET 4 | 34 | 3 | 1 | 7 weeks+3 |
1.2. 胎盘绒毛组织标本获取及染色体核型分析
排空膀胱,取膀胱截石位,常规消毒铺巾后,超声监视下置入装有穿刺针导架的带有无菌探头套的阴道探头(频率6.5 MHz), 以了解孕囊大小、位置及相互关系。采用16 G双腔穿刺针,沿超声穿刺引导线进针,穿过引导及子宫壁刺入胚体后,用20 mL注射器抽吸胚胎组织,观察有胚胎组织吸出或残留胚胎无胎心搏动之后退出穿刺针,在胚胎和子宫内膜交界处吸取胎盘绒毛组织。对照组自然妊娠双胎采用常规人工流产术获得胎盘绒毛组织。
纯化胎盘绒毛,置于热台倒置显微镜下分离去除血、胚胎组织和其他杂质,进行绒毛组织的染色体核型分析。两组染色体核型检测结果正常的绒毛组织均在取材后10 min内立即液氮保存,用于后续基因芯片检测。
1.3. RNA提取
利用Macherey Nagel NucleoSpin RNA Ⅱ Kit试剂盒(迪伦,德国)提取胎盘绒毛组织总RNA。抽提的总RNA经质检,核糖体28S/18S在1.0~1.5,符合后续基因芯片检测和实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,qRT-PCR)样本检测标准。
1.4. 基因芯片分析
分别比较两组胎盘绒毛组织FAK信号通路基因表达差异,将等量胎盘绒毛组织总RNA(2 μg)用于合成双链互补DNA(complementary DNA,cDNA),利用MessageAmp Ⅱ aRNA Amplification Kit(Ambion公司,美国)行生物素标记得到互补RNA(complementary RNA,cRNA)。根据美国Affymetrix公司的实验守则将cRNA片段化,产生35~200 bp大小的cRNA片段,用于基因芯片杂交。基因芯片杂交及数据分析所用仪器和设备均来自美国Affymetrix公司,将包含47 000 个转录本的人类基因组U133 plus 2.0芯片置于640型杂交炉,45 ℃条件下旋转杂交16 h,随后用450型基因芯片射流站完成芯片洗涤和染色(链霉亲和素-藻红蛋白),在3000型基因芯片扫描仪下对杂交数据进行扫描,将数据导入基因芯片操作系统GCOS 1.4进行分析。利用Significance Analysis of Microarrays(SAM)软件3.02版,采用二分类、非配对方法进行统计,筛选出IVF-ET组和对照组胎盘绒毛组织FAK信号通路差异表达基因。上述胎盘绒毛组织均浆、RNA提取及基因芯片相关实验均由北京博奥生物有限公司完成。
1.5. qRT-PCR检测
利用qRT-PCR技术验证基因芯片检测结果,根据FAK信号通路差异基因涉及的不同功能,选择FAK信号通路中8个具有代表性的基因进行验证。RNA 样本取自基因芯片检测后剩余标本。使用PrimeScript反转录试剂盒(Perfect Real time,宝生物工程大连有限公司)完成第一链互补合成反应。PRISM 7300 qRT-PCR仪系统(ABI公司,美国)行扩增反应,以GAPDH为内参,每个qRT-PCR检测设置3个复孔,利用ΔΔCt法分析结果。
1.6. 统计学方法
采用SPSS 16.0软件进行统计学处理,数据以均数±标准差表示,Student’s t检验分析各实验数据,P<0.05为差异有统计学意义。
2. 结果
2.1. 基因芯片结果
SAM软件分析发现,IVF-ET组与对照组妊娠7周的胎盘绒毛组织中涉及的FAK信号通路共有32个差异基因表达,差异表达倍数≥2,其中12个基因上调,20个基因下调(图1、2)。上调基因有原癌基因(jun proto-oncogene,JUN)、糖原合成激酶3β(glycogen synthase kinase 3 beta,GSK3B)、血小板衍生生长因子受体α多肽(platelet-derived growth factor receptor,alpha polypeptide,PDGFRA)、细胞周期蛋白D1(cyclin D1,CCND1)和整合素α(integrin alpha,ITGA)等,下调基因有丝裂原活化蛋白激酶9(mitogen-activated protein kinase 9,MAPK9)、表皮生长因子受体(epidermal growth factor receptor,EGFR)、纤连蛋白1(fibronectin 1,FN1)和B淋巴细胞瘤2(B-cell lymphoma 2,BCL2)等(图1)。利用无监督聚类软件对胎盘绒毛和胚胎组织FAK信号通路差异表达基因进行分析,结果显示8例样本被聚为两大类,与IVF-ET组胎盘绒毛组织和对照组的区分完全一致,差异明显(图3)。
1.
基因芯片检测FAK信号通路差异基因Heatmap分析
Gene chip detection of FAK signaling pathway differential gene Heatmap analysis
The expression of FAK signaling pathway genes in the placenta tissue of IVF-ET group and control group, the red represents gene up-regulated and the green represents gene expression down-regulated. VTN, vitronectin; ECM, extracellular matrix protein; ITGA, integrin, alpha; PDGFRA, platelet-derived growth factor receptor, alpha polypeptide; JUN, Jun proto-oncogene; PDGFC, platelet derived growth factor C; PDGFD, platelet derived growth factor D; CCND1, cyclin D1; FYN, FYN oncogene related to SRC; SHC, SHC (Src homology 2 domain containing) transforming protein 2; GSK3B, glycogen synthase kinase 3 beta; PAK, P21 protein (Cdc42/Rac)-activated kinase; BRAF, V-raf murine sarcoma viral oncogene homolog B1; ITGB3, integrin, beta 3; CAV2, caveolin 2; ERBB2, V-erb-b2 erythroblastic leukemia viral oncogene homolog 2; ITGB6, integrin, beta 6; FN1, fibronectin 1; CAV1, caveolin 1; HGF, hepatocyte growth factor; VAV3, Vav 3 guanine nucleotide exchange factor; PTEN, phosphate and tension homology deleted on chromsome ten; ITGA5, integrin, alpha 5; ITGB4, integrin, beta 4; FLT1, Fms-related tyrosine kinase 1; PAK6, P21 protein (Cdc42/Rac)-activated kinase 6; PIK3CB, phosphoinositide-3-kinase, catalytic, beta polypeptide; PGF, placental growth factor; MAPK9, mitogen-activated protein kinase 9; LAMA2, laminin, alpha 2; BCL2, B-cell lymphoma 2; EGFR, epidermal growth factor receptor.
3.
基因芯片检测FAK信号通路差异基因无监督聚类分析
Unsupervised cluster analysis of differential genes in FAK signaling pathway by gene chip
Unsupervised cluster analysis showed FAK signaling pathway differential gene expression between placental villus tissues, red for IVF-ET and green for control.
2.
基因芯片检测FAK信号通路差异基因双对数散点图
Double log scatter plot of gene chip detection FAK signaling pathway differential gene
The double-log scatter plot showed that the expression of FAK signaling pathway genes were different by gene chip in the IVF-ET group and the control group. Red pots represent up-regulation of gene expression; Green pots represent down-regulation of gene expression.
2.2. 差异基因功能分析
通过京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库进行生物信息分析,显示差异表达基因名称及其在FAK信号通路中的位置和相互调节关系。结果可见,IVF-ET技术影响早期胎盘绒毛组织FAK信号通路的上游基因表达,胎盘滋养层细胞通过基因差异表达代偿,基本可以保证FAK信号通路的基本功能(图4)。
4.
IVF-ET对胎盘绒毛FAK信号通路基因表达的影响
Effect of IVF-ET on gene expression of FAK signaling pathway in placental villi
The changes in gene expression in the FAK signaling pathway of placental villus in the IVF-ET technique and the position and mutual regulation of the genes are shown.Red represents up-regulation of gene expression and green represents down-regulation of gene expression.Abbreviations as in Figure 1.
2.3. qRT-PCR验证
采用qRT-PCR验证基因芯片对FAK信号通路差异基因的检测结果(图5),分别以GAPDH为内参与对照组相比,IVF-ET组胎盘绒毛组织中JUN、GSK3B、PDGFRA和ITGA基因表达上调,MAPK9、EGFR、FN1和BCL2基因表达下调,差异有统计学意义。qRT-PCR结果和基因芯片检测结果一致,说明基因芯片检测结果可信度较高。
5.
qRT-PCR比较IVF-ET组和对照组胎盘FAK信号通路基因差异表达
qRT-PCR comparison of differential expression of genes in FAK signaling pathway in IVF-ET and control placenta
qRT-PCR results showing differential expression of eight FAK signaling pathway genes, including JUN, GSK3B, PDGFRA, ITGA, MAPK9, EGFR, FN1 and BCL2, showing up-regulated genes (red columns), down-regulated genes (green columns) and control groups (blue column). Abbreviations as in Figure 1. All data were ±s. *P<0.05, **P<0.01.
3. 讨论
ART影响胎盘滋养层细胞发育和功能,其过程中囊胚滋养外胚层与培养基和外界环境直接接触,受到体外培养环境干扰,可能影响未来胎盘滋养层细胞分化、发育和功能[4]。动物模型研究结果符合这一假设,ART过程中的超排卵和胚胎培养无论是孤立还是相关,都可能导致几个基因座位中的囊胚表观遗传缺陷[5]。妊娠早期,小鼠胎盘中的基因表达修饰不同,这取决于培养环境的丰富度,比如使用成分简单的M16培养基和成分更复杂的G1/G2培养基会使小鼠胎盘基因表达发生明显差异[6]。不同的ART也会对胎盘滋养层细胞基因表达产生不同的影响,比如IVF-ET与人工授精相比,牛胎盘绒毛中miRNA表达会发生明显差异[7]。ART改变胎盘和胎儿生长的动力学与各种生物学途径中的修饰有关,可能会引发胎盘代偿现象。虽然调整这种胎盘功能代偿的分子机制仍然模糊,但表观遗传变化肯定在其适应机制中发挥作用[8]。根据胎盘滋养层细胞在发育中的功能变化和已知的围产期病理生理机制,滋养层细胞发育、分化、侵袭和重塑螺旋动脉的关键功能集中在胎盘绒毛发育早期阶段,本研究选用ART来源的妊娠7~8周减胎的新鲜胎盘绒毛组织,较好地反映了IVF-ET对胎盘滋养层细胞发育和功能的影响。
胎盘滋养层细胞是妊娠过程中最活跃的细胞之一,其内部复杂的结构和功能变化使滋养层细胞具有侵袭、螺旋动脉重塑、母胎屏障、物质交换、内分泌、免疫耐受等功能,妊娠早期其发育和侵袭过程受到时间和空间上的严格精细调控[9],其侵入过度会导致各种滋养层细胞疾病,而侵入不足则会导致自然流产、子痫前期、胎儿生长受限和妊娠期高血压等疾病[10],因此,滋养层细胞成为研究ART过程中妊娠相关疾病的焦点。FAK是整合素介导细胞质内非受体型酪氨酸蛋白激酶,在细胞内信号转导中具有重要作用,与细胞迁移、增殖、凋亡等生物学过程的关系密切[11]。近年来,FAK信号转导机制的研究越来越受到重视,已成为生物学领域研究的热点[12]。本研究发现,IVF-ET来源的人早期胎盘滋养层细胞FAK信号通路32个基因表达发生变化,与自然妊娠相比,上调基因12个,下调基因20个(图1、2)。无监督聚类分析将IVF-ET来源胎盘绒毛组织与自然妊娠来源组织区分为两类(图3),提示IVF-ET技术本身对胎盘滋养层细胞FAK信号通路基因表达产生比较明显的影响。
IVF-ET早期胎盘滋养层细胞FAK信号通路PDGFRA基因高表达。PDGFRA主要通过FAK信号通路,在表皮生长因子(epithelial growth factor,EGF)和血小板生长因子(platelet-derived growth factor,PDGF)刺激下,调节滋养层细胞对生长因子、应激刺激、细菌产物、炎症介质的细胞反应[13],表明IVF-ET过程本身即存在应激、细菌感染和无菌性炎症介质等可能,与自然妊娠相比,IVF-ET技术一定程度上激活了FAK的细胞外信号调节激酶信号通路。
本研究发现,IVF-ET组胎盘滋养层细胞FAK信号通路JUN基因表达上调(图4),JUN同时参与丝裂原活化蛋白激酶信号通路,该信号通路能够被生长因子、脂多糖、肿瘤坏死因子α、白细胞介素1、紫外线、射线、热休克、细胞外高渗及DNA变性剂等激活[14]。IVF-ET组来源胎盘滋养层细胞FAK信号通路JUN基因高表达,显示IVF-ET早期胎盘FAK信号通路过度激活,将转录调节因子JUN的氨基末端特定位点磷酸化,JUN是序列特异性转录激活蛋白1 (activating protein 1,AP-1)的成分之一,磷酸化的JUN通过诱导同源或异源二聚体形成,与AP-1位点的顺式作用原件结合,从而启动某些效应基因的转录[15]。本研究同时发现,IVF-ET早期胎盘MAPK9基因表达下调,显示出一定程度的代偿作用。此外,IVF-ET早期胎盘通过FAK信号通路高表达GSK3B,抑制胎盘滋养层细胞凋亡,显示出同样的代偿作用。
本研究发现,IVF-ET早期胎盘滋养层细胞FAK信号通路中抑癌基因(phosphatase and tensin homolog,PTEN)、FN1和BCL2基因表达下调。PTEN主要通过FAK信号途径调节胎盘滋养层细胞侵袭性和胰岛素敏感性,在先兆子痫和妊娠期糖尿病患者胎盘绒毛组织中PTEN基因低表达,PTEN基因低表达可引起滋养层细胞凋亡和大量细胞因子释放,参与先兆子痫的发病[16]。FN1通过FAK信号途径调节滋养层细胞粘附、生长和侵袭,也参与创伤修复和胚胎发育,FN1也是促进滋养层细胞增殖、链接和维持胎盘正常组织形态的重要粘附分子,在IVF-ET早期胎盘绒毛组织中FN1低表达,提示滋养层细胞粘附能力和胚胎植入能力下降[17]。
BCL2是抑制凋亡的基因,通过FAK信号通路调节胎盘滋养层细胞功能,进而影响胎盘成熟和妊娠期疾病,IVF-ET早期胎盘绒毛组织BCL2低表达,提示对凋亡刺激因素的抵抗力下降,当凋亡发展到一定程度就会影响胎盘功能并进入病理状态[18]。EGFR作为细胞外刺激信号,其通过FAK信号通路调节金属基质蛋白酶(matrix metalloproteinase,MMPs)、组织抑制剂(tissue inhibitors of metalloproteinases,TIMPs)基因和蛋白表达,从而影响滋养层细胞的侵袭和血管重铸过程,EGFR低表达表明在IVF-ET早期胎盘绒毛组织植入子宫内膜和重塑螺旋动脉的能力下降[19]。
综上,ART相关的潜在表观遗传风险越来越受到关注。本研究探讨了IVF-ET来源早期胎盘滋养层细胞FAK信号通路基因表达,研究结果与ART相关不良妊娠结局与异常滋养细胞侵袭相关的假说相一致[20]。IVF-ET可能影响胎盘滋养层细胞发育和功能,多数情况下胎盘滋养层细胞通过代偿增加侵袭能力,增加对炎症、凋亡和损伤的修复能力,调整细胞骨架以适应渗透压和氧化应激,最终成功维持妊娠和正常胎儿发育[21]。同时,若外界损伤过强或胎盘滋养层细胞代偿机制不堪重负,则会导致不同程度的不良妊娠结局,包括流产、胎儿宫内发育迟缓和妊娠期高血压等[22]。目前,对胎盘这种代偿机制和由此引发的风险还不十分了解,胚胎可能留存妊娠期间表观遗传适应机制的痕迹,加剧成年期的代谢性疾病风险[23]。IVF-ET改变胎盘和胎儿生长的动力学可能与各种生物学途径中的修饰有关,关于调整胎盘功能代偿的分子生物学机制仍然模糊,但表观遗传变化肯定在其适应机制中发挥了重要作用[24]。本研究提供的IVF-ET早期胎盘滋养层细胞FAK信号通路基因表达变化以及基因在通路中的定位和相互调节关系,将有助于IVF-ET技术的改善,增加IVF-ET技术的安全性,以确保整个ART过程更加接近自然妊娠过程和结局。
志谢:
感谢北京大学第三医院生殖医学中心提供病例标本来源!
Funding Statement
国家自然科学基金（81070493）
Supported by the National Natural Science Foundation of China(81070493)
Footnotes
The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。
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