Dizon-Townson D S, Lu J, Morgan T K, Ward K J
Departments of Obstetrics and Gynecology and Human Genetics, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Am J Obstet Gynecol. 2000 Sep;183(3):706-11. doi: 10.1067/mob.2000.106583.
The growth and differentiation of the embryo and the contiguous placental structures are fundamental to human reproduction and survival. Little is known, however, about the genetic control of these processes during early human development. Normal placentation is the result of a well-orchestrated sequence of events that consists of cellular adhesion and limited invasion controlled by relatively unknown genetic processes. We hypothesized that genes expressed by first-trimester chorionic villi constitute critical regulators of placentation and hence of early human development. Our objective was therefore to isolate and characterize genes, both known and unknown, expressed by the human placenta during the first trimester.
Tissues collected consisted of placental material collected during first-trimester elective pregnancy terminations. Fetal chorionic villi were separated grossly from maternal decidual and quickly frozen in liquid nitrogen for ribonucleic acid preservation. Tissues from different gestational ages were kept separate. Total ribonucleic acid was extracted, messenger ribonucleic acid was isolated, and complementary deoxyribonucleic acid was synthesized. Complementary deoxyribonucleic acid was cloned into the ZAP Express lambda vector (Stratagene, La Jolla, Calif). Automated sequencing of random plaques was done. Sequence homology was searched for with the Basic Local Assignment Search Tool to search the Genbank database (National Center for Biotechnology Institute, Bethesda, Md). In the event that a known gene sequence was derived, no further workup was undertaken. If no homology was identified, the complete complementary deoxyribonucleic acid insert was sequenced in its entirety. Novel genes were further characterized by tissue-specific patterns, cellular localization, and chromosomal location. Expression by fetal villi was confirmed by reverse transcriptase polymerase chain reaction.
We isolated a number of genes known to be expressed at the maternal-fetal interface. Seventeen of 186 random clones were >1 kilobase in length and did not display homology with known genes, and these may therefore constitute novel genes critical for placentation. One of the clones from a human chorionic villi complementary deoxyribonucleic acid library at 12 weeks' gestation is a 7-kilobase gene that is also abundantly expressed in human fetal brain, lung, liver, and kidney. In situ hybridization localized the transcript to the fetal renal glomerulus.
Our findings indicate that the placenta serves as a rich source for potential novel gene expression. Seventeen clones were >1 kilobase in length and are potential novel genes involved in early first-trimester placentation. One of these 17 potential novel genes is expressed in abundance in several fetal tissues, which suggests a role in early human fetal development. Other potential novel genes are currently being characterized. The powerful molecular techniques that we used to isolate genes expressed by early fetal chorionic villi will lead us to a better understanding of the genetic control of normal human reproduction. They also may be used to study obstetric and other human disease.
胚胎及其相邻胎盘结构的生长与分化是人类生殖与生存的基础。然而，对于人类早期发育过程中这些进程的基因调控，我们知之甚少。正常胎盘形成是一系列精心编排的事件的结果，这些事件包括细胞黏附以及由相对未知的遗传过程控制的有限侵袭。我们推测，孕早期绒毛膜绒毛所表达的基因构成胎盘形成以及人类早期发育的关键调节因子。因此，我们的目标是分离并鉴定孕早期人类胎盘所表达的已知和未知基因。
所收集的组织包括孕早期选择性终止妊娠时采集的胎盘组织。将胎儿绒毛膜绒毛与母体蜕膜大体分离，并迅速在液氮中冷冻以保存核糖核酸。来自不同孕周的组织分开保存。提取总核糖核酸，分离信使核糖核酸，并合成互补脱氧核糖核酸。将互补脱氧核糖核酸克隆到ZAP Express λ载体（Stratagene公司，加利福尼亚州拉霍亚）中。对随机噬菌斑进行自动测序。使用基本局部比对搜索工具在Genbank数据库（美国国立医学图书馆国家生物技术信息中心，马里兰州贝塞斯达）中搜索序列同源性。如果获得已知基因序列，则不再进行进一步分析。如果未鉴定到同源性，则对完整的互补脱氧核糖核酸插入片段进行全长测序。通过组织特异性模式、细胞定位和染色体定位对新基因进行进一步鉴定。通过逆转录聚合酶链反应确认胎儿绒毛的表达。
我们分离出一些已知在母胎界面表达的基因。186个随机克隆中有17个长度大于1千碱基，且与已知基因无同源性，因此这些可能构成对胎盘形成至关重要的新基因。来自妊娠12周的人绒毛膜绒毛互补脱氧核糖核酸文库的一个克隆是一个7千碱基的基因，在人类胎儿脑、肺、肝和肾中也大量表达。原位杂交将转录本定位到胎儿肾小球。
我们的研究结果表明，胎盘是潜在新基因表达的丰富来源。17个克隆长度大于1千碱基，是参与孕早期胎盘形成的潜在新基因。这17个潜在新基因中的一个在几种胎儿组织中大量表达，这表明其在人类早期胎儿发育中发挥作用。其他潜在新基因目前正在鉴定中。我们用于分离早期胎儿绒毛膜绒毛所表达基因的强大分子技术将使我们更好地理解正常人类生殖的基因调控。它们也可用于研究产科及其他人类疾病。
