Human myometrial genes are differentially expressed in labor: A suppression subtractive hybridization study
Human myometrial genes are differentially expressed in labor: A suppression subtractive hybridization study
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DOI：
10.1210/jc.87.6.2435
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发表时间：
2002-06-01
影响因子：
5.8
通讯作者：
Smith, R
中科院分区：
文献类型：
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作者：
Chan, EC;Fraser, S;Smith, R
人类分娩受到一系列因素的影响，其中许多因素是未知的。我们的目标是通过抑制消减杂交来识别人类子宫肌层中因分娩而改变的基因。我们还试图确定这些基因在子宫上段或宫底和下段的子宫肌层中是否存在差异表达。足月子宫肌组织是在获得知情同意后从接受剖腹产的产妇和非产妇身上获得的。总RNA用于抑制消减杂交（CLONTECH PCR Select），以产生两个消减cDNA文库，分别富集在分娩期间或之前、分娩和非分娩文库中表达的基因。对 400 个阳性克隆（占两个消减文库的 20%）进行点印迹筛选，发现 30 个差异表达克隆，其中 14 个通过人工上调。在 10 个已知的在分娩过程中上调的基因中，有 6 个具有明显的免疫调节和炎症作用。其中三种是众所周知的炎症介质和调节剂，以前与分娩有关：IL-8、锰超氧化物歧化酶 (MnSOD) 和金属蛋白酶-9。另外三个基因，干扰素诱导的 1-8d 基因、延伸因子 1a 和核磷蛋白，此前尚未与分娩相关。发现组成型表达的基因，包括亲环蛋白和α-肌动蛋白，会因劳动力而改变。使用 Taqman 探针的定量实时 RT-PCR 进一步证实了其中一些基因的上调。所测定的特定基因的量标准化为 18S 核糖体 RNA，并表示为平均值 +/- SEM。定量实时 RT-PCR 显示，IL-8 mRNA 从非临产样本 (n = 38) 中的 0.003 +/- 0.002 上升到胎龄匹配的自然临产妇女中的 0.24 +/- 0.11 (n = 20) (P = 0.035)。同样，孕龄匹配的女性中 MnSOD 从 0.11 +/- 0.02 (n = 24) 上升至 1.23 +/- 0.56 (n = 24) (P = 0.047)。此外，亲环蛋白（通常用作组成型或管家基因标记）在分娩期间从 0.0008 +/- 0.0002 (n = 6) 增加到 0.002 +/- 0.0004 (n = 6；P = 0.008)。值得注意的是，MnSOD mRNA 在子宫上段 (0.63 +/- 0.18) 和下段 (0.15 +/- 0.05；n = 15；P = 0.022) 之间分布存在差异，但 IL-8 则不然 (n = 17；P = 0.97)。引产进一步显示，IL-8 水平（0.63 +/- 0.21；n = 14）显着高于自然分娩（0.22 +/- 0.11；n = 20；P = 0.046），但 MnSOD 水平则不然（P = 0.1）。这项工作鉴定了以前与分娩无关的新基因和已知基因。它扩展了先前的数据，表明人类妊娠子宫内存在一些但不是全部基因的差异表达。炎症基因占已知在分娩过程中上调的基因的主要部分，这为人类分娩炎症机制的假设提供了支持。这项工作进一步表明，与人类劳动相关的许多因素及其复杂的相互作用仍有待阐明。
Human parturition is effected by a cascade of factors, of which many are unknown. We aim to identify the genes that are changed by labor in the human myometrium by suppression subtractive hybridization. We also seek to ascertain whether these genes are differentially expressed in the myometrium at the upper or fundal and lower segments of the uterus. Term myometrial tissues were obtained from laboring and nonlaboring women undergoing cesarean section after obtaining informed consent. Total RNA was used in suppression subtractive hybridization (CLONTECH PCR Select) to produce two subtracted cDNA libraries enriched for genes expressed during or before labor, labor and not-in-labor libraries, respectively. Dot blot screening of 400 positive clones, constituting 20% of the two subtracted libraries, revealed 30 differentially expressed clones, 14 of which were up-regulated by labor. Among the 10 known genes that were up-regulated in labor, 6 had apparent immune regulatory and inflammatory roles. Three are well-known inflammatory mediators and modulators that were previously linked with parturition: IL-8, manganese superoxide dismutase (MnSOD), and metalloproteinase-9. Three others, interferon-inducible 1-8d gene, elongation factor l a, and nucleophosmin, have not been previously linked with labor. Constitutively expressed genes, including cyclophilin and a-actin, were found to be altered by labor. Quantitative real-time RT-PCR using Taqman probes further confirmed the up-regulation of some of these genes. The amounts of the specific genes assayed were standardized to 18S ribosomal RNA and are expressed as mean +/- SEM. Quantitative real-time RT-PCR showed that IL-8 mRNA rose from 0.003 +/- 0.002 in nonlaboring samples (n = 38) to 0.24 +/- 0.11 (n = 20) in gestational-age-matched spontaneously laboring women (P = 0.035). Similarly, MnSOD rose from 0.11 +/- 0.02 (n = 24) to 1.23 +/- 0.56 (n = 24) in gestational-age-matched women (P = 0.047). Additionally, cyclophilin, often used as a constitutive or housekeeping gene marker, increased from 0.0008 +/- 0.0002 (n = 6) to 0.002 +/- 0.0004 (n = 6; P = 0.008) during labor. Notably, MnSOD mRNA was differentially distributed between the upper (0.63 +/- 0.18) and lower (0.15 +/- 0.05; n = 15; P = 0.022) segments of the uterus, but IL-8 was not (n = 17; P = 0.97). Induced labor further showed significantly higher levels of IL-8 (0.63 +/- 0.21; n = 14) than spontaneous labor (0.22 +/- 0.11; n = 20; P = 0.046), but not MnSOD (P = 0.1). This work identifies novel as well as known genes that were not previously associated with parturition. It extends previous data indicating that there is differential expression of some, but not all genes within the gravid human uterus. Inflammatory genes constitute a major proportion of the known genes found to be up-regulated in labor, lending support to the hypothesis of an inflammatory mechanism for human parturition. This work further indicates that many factors associated with human labor and their complex interactions remain to be elucidated.