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中华妇幼临床医学杂志(电子版) ›› 2020, Vol. 16 ›› Issue (02) : 218 -226. doi: 10.3877/cma.j.issn.1673-5250.2020.02.014

所属专题： 文献；

论著

子痫前期患者胎盘组织中Pten基因的表达及其临床价值
练蕊1, 朱宝生2,†()   
  1. 1. 大理大学临床医学院　671003；云南省第一人民医院医学遗传科，昆明　650032
    2. 云南省第一人民医院医学遗传科，昆明　650032
  • 收稿日期:2019-12-18 修回日期:2020-03-03 出版日期:2020-04-01
  • 通信作者: 朱宝生

Expressions and clinical values of Pten gene in placenta tissues of patients with preeclampsia

Rui Lian1, Baosheng Zhu2,†()   

  1. 1. Dali Medical College, Dali 671003, Yunnan Province, China; Genetic Diagnosis Center, First People′s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China
    2. Genetic Diagnosis Center, First People′s Hospital of Yunnan Province, Kunming 650032, Yunnan Province, China
  • Received:2019-12-18 Revised:2020-03-03 Published:2020-04-01
  • Corresponding author: Baosheng Zhu
  • About author:
    Corresponding author: Zhu Baosheng, Email:
  • Supported by:
    Major Science and Technology Project of Yunnan Province(2018ZF009)
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引用本文：

练蕊, 朱宝生. 子痫前期患者胎盘组织中Pten基因的表达及其临床价值[J/OL]. 中华妇幼临床医学杂志(电子版), 2020, 16(02): 218-226.

Rui Lian, Baosheng Zhu. Expressions and clinical values of Pten gene in placenta tissues of patients with preeclampsia[J/OL]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2020, 16(02): 218-226.

目的

探讨Pten基因在子痫前期(PE)孕妇胎盘组织中异常表达的临床意义。

方法

选择2018年8月1日至2019年1月1日在云南省第一人民医院产科采取择期剖宫产术分娩的60例孕妇为研究对象，纳入研究组，根据PE严重程度，将其分为轻度PE亚组(n＝30)和重度PE亚组(n＝30)。同时，采用随机数字表法，选择同期在本院采取剖宫产术分娩的30例正常孕妇纳入对照组。采用免疫组织化学染色法、实时荧光定量PCR法，检测pten蛋白及Pten mRNA在轻度与重度PE亚组及对照组孕妇胎盘组织中的表达水平。采用Pearson相关分析对研究组孕妇胎盘组织中pten蛋白染色评分和患者收缩压进行相关性分析。本研究遵循的程序符合云南省第一人民医院伦理委员会所制定的伦理学标准，并得到该伦理委员会审批(审查日期：2018年7月11日)，并且与所有研究对象均签署临床研究知情同意书。

结果

①轻度与重度PE亚组及对照组孕妇年龄、孕次、怀孕天数、胎儿出生体重、胎盘重量和新生儿身长等比较，差异无统计学意义(P>0.05)，而收缩压、舒张压、丙氨酸转氨酶(ALT)、天冬氨酸氨基转移酶(AST)、血小板计数等比较，差异有统计学意义(P<0.05)。②pten蛋白在轻度与重度PE亚组及对照组产妇的胎盘组织中均有表达，主要定位于胎盘绒毛滋养细胞核中，阳性着色呈黄褐色，正常细胞的细胞核呈蓝色。③对照组孕妇胎盘组织中pten蛋白染色评分为(7.2±0.9)分，高于研究组的(4.0±1.3)分，2组比较，差异有统计学意义(t＝2.752，P＝0.007)。轻度与重度PE亚组及对照组孕妇pten蛋白表达水平分别为(5.7±1.6)分，(2.7±1.1)分和(7.2±0.9)分，轻度与重度PE亚组及对照组孕妇胎盘组织中pten蛋白染色评分总体比较，差异均有统计学意义(F＝315.726，P<0.001)；轻度与重度PE亚组及对照组间进一步两两比较，差异亦均有统计学意义(轻度PE亚组vs重度PE亚组：LSD-t＝2.570，P＝0.013；轻度PE亚组vs对照组：LSD-t＝2.256，P＝0.031；重度PE亚组vs对照组：LSD-t＝2.483，P＝0.016)。④轻度PE亚组、重度PE亚组和对照组孕妇Pten mRNA相对表达水平分别为(0.5±0.1)、(0.4±0.1)和(1.4±0.3)，轻度与重度PE亚组及对照组比较，差异有统计学意义(F＝368.748，P<0.001)；轻度与重度PE亚组及对照组间进一步两两比较，差异亦均有统计学意义(轻度PE亚组vs重度PE亚组：LSD-t＝2.164，P＝0.039；轻度PE亚组vs对照组：LSD-t＝14.476，P<0.001；重度PE亚组vs对照组：LSD-t＝21.825，P<0.001)。⑤研究组孕妇胎盘组织中pten蛋白染色评分与收缩压呈负相关关系(r＝－0.356，P＝0.005)。

结论

PE孕妇胎盘组织中pten蛋白染色评分，较对照组明显下降，并且重度PE孕妇的下降程度更为明显。Pten基因可能参与了PE的发生，其表达水平可能成为评价PE严重程度的潜在生物标志物。

关键词: 先兆子痫, Pten基因, 胎盘, 免疫化学, 孕妇
Objective

To investigate the clinical significance of expressions of Pten gene in placenta tissues of pregnant woman with preeclampsia (PE).

Methods

From August 1, 2018 to January 1, 2019, a total of 60 pregnant women with PE who were hospitalized in the Obstetrics Department of the First People′s Hospital of Yunnan Province and underwent elective cesarean section were recruited into this study (study group). According to the severity of PE, they were divided into mild PE sub-group (n=30) and severe PE sub-group (n=30). Meanwhile, 30 healthy pregnant women who were hospitalized in the same hospital and underwent elective cesarean section were randomly selected into control group. Immunohistochemical staining and real-time fluorescence quantitative PCR were used to detect the expression levels of pten protein and Pten mRNA in the maternal placental tissues of three groups. Pearson correlation analysis was used to analyze the correlation between pten protein levels in the placental tissues of pregnant women and systolic blood pressure of study group. The procedures followed in this study were in accordance with the ethical standards established by the Ethics Committee of the cases collection hospital, and this study was approved by this committee (review date: July 11, 2018). Informed consent was obtained and signed with each subject.

Results

①There were no significant differences in age, gravidity, gestational age, birth weight of newborns, placental weight and length of newborns among three groups (P>0.05), while there were statistically significant differences in systolic blood pressure, diastolic blood pressure, alanine transaminase (ALT), aspartate aminotransferase (AST) and platelet count (P<0.05). ②Pten protein was expressed in the placental tissues of three groups, mainly localized in the nucleus of placental villus trophoblastic cells. The positive staining was yellowish-brown, and the nucleus of normal cells was blue. ③Pten protein staining scores in placenta tissues of pregnant women in control group was (7.2±0.9) scores, which was higher than that of (4.0±1.3) scores in study group, and the difference between two groups was statistically significant (t=2.752, P=0.007). Pten protein staining scores in pregnant women in mild PE sub-group, severe PE sub-group and control group were (5.7±1.6) scores, (2.7±1.1) scores and (7.2±0.9) scores, respectively. The overall comparison of pten protein staining scores in placental tissues of pregnant women among three groups showed statistically significant difference (F=315.726, P<0.001). Further comparison between each two groups showed that all the differences were statistically significant (mild PE sub-group vs severe PE sub-group: LSD-t=2.570, P=0.013; mild PE sub-group vs control group: LSD-t = 2.256, P=0.031; severe PE sub-group vs control group: LSD-t=2.483, P=0.016). ④ The relative expression levels of Pten mRNA in pregnant women with mild PE sub-group, severe PE sub-group and control group were (0.5 ± 0.1), (0.4 ± 0.1) and (1.4 ± 0.3), respectively. Compared with three groups, the differences were statistically significant (F=368.748, P<0.001). Further comparison between each two groups showed that all the differences were statistically significant (mild PE sub-group vs severe PE sub-group: LSD-t=2.164, P=0.039; mild PE sub-group vs control group: LSD-t=14.476, P<0.001; severe PE sub-group vs control group: LSD-t=21.825, P<0.001). ⑤Pten protein staining scores in placental tissues of pregnant women in study group was negatively correlated with systolic blood pressure(r=-0.356, P=0.005).

Conclusions

Pten protein staining scores in placenta tissues of PE patients was significantly lower than that of control group, and the degree of decline in pregnant women with severe PE was more significant. Pten gene may be involved in the occurrence of PE, and its expression level may become a potential biomarker for evaluating severity of preeclampsia.

Key words: Pre-eclampsia, Pten gene, Placenta, Immunochemistry, Pregnant women
表1 RevertAid cDNA反转录体系
试剂名称 体积
5反应缓冲液(reaction buffer) 10 μL
核糖核酸酶抑制剂(RiboLock Rnase inhibitor) 1 μL (40 U)
dATP、dCTP、dGTP和dTTP的混合溶液(dNTP Mix)，每种10 mol/L 5 μL
RevertAid反转录酶(M-MuLV Reverse Transcriptase) 2 μL (400 U)
多聚胸腺嘧啶(Olig dT)(0.5 μg/μL) 3 μL
RNA 3 μL
焦碳酸二乙酯(DEPC) H2O 2～4 μg
最终反应体系总体积 补足50 μL
表2 轻度与重度PE亚组及对照组孕妇一般临床资料比较
组别 例数 年龄(岁，±s) 孕次(次，±s) 怀孕天数(d，±s) 收缩压(mmHg，±s) 舒张压(mmHg，±s) ALT(U/L，±s) AST(U/L，±s)
轻度PE亚组 30 28.4±2.7 2.4±1.3 262.0±15.7 150.2±11.5 80.3±9.0 54.2±2.2 49.6±7.2
重度PE亚组 30 29.1±3.5 2.2±1.5 255.3±12.2 168.6±22.1 97.6±26.5 102.5±6.7 99.3±4.1
对照组 30 27.9±3.2 2.1±0.8 266.4±7.0 119.7±4.9 75.0±5.0 21.5±2.2 17.2±3.2
检验值 ? F＝3.096 F＝0.759 F＝5.428 F＝146.032 F＝114.547 F＝179.365 F＝182.739
P值 ? 0.732 0.249 0.624 0.014 0.016 <0.001 <0.001
组别 例数 血小板计数(×109/L，±s) 随机尿蛋白[例数(%)] 24 h尿蛋白(g，±s) 胎儿出生体重(g，±s) 胎盘重量(g，±s) 新生儿身长(cm，±s)
呈阴性 呈阳性
轻度PE亚组 30 151.9±27.4 2(6.7) 28(93.3) 0.6±0.2 3 168.3±405.4 528.0±67.6 49.4±1.6
重度PE亚组 30 94.5±23.5 0(0) 30(100.0) 1.4±0.2 2 965.0±371.7 494.0±61.9 48.6±1.5
对照组 30 199.7±27.8 30(100.0) 0(0) — 3 375.0±333.7 562.5±55.6 51.2±1.5
检验值 ? F＝191.183 χ2＝81.853 — F＝23.440 F＝38.069 F＝33.009
P值 ? <0.001 <0.001 — 0.039 0.024 0.041
图1 轻度与重度PE亚组及对照组孕妇胎盘组织pten蛋白免疫组化检测结果图(图1A：重度PE亚组孕妇胎盘组织；图1B：轻度PE亚组孕妇胎盘组织；图1C：对照组孕妇正常胎盘组织)(免疫组化染色，高倍)
表3 轻度与重度PE亚组及对照组孕妇pten蛋白染色评分和Pten mRNA相对表达水平(±s)
组别 pten蛋白染色评分(分) Pten mRNA相对表达水平
轻度PE亚组 5.7±1.6 0.5±0.1
重度PE亚组 2.7±1.1 0.4±0.1
对照组 7.2±0.9 1.4±0.3
总体比较 ? ?
? F值 315.726 368.748
? P值 <0.001 <0.001
轻度PE亚组 vs重度PE亚组 ? ?
? LSD-t值 2.570 2.164
? P值 0.013 0.039
轻度PE亚组 vs对照组 ? ?
? LSD-t值 2.256 14.476
? P值 0.031 <0.001
重度PE亚组 vs对照组 ? ?
? LSD-t值 2.483 21.825
? P值 0.016 <0.001
图2 轻度与重度PE亚组及对照组孕妇Pten mRNA相对表达水平柱状图
图3 研究组子痫前期孕妇pten蛋白染色评分与收缩压相关性分析散点图
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