Chinese Medical E-ournals Database
Adv Search
中华妇幼临床医学杂志(电子版)

Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition) ›› 2020, Vol. 16 ›› Issue (02): 218 -226. doi: 10.3877/cma.j.issn.1673-5250.2020.02.014

Special Issue: ；

Original Article

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)
HTML ( ) PDF ( )
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
[1]
中华医学会妇产科学分会妊娠期高血压疾病学组. 妊娠期高血压疾病诊治指南(2015)[J]. 中华妇产科杂志，2015, 50(10):721-728. DOI: 10.3760/cma.j.issn.0529-567x.2015.10.001.
[2]
Asiltas B, Surmen-Gur E, Uncu G. Prediction of first-trimester preeclampsia: relevance of the oxidative stress marker MDA in a combination model with PP-13, PAPP-A and beta-HCG [J]. Pathophysiology, 2018, 25(2): 131-135. DOI: 10.1016/j.pathophys.2018.02.006.
[3]
Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer [J]. Science, 1997, 275(5308): 1943-1947. DOI: 10.1126/science.275.5308.1943.
[4]
Xi Z, Jing L, Le-Ni K, et al. Evaluation of PTEN and CD4＋FOXP3＋ T cell expressions as diagnostic and predictive factors in endometrial cancer: a case control study [J]. Medicine (Baltimore), 2019, 98(30): e16345. DOI: 10.1097/MD.0000000000016345.
[5]
Sarbassov DD, Guertin DA, Ali SM, et al. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex [J]. Science, 2005, 307(5712): 1098-1101. DOI: 10.1126/science.1106148.
[6]
Soria JC, Lee HY, Lee JI, et al. Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation [J]. Clin Cancer Res, 2002, 8(5): 1178-1184.
[7]
Xue G, Hemmings BA. PKB/Akt-dependent regulation of cell motility [J]. J Natl Cancer Inst, 2013, 105(6): 393-404. DOI: 10.1093/jnci/djs648.
[8]
Qian Y, Corum L, Meng Q, et al. PI3K induced actin filament remodeling through Akt and p70S6K1: implication of essential role in cell migration [J]. Am J Physiol Cell Physiol, 2004, 286(1): C153-C163. DOI: 10.1152/ajpcell.00142.2003.
[9]
Ni J, Cozzi P, Hao J, et al. Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway [J]. Int J Biochem Cell Biol, 2013, 45(12): 2736-2748. DOI: 10.1016/j.biocel.2013.09.008.
[10]
Li W, Dong Y, Zhang B, et al. PEBP4 silencing inhibits hypoxia-induced epithelial-to-mesenchymal transition in prostate cancer cells [J]. Biomed Pharmacother, 2016, 81: 1-6. DOI: 10.1016/j.biopha.2016.03.030.
[11]
Cao Z, Liu LZ, Dixon DA, et al. Insulin-like growth factor-I induces cyclooxygenase-2 expression via PI3K, MAPK and PKC signaling pathways in human ovarian cancer cells [J]. Cell Signal, 2007, 19(7): 1542-1553. DOI: 10.1016/j.cellsig.2007.01.028.
[12]
Xu L, Brink M. mTOR, cardiomyocytes and inflammation in cardiac hypertrophy [J]. Biochim Biophys Acta, 2016, 1863(7 Pt B): 1894-1903. DOI: 10.1016/j.bbamcr.2016.01.003.
[13]
McKnight NC, Zhenyu Y. Beclin 1, an essential component and master regulator of PI3K-Ⅲ in health and disease [J]. Curr Pathobiol Rep, 2013, 1(4): 231-238. DOI: 10.1007/s40139-013-0028-5.
[14]
Wang RC, Wei Y, An Z, et al. Akt-mediated regulation of autophagy and tumorigenesis through Beclin 1 phosphorylation [J]. Science, 2012, 338(6109): 956-959. DOI: 10.1126/science.1225967.
[15]
James JL, Saghian R, Perwick R, et al. Trophoblast plugs: impact on utero-placental haemodynamics and spiral artery remodelling [J]. Hum Reprod, 2018, 33(8): 1430-1441. DOI: 10.1093/humrep/dey225.
[16]
Brkić J, Dunk C, O′Brien J, et al. MicroRNA-218-5p promotes endovascular trophoblast differentiation and spiral artery remodeling [J]. Mol Ther, 2018, 26(9): 2189-2205. DOI: 10.1016/j.ymthe.2018.07.009.
[17]
Tessier DR, Yockell-Lelièvre J, Gruslin A. Uterine spiral artery remodeling: the role of uterine natural killer cells and extravillous trophoblasts in normal and high-risk human pregnancies [J]. Am J Reprod Immunol, 2015, 74(1): 1-11. DOI: 10.1111/aji.12345.
[18]
Ding GC, Chen M, Wang YX, et al. MicroRNA-128a-induced apoptosis in HTR-8/SVneo trophoblast cells contributes to pre-eclampsia [J]. Biomed Pharmacother, 2016, 81: 63-70. DOI: 10.1016/j.biopha.2016.03.040.
[19]
Song L, Liu S, Zhang L, et al. MiR-21 modulates radiosensitivity of cervical cancer through inhibiting autophagy via the PTEN/Akt/HIF-1α feedback loop and the Akt-mTOR signaling pathway [J]. Tumour Biol, 2016, 37(9): 12161-12168. DOI: 10.1007/s13277-016-5073-3.
[20]
Allred DC, Clark GM, Elledge R, et al. Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer [J]. J Natl Cancer Inst, 1993, 85(3): 200-206. DOI: 10.1093/jnci/85.3.200.
[21]
赵莉娜，吴锦华，刘国成，等. 早发型重度子痫前期患者胎盘组织中Notch1和PPAR-γ蛋白表达的临床意义 [J/CD]. 中华妇幼临床医学杂志(电子版)，2019，15(2):164-170. DOI: 10.3877/cma.j.issn.1673-5250.2019.02.007.
[22]
Calimag-Loyola A, Lerma EV. Renal complications during pregnancy: in the hypertension spectrum [J]. Dis Mon, 2019, 65(2): 25-44. DOI: 10.1016/j.disamonth.2018.03.001.
[23]
Xue P, Fan W, Diao Z, et al. Up-regulation of PTEN via LPS/AP-1/NF-kappaB pathway inhibits trophoblast invasion contributing to preeclampsia[J]. Mol Immunol, 2020, 118:182-190.
[24]
Leavey K, Wilson SL, Bainbridge SA, et al. Epigenetic regulation of placental gene expression in transcriptional subtypes of preeclampsia [J]. Clin Epigenetics, 2018, 10: 28. DOI: 10.1186/s13148-018-0463-6.
[25]
Harris LK. Review: trophoblast-vascular cell interactions in early pregnancy: how to remodel a vessel [J]. Placenta, 2010, 31(Suppl): S93-S98. DOI: 10.1016/j.placenta.2009.12.012.
[26]
Ji L, Brkiź J, Liu M, et al. Placental trophoblast cell differentiation: physiological regulation and pathological relevance to preeclampsia [J]. Mol Aspects Med, 2013, 34(5): 981-1023. DOI: 10.1016/j.mam.2012.12.008.
[27]
Lash GE. Molecular cross-talk at the feto-maternal interface [J]. Cold Spring Harb Perspect Med, 2015, 5(12)DOI: 10.1101/cshperspect.a023010.
[28]
Salomon C, Guanzon D, Scholz-Romero K, et al. Placental exosomes as early biomarker of preeclampsia: potential role of exosomal microRNAs across gestation [J]. J Clin Endocrinol Metab, 2017, 102(9): 3182-3194. DOI: 10.1210/jc.2017-00672.
[29]
BrosensI, Puttemans P, Benagiano G. Placental bed research: I. The placental bed: from spiral arteries remodeling to the great obstetrical syndromes[J]. Am J Obstet Gynecol, 2019, 211(5): 437-456. DOI: 10.1016/j.ajog.2019.05.044.
[30]
Burton GJ, Jauniaux E. The cytotrophoblastic shell and complications of pregnancy [J]. Placenta, 2017, 60: 134-139. DOI: 10.1016/j.placenta.2017.06.007.
[31]
Milovanov AP. Cytotrophoblastic invasion is the most important mechanism of placentation and pregnancy progression [J]. Arkh Patol, 2019, 81(4): 5-10. DOI: 10.17116/patol2019810415.
[32]
Salomon C, Yee SW, Mitchell MD, et al. The possible role of extravillous trophoblast-derived exosomes on the uterine spiral arterial remodeling under both normal and pathological conditions [J]. Biomed Res Int, 2014, 2014: 693157. DOI: 10.1155/2014/693157.
[33]
Liang H, Zhang Q, Lu J, et al. MSX2 induces trophoblast invasion in human placenta [J]. PLoS One, 2016, 11(4): e0153656. DOI: 10.1371/journal.pone.0153656.
[34]
Roberts DJ, Post MD. The placenta in pre-eclampsia and intrauterine growth restriction [J]. J Clin Pathol, 2008, 61(12): 1254-1260. DOI: 10.1136/jcp.2008.055236.
[35]
Lala PK, Chakraborty C. Factors regulating trophoblast migration and invasiveness: possible derangements contributing to pre-eclampsia and fetal injury [J]. Placenta, 2003, 24(6): 575-587. DOI: 10.1016/s0143-4004(03)00063-8.
[36]
Song MS, Carracedo A, Salmena L, et al. Nuclear PTEN regulates the APC-CDH1 tumor-suppressive complex in a phosphatase-independent manner [J]. Cell, 2011, 144(2): 187-199. DOI: 10.1016/j.cell.2010.12.020.
[37]
Oudit GY, Sun H, Kerfant BG, et al. The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease [J]. J Mol Cell Cardiol, 2004, 37(2): 449-471. DOI: 10.1016/j.yjmcc.2004.05.015.
[38]
Morello F, Perino A, Hirsch E. Phosphoinositide 3-kinase signalling in the vascular system [J]. Cardiovasc Res, 2009, 82(2): 261-271. DOI: 10.1093/cvr/cvn325.
[39]
Roberts JM, Escudero C. The placenta in preeclampsia [J]. Pregnancy Hypertens, 2012, 2(2): 72-83.
[40]
Laguё MN, Detmar J, Paquet M, et al. Decidual PTEN expression is required for trophoblast invasion in the mouse [J]. Am J Physiol Endocrinol Metab, 2010, 299(6): E936- E946. DOI: 10.1152/ajpendo.00255.2010.
[41]
Xiao J, Tao T, Yin Y, et al. miR-144 may regulate the proliferation, migration and invasion of trophoblastic cells through targeting PTEN in preeclampsia [J]. Biomed Pharmacother, 2017, 94: 341-353. DOI: 10.1016/j.biopha.2017.07.130.
[42]
Lou CX, Zhou XT, Tian QC, et al. Low expression of microRNA-21 inhibits trophoblast cell infiltration through targeting PTEN [J]. Eur Rev Med Pharmacol Sci, 2018, 22(19): 6181-6189. DOI: 10.26355/eurrev_201810_16023.
[43]
Chen H, Ye D, Xie X, et al. PTEN promoter methylation and protein expression in normal early placentas and hydatidiform moles [J]. J Soc Gynecol Investig, 2005, 12(3): 214-217. DOI: 10.1016/j.jsgi.2005.01.009.
[1] Qianmei Li, Guannan He, Jing Zhao, Xi Chen, Yuying Tang, Liqiong Ma, Rong Liang, Tao Yuan, Mingxing Li. Placental microvascular flow imaging characteristics and prognosis of early low-risk and high-risk pregnancies[J]. Chinese Journal of Medical Ultrasound (Electronic Edition), 2024, 21(07): 726-732.
[2] Jingyu Qian, Mingming Zheng. Interpretation of the Italian guidelines on non-invasive and invasive prenatal diagnosis：executive summary of recommendations for practice the Italian Society for Obstetrics and Gynecology（SIGO）[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 486-492.
[3] Xialin Li, Fang He. Risk assessment and early warning system for postpartum hemorrhage[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 498-503.
[4] Ziyang Liu, Jianjian Cui, Yin Zhao. Current research status on obstetric disseminated intravascular coagulation and its scoring system[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 511-518.
[5] Fanying Zeng, Jie Ruan, Xinghui Liu, Guolin He. Current status of perinatal medicine advances under the new reproductive situation and coping strategies in prenatal care[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 519-524.
[6] Xiaofei Li, Hongli Liu, Qiuling Shi, Jing Tian, Li Li, Hongbo Qi, Xin Luo. A prospective randomized controlled study of low intensity focused ultrasound uterine involution treatment for prevention and treatment of postpartum hemorrhage in natural childbirth women[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 534-539.
[7] Rong Huang, Ziyu Liang, Wenjin Qi. Expression and significance of NLRP3 inflammasome in serum of pregnant women with premature rupture of membranes[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 540-548.
[8] Xia He, Rong Huang, Wenjin Qi. High-throughput sequencing study on the abundance of placenta and fetal membrane flora in pregnant women with premature rupture of membranes[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 549-555.
[9] Jiangyan Xie, Yafei Wang, Fang He. Pregnancy complicated with thrombotic thrombocytopenic purpura：two cases report and literature review[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(05): 556-563.
[10] Xiaoyan Han, Hua Yang. Relationship between low level of serum placental growth factor in the second trimester pregnancy women and adverse fetal prognosis[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(04): 398-402.
[11] Jiali Du, Rui Bao, Chunhong Qiao, Wei Han. Construction of a prediction model for adverse pregnancy outcomes after emergency cervical cerclage in pregnant women with cervical incompetence during the second trimester[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(04): 403-409.
[12] Tingting Xu, Yongchi Zhan, Xiaodong Wang, Xiaodong Wang. Perinatal outcomes of fetomaternal hemorrhage syndrome pregnant women with sinusoidal fetal heart tracing[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(04): 382-389.
[13] Juan Tan, Jianxin Tan, Binbin Shao, Yan Wang, Zhengfeng Xu. Current research status on non-invasive prenatal testing for fetal with single gene inheritance diseases[J]. Chinese Journal of Obstetrics & Gynecology and Pediatrics(Electronic Edition), 2024, 20(03): 245-250.
[14] Fan Hong, Dunjin Chen, Yang Fu, Xinyue Liang, Yi Wu, Xiaoyi Wang. Clinical study of in vitro fertilization-embryo transfer pregnancy complicated with placenta previa[J]. Chinese Journal of Obstetric Emergency(Electronic Edition), 2024, 13(03): 176-182.
[15] Miao Hu, Lili Du, Lizi Zhang, Lin Lin, Yuliang Zhang, Shifeng Gu, Zhongjia Gu, Siying Lai, Jingying Liang, Yu Liu, Minshan Huang, Yuanyuan Huang, Qingqing Huang, Shijun Luo, Dunjin Chen. A study on placenta accreta grading and perinatal outcomes in patients conceived through in vitro fertilization/intracytoplasmic sperm injection[J]. Chinese Journal of Obstetric Emergency(Electronic Edition), 2024, 13(03): 183-189.
Viewed
Full text


Abstract

Copyright © Chinese Medical Association, Chinese Medical Multimedia Press  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号