左、右归丸对控制性超排卵小鼠子宫内膜VEGF及Ang影响的研究
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摘要
目的:基于VEGF及Ang探讨补肾复方左、右归丸对控制性超排卵小鼠子宫内膜容受性的影响。方法:采用GnRH-a+PMSG+HCG建立控制性超排卵小鼠模型,将其分为模型组、左归丸组、右归丸组、阿司匹林对照组、同时设正常对照组。在造模的同时,左、右归丸组分别按6、3、1.5g/kg左、右归丸灌胃阿司匹林对照组按0.2g/kg阿司匹林灌胃,模型组、正常对照组以等体积生理盐水灌胃,每天1次,连用11天。采用免疫组化法对子宫进行病理切片制作,通过IPP7.0图像分析系统检测子宫内膜VEGF、VEGF-R、Ang-1、Ang-2及Tie-2蛋白表达水平,采用荧光定量PCR技术检测阴道组织VEGF、VEGF-R、Ang-1、Ang-2及Tie-2 mRNA表达水平。结果:正常对照组VEGF/VEGF-R蛋白表达、VEGF-R mRNA表达、Ang/Tie蛋白表达和Ang-1 mRNA表达均明显高于模型组,与模型组比较,右归丸3g/kg剂量组及6g/kg剂量组VEGF蛋白表达明显增高;右归丸3g/kg剂量组VEGF、VEGF-1 mRNA表达(2.02±0.92、2.10±0.39),6g/kg剂量组VEGF、VEGF-1 mRNA表达(1.52±0.33、1.89±0.22)明显升高;右归丸3g/kg剂量组及6g/kg剂量组Ang-1、Ang-2蛋白表达明显增高,左、右归丸1.5g/kg剂量组、3g/kg剂量组、6g/kg剂量组tie-2明显增高;右归丸1.5g/kg剂量组Ang-1、Ang-2、Tie-2 m RNA表达(3.52±0.98、1.84±0.23、2.68±0.82)升高;3g/kg剂量组Ang-1、Ang-2、Tie-2 m RNA表达(3.48±0.89、1.88±0.26、2.69±0.71)、6g/kg剂量组Ang-1、Ang-2、Tie-2mRNA表达(3.57±0.98、1.96±0.32、2.64±0.66)明显升高。结论:补肾复方左、右归丸可通过调节控制性超排卵小鼠子宫内膜VEGF/VEGF-R及Ang/Tie蛋白和mRNA表达而改善子宫内膜容受性,其中补肾阳的右归丸比补肾阴的左归丸效果明显。
Objective: To explore the mechanism of Zuo Gui Wan/You Gui Wan in improving endometrial receptivity in COH mice by VEGF and Ang. Methods: COH mice model was established by using GnRHa + PMSG + HCG,and they were divided them into 8 groups i. e. the model group,Zuo Gui Wan/You Gui Wan groups,aspirin group,in addition the control group was set. During mice model was made,mice in Zuo Gui Wan/You Gui Wan(6 g,3 g,1 g/kg) groups,0. 2 g/kg aspirin group and,the model group(saline) and control group(saline)were administered(ig),respectively,once a day for 11 days. Uterine pathological sections were observed by immunohistochemical method,endometrial VEGF,VEGF-R Ang-1,Ang-2 and Tie-2 protein,and protein expression level were detected by IPP7. 0 image analysis system,endometrial VEGF,VEGF-R Ang-1,Ang-2 and Tie-2 protein mRNA expression level were detected by fluorescence quantitative PCR technique. Results: The expression of VEGF/VEGF-R protein,expressions of VEGF-R and mRNA,expressions of Ang/Tie protein and expressions of Ang-1 and mRNA in the control group were higher than those in the model group(P < 0. 01). Compared with the model group,expressions of VEGF protein and mRNA(2. 02 ± 0. 92,2. 10 ± 0. 39) in 3 g/kg You Gui Wan group,(1. 52 ± 0. 33、1. 89 ± 0. 22) in 6 g/kg group was significantly increased(P < 0. 05); Tie-2 in Zuo Gui Wan/You Gui Wan(1. 5 g,3 g,6 g/kg) groups significantly increased(P < 0.05); Ang-1,Ang-2 and tie-2 mRNA expression(3. 52 ± 0. 98,1. 84 ± 0. 23,2. 68 ± 0. 82) in 1. 5 g/kg You Gui Wan group,(3. 48 ± 0. 89,1.88 ± 0. 26,2. 69 ± 0. 71) in 3 g/kg group,(3. 57 ± 0. 98,1. 96 ± 0. 32,2. 64 ± 0. 66) in 6 g/kg group were significantly increased(P < 0. 05).Conclusion: Zuo Gui Wan/You Gui Wan improve endometrial receptivity through regulating expressions of VEGF/VEGF-R and Ang/Tie protein and mRNA in the endometrium of COH mice.
Objective: To explore the mechanism of Zuo Gui Wan/You Gui Wan in improving endometrial receptivity in COH mice by VEGF and Ang. Methods: COH mice model was established by using GnRHa + PMSG + HCG,and they were divided them into 8 groups i. e. the model group,Zuo Gui Wan/You Gui Wan groups,aspirin group,in addition the control group was set. During mice model was made,mice in Zuo Gui Wan/You Gui Wan(6 g,3 g,1 g/kg) groups,0. 2 g/kg aspirin group and,the model group(saline) and control group(saline)were administered(ig),respectively,once a day for 11 days. Uterine pathological sections were observed by immunohistochemical method,endometrial VEGF,VEGF-R Ang-1,Ang-2 and Tie-2 protein,and protein expression level were detected by IPP7. 0 image analysis system,endometrial VEGF,VEGF-R Ang-1,Ang-2 and Tie-2 protein mRNA expression level were detected by fluorescence quantitative PCR technique. Results: The expression of VEGF/VEGF-R protein,expressions of VEGF-R and mRNA,expressions of Ang/Tie protein and expressions of Ang-1 and mRNA in the control group were higher than those in the model group(P < 0. 01). Compared with the model group,expressions of VEGF protein and mRNA(2. 02 ± 0. 92,2. 10 ± 0. 39) in 3 g/kg You Gui Wan group,(1. 52 ± 0. 33、1. 89 ± 0. 22) in 6 g/kg group was significantly increased(P < 0. 05); Tie-2 in Zuo Gui Wan/You Gui Wan(1. 5 g,3 g,6 g/kg) groups significantly increased(P < 0.05); Ang-1,Ang-2 and tie-2 mRNA expression(3. 52 ± 0. 98,1. 84 ± 0. 23,2. 68 ± 0. 82) in 1. 5 g/kg You Gui Wan group,(3. 48 ± 0. 89,1.88 ± 0. 26,2. 69 ± 0. 71) in 3 g/kg group,(3. 57 ± 0. 98,1. 96 ± 0. 32,2. 64 ± 0. 66) in 6 g/kg group were significantly increased(P < 0. 05).Conclusion: Zuo Gui Wan/You Gui Wan improve endometrial receptivity through regulating expressions of VEGF/VEGF-R and Ang/Tie protein and mRNA in the endometrium of COH mice.
引文
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12 谭惠予,王学乾,梁路,黄德清.辅助生殖技术失败女性患者中医症候分析.亚太传统医药,2016;12(19)∶55~56
13 李小妮,李雅璇,周吉海,等.补肾助孕汤对小鼠生殖能力影响的实验研究.中国中西医结合杂志,2013;33(3)∶365~369
14 丁青,欧阳进,伍参荣,等.右归丸对卵巢早衰模型小鼠Th17/Treg表达的影响.中华中医药杂志,2013;28(4)∶1091~1093
2 徐慧颖,李娜,张云山.胚胎植入-子宫内膜容受性是关键.生殖医学杂志,2014;23(3)∶198~201
3 Jarvel IY,Sladkevicins P,Kelly S,et al.Evaluation of endometrial receptivity during in vitro fertilization using three dimensional power Doppler ultrasound.Ultrasound Obstet Gyncol,2005;26∶765~769
4 杜秀雅,马华刚.子宫内膜血管新生相关因子对内膜容受性影响的研究进展,生殖医学杂志,2014;23(6)∶518~521
5 王振迎,陈晓勇,陈瑾等.寿胎丸加减方对超排卵小鼠种植窗期子宫内膜整合素β3蛋白表达的影响.南昌大学学报(医学版),2010;50(3)∶33~37
6 Kenneth J.Livak and Thomas D.Schmittgen.Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-△△CTMethod.Methods,2001;25(4)∶402~408
7 Raine-Fenning N.Doppler assessment of uterine artery blood flow for the prediction of pregnancy after assisted reproduction treatment.Ultrasound Obestet Gynecol.2008;31∶371~375
8 Rowe AJ,Wulff C,Fraser HM.Localization of mRNA for vascular endothelial growth factor(VEGF),angiopoietins and their receptors during the peri-implantation period and early pregnancy in marmosets(Callithrix jacchus).Reproduction,2013;126∶227~228
9 高晓妹,陈蓉,张荣宜.血管内皮生长因子在围植入期小鼠子宫内膜中的表达.安徽医科大学学报,2010;45(1);31~33
10 Sagsoz H,Liman N,Kucukaslan I,et al.Immunolocalization of vascular endothelial growth factor,its receptors(flt1/fms,flk1/KDR,flt4)and vascular endothelial growth inhibitor in the bitch uterus during the sexual cycle.Anim Reprod,2013;140∶378~384
11 江媚,刘雁峰,黄羚.子宫内膜容受性低中医证型特点初探.中华中医药杂志,2015;30(11)∶3836~3839
12 谭惠予,王学乾,梁路,黄德清.辅助生殖技术失败女性患者中医症候分析.亚太传统医药,2016;12(19)∶55~56
13 李小妮,李雅璇,周吉海,等.补肾助孕汤对小鼠生殖能力影响的实验研究.中国中西医结合杂志,2013;33(3)∶365~369
14 丁青,欧阳进,伍参荣,等.右归丸对卵巢早衰模型小鼠Th17/Treg表达的影响.中华中医药杂志,2013;28(4)∶1091~1093