摘要
目的阻断Wnt/β-catenin信号通路对补肾活血中药作用后薄型子宫内膜干细胞标志物的影响。方法选取薄型子宫内膜患者在确定排卵日后第7~9天,用一次性子宫内膜取样器吸取子宫内膜组织约100mg,处理固定。将取得的内膜组织分离培养子宫内膜干细胞。制备药物血清。将子宫内膜干细胞分为对照组、补肾活血中药高、中、低剂量组、西药组。予药物干预后,将转染T细胞因子(T-cell factor,TCF)的显性负性突变体(dominant negative TCF,dn TCF)表达质粒转染子宫内膜干细胞阻断Wnt/β-catenin信号通路,免疫荧光法检测CD34/CD117蛋白的表达、RT-PCR法检测Oct-4基因、Western blot法检测ABCG2蛋白的表达。结果转染dn TCF-3阻断Wnt/β-catenin信号通路后,免疫荧光检测结果显示,CD34蛋白的阳性面积与总面积100%相比,中剂量组、高剂量组及西药组表达率较阻断前分别下降20. 11%、25. 78%、28. 89%,差异具有统计学意义(P <0. 05);CD117蛋白的阳性面积与总面积100%相比,中剂量组、高剂量组及西药组表达率较阻断前分别下降18. 11%、24. 78%、26. 89%,差异具有统计学意义(P <0. 05)。RT-PCR检测结果显示,Oct-4 mRNA的表达率与总mRNA100%相比,高剂量组、西药组表达率较阻断前分别下降24. 96%、24. 84%。:Western blot检测结果显示,ABCG2蛋白的表达率与总蛋白100%相比,高剂量组、西药组表达率较阻断前分别下降15. 06%、13. 24%,差异具有统计学意义(P <0. 05)。结论补肾活血中药可能是通过作用于Wnt/β-catenin信号通路来作用于薄型子宫内膜干细胞的。
Objective To investigate the effect of blocking Wnt/ β - catenin signaling pathway on the markers of thin endometrium stem cells after treated by Bushen huoxue recipe. Methods We assimilated the thin endometrium endometrial tissue for about 100mg from patients on the 7- 9th day after ovulation with a one- time endometrial sampler,then conducted saline rinse,put it into 4% paraformaldehyde.Endometrium stem cells were then isolated and cultured.We then prepared the drug serum.The endometrial stem cells were divided into the control group （blank serum ＋ saline）,bshxr high,medium and low dose group （bshxr high,medium and low dose serum ＋ saline）,and also the western medicine group （blank serum ＋ progynova）.For drug intervention,the transfection of T cytokines （T- cell facter,TCF）the dominant negative mutant （dominant negative,TCF,dnTCF）expression plasmid was transfected into endometrial stem cells to block Wnt/ beta - catenin signaling pathway.We tested the expression of CD34/CD117 protein by immunofluorescence and Oct- 4 gene by RT- PCR and ABCG2 protein by Western blot. Results After the transfection of dnTCF- 3 to block Wnt/ beta - catenin signaling pathway,immunofluorescence assay showed that,compared between the positive area of CD34 protein and 100% of the total area,the middle dose group,high dose group and western medicine group decreased by 20.11%,25.78%,and 28.89%,（ P 〈0.05）; compared between the positive area of the CD117 protein and the total area of 100%,the middle dose group,high dose group and western medicine group decreased by 18.11%,24.78%,and 26.89%,（ P 〈0.05）.The results of RT- PCR detection showed that the expression rate of Oct- 4 mRNA were 24.96% and 24.84% compared with that of the total mRNA 100%,and the expression rate of high dose group and western medicine group decreased compared with that of before the blocking.Western blot test results showed that the expression rate of ABCG2 protein was 15.06% and 13.24%,lower than that of the total protein 100% i
作者
尹晓丹
薛晓鸥
杨维
辛明蔚
何军琴
YIN Xiao-dan;XUE Xiao-ou;YANG Wei;XIN Ming-wei;HE Jun-qin(Department of Traditional Chinese Medicine,Beijing Obstretrics and Gynecology Hospital,Capital Medical University,Beijing 100026,China;Department of Gynaecology,Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 100700,China)
出处
《标记免疫分析与临床》
CAS
2018年第10期1425-1430,共6页
Labeled Immunoassays and Clinical Medicine
基金
北京市自然科学基金(编号:7164260)
