摘要
目的探讨补肾活血方与富血小板血浆介导Wnt/β-catenin信号通路对共培养体系中脂肪间充质干细胞(ADSCs)增殖活性和功能表达作用的机制研究。方法取手术患者皮下脂肪组织及退变髓核组织,采用多次酶消化法原代分离、培养ADSCs及NPCs,建立细胞共培养体系。纳入腰椎间盘退变患者,连续服用补肾活血方2周,分别于初次服药前和末次服药后1 h抽取静脉血各50 mL,采用二次离心法制备富血小板血浆,以不同血浆干预培养,实验共分为5组:富血小板血浆组(PRP)、贫血小板血浆组(PPP)、含药富血小板血浆组(M-PRP)和含药贫血小板血浆组(M-PPP)、胎牛血清组(FBS)。倒置相差显微镜下观察共培养体系中细胞的形态变化;利用RT-PCR方法检测WNT3A、CTNNB1和MMP3 mRNA的表达情况;Western blotting法检测Wnt3a、β-catenin蛋白表达。结果倒置相差显微镜下显示,M-PRP组细胞以多角形或短梭形为主,细胞轮廓清晰、折光度较好。RT-PCR结果显示:与FBS组相比:PRP组WNT3A、CTNNB1表达量显著提高(P<0.05),MMP3达量显著下调(P<0.05);M-PRP组CTNNB1、MMP3表达量显著下调(P<0.05);PRP组基因表达量显著高于M-PRP组(P<0.05)。Western blotting法检测结果:与FBS组相比:PRP组、M-PRP组和M-PPP组β-catenin蛋白表达量显著下调(P<0.05)。结论补肾活血方与富血小板血浆联合应用可能通过抑制Wnt信号通路而发挥提高ADSCs细胞活性和功能的作用。
Objective To investigate the mechanism of Kidney-Tonifying and Blood Circulation-Promoting Recipe(补肾活血方) and platelet-rich plasma mediating Wnt/beta-catenin signal pathway on the proliferation and functional expression of adipose mesenchymal stem cells(ADSCs)in the co-culture system.Methods The subcutaneous adipose tissue and degenerative nucleus pulposus tissue were taken out from surgical patients,and primary ADSCs and NPCs were isolated and cultured by multiple enzyme digestion to establish a cell co-culture system.In patients with lumbar disc degeneration,taking Kidney-Tonifying and Blood Circulation-Promoting Recipe for 2 weeks.In the first and the last one hour after taking this medicine,before taking the medicine,the venous blood 50 mL was taken out for adopting quadratic centrifugation preparation of platelet-rich plasma.The experiment was divided into five groups:group platelet rich plasma(PRP),poor plasma(PPP),platelet platelet rich plasma medicated group(M-PRP)and poor platelet plasma medicated group(M-PPP),fetal bovine serum(FBS).The morphological changes of the cells in the co-culture system were observed under an inverted phase contrast microscope.RT-PCR was used to detect the expressions of WNT3 A,CTNNB1 and MMP3 mRNA.The expressions of Wnt3 a and beta-catenin were detected by Western blotting.Results In this study,the inverted phase contrast microscope showed that the cells in M-PRP group were mainly polygonal or short spindle,with clear contour and good refraction.RT-PCR results showed that compared with FBS group,WNT3 A and CTNNB1 expressions in PRP group were significantly increased(P<0.05),and MMP3 levels were significantly decreased(P<0.05).The expression levels of CTNNB1 and MMP3 in M-PRP group were significantly decreased(P<0.05).The gene expression in PRP group was significantly higher than that in M-PRP group(P<0.05).Western blotting results:Compared with FBS group,the protein expression of beta-catenin in PRP group,M-PRP group and M-PPP group was significantly decreased(P<0.05)
作者
李凯明
朱立国
李玲慧
张清
杨少锋
王尚全
陈明
梁龙
展嘉文
LI Kaiming;ZHU Liguo;LI Linghui;ZHANG Qing;YANG Shaofeng;WANG Shangquan;CHEN Ming;LIANG Long;ZHAN Jiawen(Department of Orthopaedics,Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China;Beijing key laboratory of orthopedics of traditional Chinese Medicine,Beijing 100102,China;The First Affiliated Hospital of Hunan University of Traditional Chinese Medicine,Changsha 410007,Hunan,China;Department of Spinal Surgery,Wangjing Hospital,China Academy of Chinese Medical Sciences,Beijing 100102,China)
出处
《辽宁中医杂志》
CAS
2021年第2期145-149,共5页
Liaoning Journal of Traditional Chinese Medicine
基金
国家自然科学基金(81674005,81904230,81804120,81302992)
中央本级重大增减支项目“名贵中药资源可持续利用能力建设项目”(2060302)
中央级公益性科研院所基本科研业务费专项资金资助(ZZ10-015)
北京市自然科学基金(7164313)
中国博士后科学基金(2017M611125,2016M591364)。
关键词
补肾活血方
富血小板血浆
脂肪间充质干细胞
WNT信号通路
Kidney-Tonifying and Blood Circulation-Promoting Recipe(补肾活血方)
platelet-rich plasma
mesenchymal stem cells
Wnt signal pathway
