补肾活血方联合宫腔灌注治疗对宫腔粘连相关机制TGF-β1、Smad2和Smad3的影响分析

目的:探讨补肾活血方及宫腔灌注治疗对宫腔粘连患者临床症状的改善及对转化生长因子-β1(transforming growth factor-beta1,TGF-β1)、Smad2和Smad3的影响。方法:选择2017年7月至2018年7月重庆市中医院妇科收治的宫腔粘连患者60例,依据随机数字表法分为实验组和对照组,每组各30例。对照组采用戊酸雌二醇治疗,观察组采用补肾活血方联合丹参注射液宫腔灌注治疗。观察2组患者临床症状改善情况。采用ELISA法检测2组基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)、基质金属蛋白酶-3(matrix metalloproteinases-3,MMP-3)及基质金属蛋白酶-9(matrix metalloproteinases-9,MMP-9)水平的变化。采用Western blot和Q-RT-PCR法检测2组患者治疗前后子宫内膜组织TGF-β1、Smad2和Smad3蛋白水平及mRNA水平。结果:与治疗前比较,2组患者子宫内膜厚度、MMP-2、MMP-3及MMP-9水平明显增加,子宫内膜血流参数(pulsatility index,PI)、阻力指数(resistance index,RI)、粘连类型、粘连范围、月经情况及总分均有所降低。实验组子宫内膜厚度、MMP-2、MMP-3及MMP-9水平高于对照组,PI、RI、粘连类型、粘连范围、月经情况及总分均低于对照组(均P<0.05)。与治疗前比较,2组患者TGF-β1、Smad2和Smad3蛋白水平及mRNA水平均有所降低。实验组TGF-β1、Smad2和Smad3蛋白水平及mRNA水平均低于对照组(P<0.05)。结论:补肾活血方及宫腔灌注治疗可改善宫腔粘连血流参数,预防宫腔粘连的再次发生,其机制可能与上调MMP-2、MMP-3及MMP-9的表达,下调TGF-β1、Smad2和Smad3表达水平有关。

Promotion of Chondrogenesis of Marrow Stromal Stem Cells by TGF-β3 Fusion Protein In Vitro

The purpose of this study was to investigate the repair of the osteoarthritis（OA）-induced car- tilage injury by transfecting the new TGF-β3 fusion protein （LAP-MMP-mTGF-β3） with targeted ther- apy function into the bone marrow-derived mesenchymal stem cells （MSCs） in rats. The recombinant of plRES-EGFP-MMP was constructed by combination of DNA encoding MMP enzyme cutting site and eukaryotic expression vector plRES-EGFP. LAP and mTGF-β3 fragments were obtained from rat em- bryos by RT-PCR and inserted into the upstream and downstream of MMP from plRES-EGFP-MMP respectively, so as to construct the recombinant plasmid ofplRES-EGFP-LAP-MMP-mTGF-β3, plRES- EGFP-LAP-MMP-mTGF-β3 was transfected into rat MSCs. The genetically modified MSCs were cul- tured in medium with MMP-1 or not. The transfected MSCs were transplanted in the rat OA models. The OA animal models were surgically induced by anterior cruciate ligament transaction （ACLT）. The pathological changes were observed under a microscope by HE staining, Alcian blue, Safranin-fast Gre- en and graded by Mankin＇s scale, plRES-EGFP-LAP-MMP-mTGF-β3 was successfully constructed by means of enzyme cutting and sequencing, and the mTGF-β3 fusion protein （39 kD） was certified by Western blotting. Those genetically modified MSCs could differentiate into chondrocytes induced by MMP and secrete the relevant-matrix. The transfected MSCs could promote chondrogenesis and matrix production in rat OA models in vivo. It was concluded that a new fusion protein LAP-MMP-mTGF-β3 was constructed successfully by gene engineering, and could be used to repair the OA-induced cartilage injury.