电针联合骨髓间充质干细胞移植治疗大鼠宫腔粘连的协同效应观察

Synergistic effects of electroacupuncture and bone marrow mesenchymal stem cell transplantation on rat intrauterine adhesion:an observation

目的:本研究旨在探讨电针联合骨髓间充质干细胞(BMSCs)移植对宫腔粘连(IUA)大鼠子宫内膜的影响,以及两者联合治疗修复子宫内膜的可能机制。方法:成年未交配雌性SD大鼠随机分为空白组、模型组、细胞组和联合组,每组10只。采用机械搔刮联合脂多糖感染的双重损伤法建立大鼠IUA模型。造模成功后第1、3、7天,模型组大鼠尾静脉注射磷酸盐缓冲液,细胞组仅尾静脉注射BMSCs悬液进行BMSCs移植,联合组大鼠移植与细胞组等量的BMSCs并进行电针治疗,每日针刺“关元”、电针双侧“足三里”和“三阴交”20 min,持续3个动情周期。每组5只大鼠在干预结束后取子宫组织,采用HE染色检测大鼠子宫内膜厚度以及腺体数目;采用Masson染色检测子宫内膜纤维化面积;采用免疫组织化学法检测血管生成标志物血管内皮生长因子(VEGF),增殖细胞核抗原(PCNA),雌激素受体(ER)的表达;Western blot法检测容受性相关分子同源框蛋白A10(HoxA10)、白血病抑制因子(LIF)的蛋白表达量。每组剩余的5只大鼠在干预后合笼并通过胚胎着床数来评估子宫功能的恢复情况。结果:与空白组相比,模型组大鼠的子宫内膜变薄(P<0.001)、腺体数量减少(P<0.001),子宫内膜纤维化面积增加(P<0.001),子宫内膜组织中的VEGF、PCNA、ER阳性表达,子宫内膜容受性相关分子HoxA10和LIF的表达,子宫损伤侧的胚胎着床数均降低(P<0.001)。与模型组相比,联合组以上指标均逆转(P<0.001,P<0.01);细胞组的子宫内膜增厚(P<0.001)、子宫内膜纤维化面积减少(P<0.001)。与细胞组相比,联合组子宫内膜厚度增加(P<0.01)、腺体数量增多(P<0.05),子宫内膜纤维化面积减少(P<0.05),子宫内膜中的VEGF、PCNA和ER阳性表达,子宫内膜HoxA10和LIF的表达,子宫损伤侧的胚胎着床数量均显著升高(P<0.001,P<0.05,P<0.01),治疗效果较细胞组更好。结论:电针联合BMSCs协同修复受损子宫内膜,促进子宫内膜形态的改善,减轻纤维化,促进血管生成和基质细胞增殖,提高子宫内膜的容受性,并最终有利于胚胎的着床。

ObjectiveTo investigate the effects of electroacupuncture(EA)com bined with bone m arrow m esen-chym al stem cells(BM SCs)transplantation on the endom etrium of rats with intrauterine adhesions(IUA),so as to ex⁃plore the possible m echanism s underlying their com bined therapeutic effects.MethodsForty adult fem ale SD rats were random ly divided into control,m odel,cell,and com bined groups.The IUA rat m odel was established using a dual injury m ethod of m echanical scratching and lipopolysaccharide infection.After successful m odeling,on days 1,3,and 7,rats in the m odel group received tail vein injection of phosphate buffered solution,while rats in the cell group re⁃ceived tail vein injection of BM SCs suspension for BM SCs transplantation;and rats in the com bined group received BM SCs transplantation com bined with EA treatm ent(2 Hz/15 Hz,1—2 m A),targeting the“Guanyuan”(CV4),bilateral“Zusanli”(ST36)and“Sanyinjiao”(SP6)for 20 m in daily for 3 consecutive estrous cycles.After intervention,uterine tis⁃sue was collected from 5 rats in each group.Histological analysis was perform ed using hem atoxylin and eosin staining to evaluate endom etrial thickness and glandular num ber.M asson staining was used to assess endom etrial fibrosis area.Im m unohistochem istry was perform ed to detect the positive expressions of vascular endothelial growth factor(VEGF),proliferating cell nuclear antigen(PCNA),and estrogen receptor(ER).W estern blot analysis was conducted to deter⁃m ine the protein expressions of hom eobox A10(HoxA10)and leukem ia inhibitory factor(LIF),both key regulators of en⁃dom etrial receptivity.The rem aining 5 rats in each group were co-housed with m ale rats,and the uterine function recoverywas evaluated by assessing the num ber of em bryo im plantations.ResultsCom pared with the control group,the m odel group showed thinning endom etrium(P<0.001),decreased glandular num ber(P<0.001),increased endom etrial fibrosis area(P<0.001),reduced positive expressions of VEGF,PCNA,ER,expressions of HoxA10 and LIF,and de⁃creased em bryo im plantation num ber(P<0.001)on the injured side of the uterus.Com pared with the m odel group,the com bined group showed a reversal of the aforem entioned indicators(P<0.001,P<0.01);the cell group exhibited thicker endom etrium(P<0.001)and reduced endom etrial fibrosis area(P<0.001).Com pared with the cell group,the com bined group showed increased endom etrial thickness(P<0.01),elevated glandular num ber(P<0.05),significantly decreased endom etrial fibrosis area(P<0.05),enhanced positive expressions of VEGF,PCNA and ER,expressions of HoxA10and LIF in the endom etrium,and a significant increase in em bryo im plantation num ber(P<0.001,P<0.05,P<0.01)on the injured side of the uterus,indicating better results than the cell group.ConclusionThe com bination of EA and BM SCs synergistically prom otes the repair of dam aged endom etrium,im proves endom etrial m orphology,reduces fibro⁃sis levels,enhances vascular regeneration and m atrix cell proliferation,im proves endom etrial receptivity,which ulti⁃m ately facilitates em bryo im plantation.