电针联合骨髓间充质干细胞移植通过调节SDF-1/CXCR4轴促进薄型子宫内膜修复的机制研究

Electroacupuncture combined with bone marrow mesenchymal stem cell transplantation promotes repair of thin endometrium by regulating SDF-1/CXCR4 signaling

目的:观察电针联合骨髓间充质干细胞(BMSCs)移植对薄型子宫内膜形态、子宫内膜组织基质细胞衍生因子-1(SDF-1)/CXC趋化因子受体4(CXCR4)轴相关蛋白及mRNA的影响,探讨其修复薄型子宫内膜的机制。方法:将健康SPF级性成熟雌性SD大鼠按随机数字表法分为空白组、模型组、BMSCs组、BMSCs+AMD3100组、BMSCs+电针组(联合组)、联合+AMD3100组,每组5只。除空白组外,其余5组采用95%乙醇宫腔注射的方法于大鼠动情期制备薄型子宫内膜模型。BMSCs组于造模第1、3、7天给予尾静脉注射BMSCs。BMSCs+AMD3100组在BMSCs组的基础上,以5mg/kg的剂量给予腹腔注射AMD3100,每日1次;联合组在BMSCs组的基础上取“子宫”“关元”“三阴交”,每日给予电针刺激15 min;联合+AMD3100组在联合组干预基础上,以5 mg/kg的剂量给予腹腔注射AMD3100,每日1次。以上3组均干预3个动情周期。通过HE染色法观察大鼠子宫内膜形态,免疫组织化学法检测子宫内膜组织波形蛋白、角蛋白19的表达,Western blot法检测子宫内膜组织同源框(HOXA)10、白血病抑制因子(LIF)、SDF-1和CXCR4的蛋白表达水平,实时荧光定量PCR法检测大鼠子宫内膜组织SDF-1和CXCR4 mRNA的表达。结果:与空白组比较,模型组大鼠子宫内膜腺体数目及子宫内膜组织波形蛋白、角蛋白19的阳性表达均减少(P<0.01),HOXA10、LIF、CXCR4蛋白及CXCR4mRNA表达水平均降低(P<0.01),SDF-1蛋白及mRNA表达水平均升高(P<0.05)。与模型组比较,BMSCs组和联合组大鼠子宫内膜腺体数目及子宫内膜组织中波形蛋白、角蛋白19的阳性表达增多(P<0.05,P<0.01),HOXA10、LIF、CXCR4蛋白及CXCR4mRNA表达水平均升高(P<0.01,P<0.05);联合组SDF-1蛋白及mRNA表达水平升高(P<0.05)。与BMSCs组比较,联合组子宫内膜腺体数目增多(P<0.01),LIF、CXCR4蛋白及CXCR4 mRNA表达水平均升高(P<0.01,P<0.05);BMSCs+AMD3100组子宫内膜腺体数目及子宫内膜组织中波形蛋白、角蛋白19的阳性表达减少(P<0.01),HOXA10、LIF、CXCR4蛋白及mRNA表达水平降低(P<0.01)。与联合组比较,联合+AMD3100组大鼠子宫内膜腺体数目及子宫内膜组织波形蛋白、角蛋白19阳性表达均减少(P<0.01),HOXA10、LIF、CXCR4蛋白及CXCR4 mRNA表达水平均降低(P<0.01)。结论:电针可增强与干细胞归巢相关的SDF-1和CXCR4信号分子的表达,促进移植的BMSCs向受损部位转移,从而促进薄型子宫内膜再生,修复子宫内膜损伤,改善子宫内膜容受性。

Objective It is to explore,based on stromal cell derived factor 1(SDF-1)/CXC chemokine recep⁃tor 4(CXCR4)signal axis,whether the electroacupuncture(EA)combined with bone marrow mesenchymal stem cells(BMSCs)transplantation can promote thin endometrium regeneration and improve endometrial receptivity,so as to fur⁃ther study its mechanisms underlying improvement of promoting BMSCs homing to repair thin endometrium.Methods Thirty matured female SD rats were randomly divided into normal control,model,BMSCs transplantation(BMSCs),BMSCs+AMD3100(a specific antagonist of CXCR4,BMSCs+AMD3100),BMSCs+EA,and BMSCs+EA+AMD3100 groups,with 5 rats in each group.The thin endometrial model was established by intrauterine injection of 95%ethanol during the period of estrus.Rats of the model group received intravenous injection of PBS solution(tail vein)on day 1,3 and 7 of modeling and intraperitoneal injection of normal saline once daily for 3 estrous cycles.Rats of the BMSCs group received intravenous injection of BMSCs suspension on day 1,3 and 7 of modeling,and those of the BMSCs+EA group received BMSCs transplantation and EA stimulation.EA(2 Hz/15 Hz,1 mA)was applied to“Guanyuan”(CV4)and bilateral“Sanyinjiao”(SP9),“Zigong”(EX-CA1)for 15 min,once daily for 3 estrous cycles.Rats of the BMSCs+AMD3100 group received intravenous injection of BMSCs suspension(1×10^(6)/mL)and intraperitoneal injection of AMD3100(5 mg/kg),and those of the BMSCs+EA+AMD3100 group received administration of BMSCs,AMD3100 and EA,with both groups being once daily for 3 estrous cycles.H.E.staining was used to observe histopathological changes of endometrium tissues,and immunohistochemistry was used to detect the expressions of cytokeratin(CK19)and vi⁃mentin in endometrium(for evaluating the damage and repair of endometrium).The expression levels of homeobox A10(HOXA10),leukemia inhibitory factor(LIF),SDF-1 and CXCR4 proteins were detected by Western blot,and those of SDF-1 and CXCR4 mRNAs in the endometrium detected by real-time PCR.Results In comparison with the normal control group,the number of endometrial glands,the immunoactivity of CK19 and vimentin,the expression levels of HOXA10,LIF and CXCR4 proteins and CXCR4 mRNA were significantly down-regulated(P<0.01),and the ex⁃pression levels of SDF-1 protein and mRNA significantly up-regulated(P<0.05)in the model group.Compared with the model group,the number of endometrial glands,the immunoactivity of CK19 and vimentin,and the expression levels of HOXA10,LIF,CXCR4 proteins and CXCR4 mRNA in the BMSCs group,and the number of endometrial glands,the immunoactivity of CK19 and vimentin,the expression levels of HOXA10,LIF,CXCR4 proteins and CXCR4 mRNA,and SDF-1 protein and mRNA in the BMSCs+EA group were significantly up-regulated(P<0.05,P<0.01).Compared to the BMSCs group,the number of endometrial glands,and the expression levels of LIF,CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA group were up-regulated(P<0.01,P<0.05);the number of endometrial glands,the immuno⁃activity of CK19 and vimentin,the expression levels of HOXA10,LIF,CXCR4 proteins and CXCR4 mRNA in the BMSCs+AMD3100 group were down-regulated(P<0.01).Compared to the BMSCs+EA group,the number of endome⁃trial glands,the immunoactivity of CK19 and vimentin,the expression levels of HOXA10,LIF,CXCR4 proteins and CXCR4 mRNA in the BMSCs+EA+AMD3100 group were down-regulated(P<0.01).Results of H.E.staining showed thin endometrium with absence of epithelial cells,and sparse glands and blood vessels,with smaller glandular cavity in the model group,which was relative milder in BMSCs and BMSCs+EA groups.Conclusion EA can promote the transfer of transplanted BMSCs to the damaged site through SDF-1/CXCR4 signaling related stem cell homing,thereby promoting thin endometrial regeneration,repairing endometrial injury,and improving endometrial tolerance in rats with thin endometrium.