Cx43修饰人脐血源基质细胞输注阻抑白血病MRD小鼠造血干细胞移植后复发

Cx43 modified human umbilical cord blood-derived stromal cells prevent relapse in minimal residual disease model of mice after hematopoietic stem cells transplantation

目的观察间隙连接蛋白43(connexin 43,Cx43)修饰人脐血源基质细胞(human umbilical cord blood-derived stromal cells,h UCBDSCs)体外对L615小鼠白血病细胞株凋亡以及在体对白血病微小残留病(minimal residual disease,MRD)小鼠疾病进展的影响。方法通过Cx43过表达腺病毒(Ad-Cx43-GFP)上调h UCBDSCs中Cx43表达,体外构建L615+Cx43+h UCBDSCs共培养模型,检测其对L615细胞凋亡的影响。建立L615细胞低瘤负荷的MRD小鼠模型,分为骨髓(bone marrow,BM)移植组和Cx43+h UCBDSCs+BM移植组进行移植,以正常L615小鼠作为对照,检测移植后外周血象、骨髓涂片、组织病理及骨髓Cx43表达变化等。结果 Ad-Cx43-GFP能够在mRNA和蛋白水平显著上调h UCBDSCs中Cx43表达。L615+Cx43+h UCBDSCs移植组L615细胞凋亡比例较对照组显著升高[(8.93±1.24)%vs(3.53±0.13)%,P<0.01]。对MRD小鼠移植后,Cx43+h UCBDSCs+BM移植组外周血WBC和PLT恢复更快,17 d时接近正常水平,而BM移植组外周血WBC和PLT恢复延迟,17 d时低于正常水平;17 d时,Cx43+h UCBDSCs+BM移植组骨髓涂片原始细胞比例较BM移植组显著降低[(7.67±1.25)%vs(56.33±1.25)%,P<0.01];与BM移植组比较,Cx43+h UCBDSCs+BM移植组肝、脾、骨髓的白血病浸润程度较低,同时骨髓中Cx43蛋白表达增加。结论上调h UCBDSCs中Cx43表达能在体外促进L615细胞凋亡,Cx43+h UCBDSCs+BM联合移植能够促进MRD小鼠外周血WBC和PLT恢复,阻抑MRD小鼠移植后复发。

Objective To investigate the effect of connexin 43 （Cx43） modified human umbilical cord blood-derived stromal cells （hUCBDSCs） on the apoptosis of mouse leukemia cell line L615s and on pathoprogression in minimal residual disease （MRD） model of mice. Methods Adenoviral plasmid Ad- Cx43-GFP was used to up-regulate the Cx43 expression in hUCBDSCs. The apoptotic rate of L615 cells co- cultured with Cx43-hUCBDSCs was determined by Annexin V/PI apoptosis detection kit. A mouse MRD model was generated through tail venous injection of with 1 x 105 GFP-L615 cells. Bone marrow （BM） and Cx43 ＋ hUCBDSCs ＋ BM transplantation were performed in the MRD mice, and the model mice served as control. Peripheral blood cell count, BM pathological smears, histopathology and the expression of Cx43 in the BM were examined. Results Ad-Cx43-GFP transfection significantly increased the expression of Cx43 in the hUCBDSCs at both mRNA and protein levels. The apoptotic rate of L615 cells was significantly higher in the Cx43 ＋ hUCBDSCs co-culture model than L615 cells cultured alone [ （8.93 ± 1.24）% vs （3.53 ± 0. 13）%, P 〈0. 01 ]. Cx43 ＋ hUCBDSCs ＋ BM transplantation resuhed in obvious recoveries in the peripheral white blood cell count （WBC） and platelet count （PLT）, with the counts almost reaching normal values in 17 d later. Whereas, the BM transplantation group showed delayed recoveries in peripheral WBC and PLT, and the counts were still lower than those of normal group in 17 d. The count of different bone marrow cells in BM pathological smears showed that the percentage of original cells was obviously decreased in the Cx43 ＋ hUCBDSCs＋BM group than the BM group [（7.67 ±1.25）% vs （56.33 ±1.25）%, P〈0.01]. The pathological observation showed an obviously lower infiltration of leukemia cells in the liver, spleen and BM of the Cx43 ＋ hUCBDSCs ＋ BM group than that in the BM group. Furthermore, increased Cx43 in the BM was observed in the Cx43 ＋ hUCBDSCs ＋ BM group than with the BM group. The expression of Cx43 in the bone marrow was enhanced in the Cx43 ＋ hUCBDSCs ＋ BM group. Conclusion Up-regulation of Cx43 in the hUCBDSCs can induce apoptosis in L615 cells. Combined Cx43 modified hUCBDSCs and hematopoietic stem cells transplantation can promote peripheral WBC and PLT recovery and prevent the relapse of MRD mice.