脐带间充质干细胞对急性卵巢缺血再灌注损伤小鼠卵巢组织内血管内皮生长因子及肿瘤坏死因子-α的影响

Effects of Umbilical Cord Mesenchymal Stem Cells on Vascular Endothelial Growth Factor and Tumor Necrosis Factor-α in Ovarian Tissue of Mice with Acute Ovarian Ischemia/Reperfusion Injury

目的探讨脐带间充质干细胞(UC-MSCs)对急性卵巢缺血再灌注损伤卵巢组织内血管内皮生长因子(VEGF)和肿瘤坏死因子-α(TNF-α)的影响。方法30只C57BL/6NCrl雌性小鼠,采用随机数字法分为三组:假手术组、卵巢缺血+再灌注损伤组(模型组)、卵巢缺血+再灌注损伤+UC-MSCs干预组(干细胞组),每组10只。操作如下:假手术组在卵巢动脉仅穿线,不阻断血液灌流,缝合肌肉、皮肤;模型组结扎卵巢动脉(余同假手术组),阻断卵巢血液供应30 min,而后剪断结扎线恢复血流;干细胞组为结扎30 min后恢复血流灌注,并给予干细胞干预治疗,即将1.0×106UC-MSCs通过尾静脉注射途径输注至小鼠体内。采用实时定量聚合酶链反应和Western blot技术等检测各组缺血卵巢组织内VEGF和TNF-α的变化,利用酶联免疫吸附试剂盒测定血清TNF-α水平,流式细胞术检测体内UC-MSCs数量变化。结果与假手术组比较,模型组小鼠卵巢组织的TNF-αmRNA以及蛋白的表达均升高(0.57±0.19比0.19±0.04,0.65±0.21比0.23±0.10)(P<0.05);与模型组比较,干细胞组TNF-αmRNA以及蛋白(分别为0.31±0.16,0.21±0.07)均下降(P<0.05);与假手术组比较,模型组小鼠卵巢组织的VEGF mRNA以及VEGF蛋白的表达均升高(0.29±0.13比0.26±0.11,0.22±0.09比0.19±0.09)(P<0.05);而与模型组比较,干细胞组VEGF mRNA及VEGF蛋白表达水平(分别为0.70±0.23,0.58±0.24)均上调(P<0.05)。模型组、干细胞组TNF-α蛋白水平较假手术组升高[(5.232±1.320)pg、(1.439±0.508)pg比(0.435±0.012)pg](P<0.01);干细胞组TNF-α水平较模型组降低(P<0.01)。模型组大鼠UC-MSCs的数量与假手术组比较,差异无统计学意义(P>0.05);干细胞组UC-MSCs的数量高于假手术组和模型组[(3.176±0.936)%比(0.091±0.034)%、(0.134±0.081)%](P<0.05)。结论行UC-MSCs移植术治疗小鼠急性卵巢缺血再灌注损伤可有效提高VEGF的表达,改善损伤区域炎症反应,通过降低TNF-α的表达水平有效抑制炎症进展。

Objective To investigate the effects of umbilical cord mesenchymal stem cells(UC-MSCs)on vascular endothelial growth factor(VEGF)and tumor necrosis factor-α(TNF-α)in ovarian tissue of mice with acute ovarian ischemia/reperfusion injury.Methods A total of 30 C57 BL/6 NCrl female mice were randomly divided into a sham group,an ovarian ischemia-reperfusion group(model group)and an ischemia-reperfusion+UC-MSCs group(stem cell group),10 in each.In the sham operation group,the ovarian artery was only threated without blocking blood perfusion,and muscles and skin were sutured;in model group,ovarian artery was ligated(rest procedures were the same as sham operation group),and ovarian blood supply was blocked for 30 min,and then the ligation line was cut to restore blood flow;in stem cell group,blood perfusion was restored 30 min after ligation,and stem cell intervention was given,i.e.,1.0×10^6 UC-MSCs were injected into mice through tail vein.RT-PCR and Western blotting were used to detect the changes of VEGF and TNF-α,and serum TNF-αwas determined by enzyme linked immunosorbent assay(ELISA)kit.The flow cytometry method were used to detect the changes of stem cells in vivo.Results Compared with the sham group,the levels of TNF-αmRNA and TNT-αprotein in the model group were significantly increased(0.57±0.19 vs 0.19±0.04,0.65±0.21 vs 0.23±0.10)(P<0.05);compared with the model group,the levels of TNF-αmRNA and TNT-αprotein of the stem cell group(0.31±0.16,0.21±0.07)were decreased(P<0.05);compared with the sham group,VEGF mRNA,VEGF protein in the model group were increased(0.29±0.13 vs 0.26±0.11,0.22±0.09 vs 0.19±0.09)(P<0.05);compared with the model group,VEGF-αmRNA and VEGF protein in the stem cell group(0.70±0.2,0.58±0.24)were increased(P<0.05).The levels of TNF-αprotein in the model group and the stem cell group were significantly higher than those in the sham group[(5.232±1.320)pg,(1.439±0.508)pg vs(0.435±0.012)pg](P<0.01).TNF-αlevel in the stem cell group was lower than that in the model group(P<0.01).There was no significant difference in the number of UC-MSCs between the model group and sham operation group(P>0.05).The number of UC-MSCs in the stem cell group was higher than that in the sham operation group and model group[(3.176±0.936)%vs(0.091±0.034)%,(0.134±0.081)%](P<0.05).Conclusion UC-MSCs transplantation for acute ovarian ischemia-reperfusion injury in mice can effectively increase the expression of VEGF,improve the inflammatory response in the injured area,and effectively inhibit the progress of inflammation by reducing the expression level of TNF-α.