人脐带间充质干细胞对糖尿病足溃疡模型大鼠血清炎性因子及相关细胞生长因子的影响

The impact of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells for model rats with diabetic foot ulcer

目的观察人脐带间充质干细胞对糖尿病足溃疡模型大鼠血清炎性因子及相关细胞生长因子的影响,探讨其治疗糖尿病足溃疡的作用机制.方法 50只无特定病原体(SPF)级雄性大鼠使用高脂饮料喂养4周后,采用腹腔注射1%链脲佐菌素(40 mg/kg)、水蒸汽熏蒸的方法建立糖尿病足溃疡模型,取建模成功30只大鼠分为模型组、高剂量人脐带间充质干细胞治疗组(高剂量治疗组:股动脉注射人脐带间充质干细胞2.0×10^6/0.2 ml)、低剂量人脐带间充质干细胞治疗组(低剂量治疗组:股动脉注射人脐带间充质干细胞1.0×10^6/0.2 ml)各10只,另取10只SPF级雄性大鼠大鼠为空白对照组(空白组).比较溃疡面积愈合率、血清炎性因子、相关细胞生长因子、肉芽组织成纤维细胞数及新生毛细血管数等指标.应用SPSS 21.0统计软件分析,符合正态分布的计量资料用均值±标准差(Mean±SD)表示,多组间比较采用方差分析,两两比较采用配对t检验,不符合正态分布的计量资料用四分位数间距表达,多组间比较采用Kruskal-WallisH检验,两组比较采用Manne Whitnery U检验.结果(1)创面愈合率:模型组、低剂量治疗组、高剂量治疗组分别为(26.31±4.32)%、(66.92±7.24)%、(74.74±8.32)%.低剂量治疗组、高剂量治疗组创面愈合率明显高于模型组(t=8.424、11.201,P<0.01),高剂量治疗组创面愈合率明显高于低剂量治疗组(t=3.148,P<0.05).(2)血清炎性因子:模型组、低剂量治疗组、高剂量治疗组血清白细胞介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)、超敏C-反应蛋白(hs-CRP)含量分别为(6.12 ±0.25)、(4.54±0.36)、(4.20±0.33) ng/L;(29.45±3.12)、(21.13±3.24)、(18.20±2.65) ng/L;(0.61±0.08)、(0.45±0.07)、(0.37±0.06) ng/L.低剂量治疗组、高剂量治疗组大鼠血清IL-1、TNF-α、hs-CRP含量明显低于模型组(t=11.400、5.849、5.111、14.666、8.691、8.222,P<0.05,P<0.01);高剂量治疗组大鼠IL-1、TNF-α、hs-CRP含量明显低于低剂量组(t=2.202、2.214、2.744,P<0.05).(3)相关细胞生长因子:模型组、低剂量治疗组、高剂量治疗组大鼠血清碱性成纤维因子(bFGF)、表皮细胞生长因子(EGF)、血管内皮生长因子(VEGF)含量分别为(46.42±6.36)、(61.35±7.20)、(68.24±8.12) ng/L;(546.32±30.24)、(722.36±32.17)、(772.45±34.26)μg/L;(2.24±0.32)、(3.65±0.41)、(4.26±0.55)μg/L.低剂量治疗组、高剂量治疗组大鼠血清bFGF、EGF、VEGF含量明显高于模型组(t=4.915、12.609、8.573、6.690、15.648、10.039,P<0.05,P<0.01);高剂量治疗组大鼠血清bFGF、EGF、VEGF含量明显高于低剂量组(t=2.031、3.740、2.812,P<0.05).(4)成纤维细胞数与毛细血管数密度:模型组、低剂量治疗组、高剂量治疗组大鼠肉芽组织成纤维细胞数、毛细血管数密度分别为(155.24 ±8.45)、(278.65±9.12)、(305.12±10.25)个/视野;(4.36±0.45)、(6.45±0.62)、(7.24±0.60)×10/μm^2.低剂量治疗组、高剂量治疗组大鼠肉芽组织成纤维细胞数、毛细血管数密度明显高于模型组(t=31.389、8.627、35.679、12.143,P<0.01);高剂量治疗组大鼠肉芽组织成纤维细胞数、毛细血管数密度明显高于低剂量组(t =6.101、2.896,P<0.05).结论人脐带间充质干细胞有助于促进糖尿病足溃疡模型大鼠溃疡愈合.

Objective To study effect of inflammatory factors and related cell growth factors of human umbilical cord mesenchymal stem cells (HUCMSCs) for model rats with diabetic foot ulcer (DFU), and analyzed the possible action mechanism of HUCMSCs therapy DFU. Methods50 specific pathogen free (SPF)-level male rats were fed with high-fat beverage for 4 weeks. The DFU models were established by means of intraperitoneal injection of 1% streptozotocin (40 mg/kg) and steam fumigation. 30 successful model rats were divided into model group, high-dose HUCMSCs group (high-dose group: femoral artery injection with HUCMSCs 2.0×10^6/0.2 ml) and low-dose HUCMSCs group (low-dose group: femoral artery injection with HUCMSCs 1.0×10^6/0.2 ml), 10 rats/each group. Another 10 SPF-level male rats were selected and set as blank control group (blank group). Then wound healing rate, serum inflammatory factors, related cell growth factors, granulation tissue fibroblasts and newly born capillaries were compared between the groups. Results(1) Wound healing rate: The model group, low-dose group, high-dose group respectively were (26.31±4.32)%,(66.92±7.24)%,(74.74±8.32)%. low-dose group and high-dose group wound healing rate were significantly higher than model group (t=8.424, 11.201, P<0.01), high-dose group wound healing rate were significantly higher than low-dose group (t=3.148, P<0.05).(2) Serum inflammatory factors: model group, low-dose group and high-dose group interleukin (IL)-1, tumor necrosis factor-α(TNF-α), hs-CRP respectively were (6.12±0.25),(4.54±0.36),(4.20±0.33) ng/L;(29.45±3.12),(21.13±3.24),(18.20±2.65) ng/L;(0.61±0.08),(0.45±0.07),(0.37±0.06) ng/L. low-dose group and high-dose group IL-1, TNF-α, hs-CRP were significantly lower than model group (t=11.400, 5.849, 5.111, 14.666, 8.691, 8.222, P<0.05, P<0.01), high-dose group were significantly lower low-dose group (t=2.202, 2.214, 2.744, P<0.05);(3) Related cell growth factors: model group, low-dose group and high-dose group basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) respectively were (46.42±6.36),(61.35±7.20),(68.24±8.12) ng/L;(546.32±30.24),(722.36±32.17),(772.45±34.26)μg/L;(2.24±0.32),(3.65±0.41),(4.26±0.55)μg/L. Low-dose group and high-dose group bFGF, EGF, VEGF were significantly higher than model group (t=4.915, 12.609, 8.573, 6.690, 15.648, 10.039, P<0.05, P<0.01). high-dose group bFGF, EGF and VEGF were significantly higher than low-dose group (t=2.031, 3.740, 2.812, P<0.05);(4) Granulation tissue fibroblasts and newly born capillaries: model group, low-dose group and high-dose group granulation tissue fibroblasts and newly born capillaries were respectively (155.24±8.45),(278.65±9.12),(305.12±10.25) cells/visual field;(4.36±0.45),(6.45±0.62),(7.24±0.60)×10/μm2. The low-dose group and high-dose group were significantly higher than model group (t=31.389, 8.627, 35.679, 12.143, P<0.01), high-dose group were significantly higher than low-dose group (t=6.101, 2.896, P<0.05). ConclusionHUMSCs help to promote the ulcerative healing of DFU rats.