【作者】 万江波；

【导师】 蔡黔；

【作者基本信息】 兰州大学 ， 外科学， 2013， 硕士

【摘要】 【目的】：评价异体骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells, BM-MSCs)经创缘皮下和小腿肌肉内移植对大鼠糖尿病足溃疡(diabetic foot ulcers, DFUs)的治疗效果以及可能的生物学机制。【方法】：大鼠糖尿病经空腹腹腔注射链脲佐菌素(streptozotocin, STZ)诱导,而后在其双后足背做一大小为3mm×7mm矩形全层皮肤缺损制成大鼠DFUs模型。全骨髓贴壁法体外培养大鼠BM-MSCs至第三代,经4,6-联脒-2-苯基吲哚(4,6-diamino-2-phenylindole,DAPI)体外标记。48只雄性Wistar大鼠随机分成A组(正常大鼠对照组,n=12)、B组(DFUs对照组,n=12)、C组(BM-MSCs创缘皮下移植组,n=12)和D组(BM-MSCs小腿肌肉移植组,n=12)。于移植后第2、5、8、11天评估各组大鼠的创面愈合率,冰冻切片观察DAPI标记的BM-MSCs在创伤组织中的示踪,HE染色法检测早期肉芽组织的厚度,免疫组化法检测CD31和Ki-67在创伤组织中的表达,血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)的表达采用ELISA和RT-PCR检测。【结果】：A组的创面愈合最快,第11天D组的创面愈合率大于C组(P<0.05)。第2天和第5天的冰冻切片上D组的荧光强度及区域大小均大于C组。而HE染色显示第5天D组的肉芽厚于C组。CD31的免疫组化显示第5天和第8天D组的平均小血管数目均多于C组(分别为P＜0.05和P＜0.05),而Ki-67的免疫组化结果显示各时间点C、D两组之间无差异,但分别与B组比较均有显著差异(P＜0.01)。ELISA和RT-PCR的结果均显示第8天和第11天D组的VEGF表达水平高于C组(分别为P＜0.05和P＜0.001),而B组的表达水平始终最低。[结论】：BM-MSCs经两种移植方式均促进了大鼠DFUs的愈合,但小腿肌肉内移植比创缘皮下移植更显著地促进了肉芽组织的形成、细胞增殖、血管化及VEGF持续稳定的表达,从而加速了上皮化进程,即加快了创面的愈合。因此,小腿肌肉内移植BM-MSCs治疗大鼠DFUs疗效更优。更多还原

【Abstract】 Objective:To evaluate if transplantation of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) could enhance the wound healing in rat diabetic foot ulcers (DFUs) and the therapeutic effect between intramuscular transplantation and subcutaneous transplantation and to investigate the possible underlying biological mechanisms involved in this process.Methods:An animal model of rat DFUs was established by injecting intraperitoneally with streptozotocin (STZ) and making a rectangular wound (3mmx7mm) on both sides of the hind feet of diabetic rats. BM-MSCs from male Wistar rats were cultured by the whole bone marrow adherence method until the third generation and were labeled with4,6-diamino-2-phenylindole (DAPI) in vitro. Forty-eight male Wistar rats were randomly divided into four groups:group A, non-diabetic rats with PBS treatment; group B, DFUs rats with PBS treatment; group C, DFUs rats with subcutaneous transplantation of BM-MSCs; and group D, DFUs with intramuscular transplantation of BM-MSCs. On days2,5,8and11post-transplantation, trace of DAPI labeled-BM-MSCs in wound tissues were detected on frozen sections and the effect of wound healing was evaluated by the rate of wound contraction, granulation tissue formation and the expression of CD31, Ki-67as well as vascular endothelial growth factor (VEGF) in wound tissues.Results:Compared to rats in group B (DFUs controls), which were treated with PBS, transplantation of BM-MSCs significantly promoted the wound healing in groups C and D. While the group D demonstrated better efficacy than group C, in which a higher rate of wound contraction (on day11, P<0.05), stronger and broader area of fluorescence on days2and5, thicker granulation tissue on day5, more CD31-positive small blood vessels (on day5and day8, P<0.05and P<0.05respectively), and a significantly higher expression of VEGF (on day8and day11, P<0.05and P<0.001respectively) were detected. While there were no differences on cellular proliferation at every time point between group C and group D. But there are significant differences when the two groups compared with group B respectively at every time point (P<0.01).Conclusions:Both the subcutaneous transplantation and intramuscular transplantation of BM-MSCs promoted the wound healing of DFUs in rats. But the intramuscular transplantation of BM-MSCs significantly enhanced the wound healing of DFUs in rats. This effect is associated with better granulation tissue formation, increased angiogenesis, and higher lever of cellular proliferation and VEGF expressions in wound tissues.更多还原