摘要
目的：探讨骨髓间充质干细胞（BMSCs）在体外培养扩增过程中引起的老化对其修复糖尿病慢性创面功能的影响。方法：从大鼠股骨骨髓中分离培养BMSCs,流式细胞分析仪鉴定细胞表型,成骨、成脂诱导鉴定其分化能力。取第5、15、25、35和45代（P5、P15、P25、P35和P45）BMSCs,0.25%的胰酶消化后制备1×10^6/m1单细胞悬液。腹腔注射65 mg/kg链脲佐菌素建立糖尿病SD大鼠模型,8周后,在SD大鼠背部左右两侧用打孔器制备直径1.2 cm的圆形创面。将70只糖尿病慢性创面模型大鼠随机分为7组,分别经尾静脉注射1 ml的P5、P15、P25、P35和P45 BMSCs单细胞悬液（1×10^6/m1）,1 ml成纤维细胞（Fb）悬液（1×10^6/m1）和1 ml生理盐水。分别于伤后3、7、14、21 d计算创面愈合率,并取创面组织制备切片,苏木精-伊红（HE）染色,光学显微镜下观察组织病理学变化及血管生成情况。结果：糖尿病SD大鼠创面较正常大鼠创面愈合延迟（P〈0.05）。伤后14 d,各BMSCs移植组与Fb组和生理盐水组比较,创面收缩与再上皮化明显,镜下可见成纤维细胞生长活跃、数量多,各BMSCs移植组间无明显差异。伤后14 d,各BMSCs移植组创面愈合率较Fb组和生理盐水组增加,以P5 BMSCs移植组变化最显著（P〈0.05）。伤后7 d,各BMSCs移植组微血管密度均显著高于Fb组和生理盐水组（P〈0.01）,以P5 BMSCs移植组效果最为显著。结论：全身注射异体BMSCs可促进糖尿病创面愈合。BMSCs在体外长期扩增传代可减少其创面修复能力,其对糖尿病创面的修复作用以P5以内干细胞较好。
Objective：To evaluate the effect of transplantation of different generation of allogeneic bone marrow mesenchymal stem cells（BMSCs） cultivated in vitro on chronic diabetic wound.Methods：BMSCs were isolated from rat femur bone marrow,and cell phenotype was identified by flow cytometry（FACs）,and their potential differentiating capacity was confirmed by osteogenic and adipogenic induction.Cell suspensions of BMSCs of 5 th,15 th,25th,35 th and 45 th generations（P5,P15,P25,P35 and P45） were prepared with the concentration of 1×10^6/ml with 0.25%trypsin digestion.Eight weeks after reproduction of rat diabetic model by intraperitoneal injection of 65 mg/kg of streptozotocin,circular wounds with the diameter of 1.2 cm were created on the back of left and the right sides with a hole punch to replicate the chronic wound model.Seventy diabetic rats with chronic wounds were randomly divided into 7 groups,and they received injection of 1 ml of P5,P15,P25,P35 and P45 BMSCs cell suspension（1 × 10^6/ml）,1 ml fibroblast（Fb） suspension（1×10^6/ml） respectively via tail vein.The rats in control received i.v.injection of 1 ml of normal saline.At 3,7,14,21 days after wounding,the wound healing rate was calculated,and wound tissues were harvested for pathological examination and observation of the state pi vascular regeneration with hematoxylin and eosin（HE） staining.Results：Wound healing in diabetic rats was delayed compared to that of the normal rats（P0.05）.At 14 days after wounding,contraction and re-epithelialization of wounds were more obvious in every BMSCs transplantation group than that of Fb and normal saline groups,with active proliferation and larger quantity of Fb,and the changes were similar in every BMSCs transplantation group.Wound healing rate in each BMSCs transplantation group[P5：（93.18±1.16）%;P15：（81.26±1.25）%;P25：（80.03±3.14）%;P35：（79.24±2.46）%;P45：（83.17±2.79）%]was higher than that in Fb group[（72.17±1.59）%]and normal saline group[（73.17±2.79）%]on day 14 after wounding,but the difference was statistically significant only in P5 BMSCs transplantion group compared to the other BMSCs-transplanted groups（P0.05）.At 7 days after injury,microvessel density in BMSCs transplantation groups[P5：8.9±1.4/HP;P15：7.9±2.0/HP,P25：7.5±1.8/HP;P35：6.9±0.9/HP;P45：7.0±1.1/HP]was much higher than that in Fb group（5.2±1.5/HP） and normal saline group（5.8±1.6/HP）,especially in P5 BMSCs transplantion group（P0.05）.Conclusions：The transplantation of allogeneic BMSCs can promote wound healing in rats with diabetes mellitus.Cultured in vitro for a prolonged time can reduce the wound repair function of BMSCs.Thererfore,The stem cells of less passage,such as BMSCs undergoing 5 passages or less should be chosen for clinical transplantation to obtain optimal effects in vivo.
出处
《感染．炎症．修复》
2014年第3期148-153,F0002,共7页
Infection Inflammation Repair
基金
国家自然科学基金资助项目(81171830
30973123)
关键词
骨髓间充质干细胞
创面愈合
慢性创面
糖尿病
Marrow mesenchymal stem cells
Wound healing
Chronic wound
Diabetic mellitus
