Objective:To explore repairing results of VEGF165 gene modified adipose-derived stem cells for diabetic rats with bone defect.Methods:Seventy-eight male Wistar rats weighted 180 to 220 g were selected,72 rats were established diabetic animal models by streptozotocin inducement method,blood glucose level was more than 16.7 mmoL/L.Experimental animals were randomly divided into 5 groups,6 rats in normal group and each 18 rats in other groups.VEGF165 gene modified adipose-derived stem cells were implanted into normal group with bone defect;single diabetic rats with bone defect were named as diabetic group;vascular endothelial growth factor implanted into single diabetic rats with bone defect named as growth factor group;adipose-derived stem cells implanted into diabetic rats with bone defect names as stem cell group;VEGF165 gene modified adipose-derived stem cells implanted diabetic rats with bone defect named as experimental group.After combination of VEGF165-ADSCs (5×106) cells combined with gel sponge,implanted into diabetic rats with bone defect.On the forth week,general form of defect repairing tissue were observed by optical microscopy;local density of micro-vessel were detected by immunohistochemistry method;content of Ca,P and ALP of repairing callus were detected by IRIS Intrepid XSP inductively coupled plasma emission spectrometer.Efficacy of the VEGF165-ADSCs repairing function was evaluated by SPSS statistic software.Results:Fluorescent staining results showed that expression of VEGF165 located on cytoplasm of ADSCs,expression percentage was more than 87％;general histology results showed that callus formation and quality was near to normal group,repairing results in diabetes group,growth factor group and stem cell group were poor.On the Forth week after implantation,content of Ca,P and ALP of repairing callus in experimental group were higher than those in growth group and stem cell group,and without significant differences compared with normal group;blood vessel density in experimental group was lower than normal group,but higher than other groups.Conclusion:VEGF165 gene modified adipose-derived stem cells for repairing diabetic rats with bone defect has advantages of osteogenesis and angiogenesis,and should be one of the effective method for repairing diabetic rats with bone defect.%目的:探讨采用基因转染大鼠脂肪干细胞构建血管化组织工程的方法对糖尿病骨质疏松性骨缺损的修复效果.方法:选取雄性Wistar大鼠78只,体重180～220 g,其中72只通过化学药物(STZ)诱导法建立糖尿病动物模型,成模大鼠血糖值均≥16.7 mmol/L.将实验动物随机分为5组,正常对照组6只,其他实验组各18只.正常对照组:在正常大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞;糖尿病组:单纯糖尿病骨缺损大鼠;生长因子组:在糖尿病大鼠骨缺损内单纯植入VEGF生长因子;干细胞组:在糖尿病大鼠骨缺损内单纯植入脂肪干细胞;实验组:在糖尿病大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞.将5×106个VEGF165-ADSCs细胞与凝胶海绵结合后,植入到糖尿病大鼠骨缺损模型中,在植入后第4周时,采用光学显微镜观察缺损修复组织大体形态;采用免疫组化SP法测定骨缺损区修复后局部微血管密度;应用美国IRIS IntrepidⅡXSP电感耦合等离子体发射光谱仪对修复骨痂内钙/磷含量和碱性磷酸酶(ALP)含量测定;统计分析上述测量结果验证VEGF165-ADSCs对糖尿病大鼠骨缺损的修复作用.结果:荧光染色结果显示,VEGF165表达定位于ADSCs的细胞浆,表达率在87％以上;大体组织学观察结果显示:实验组修复区内骨痂生成范围和质量接近正常组,糖尿病组、生长因子组、于细胞组修复效果欠佳.植入后第4周,实验组单位体积的修复组织钙、磷含量和ALP含量明显高于生长因子组、干细胞组(P＜0.05),与正常对照组组比较差异无统计学意义(P＞0.05);第4周时,实验组修复局部的血管密度低于正常对照组(P＜0.05),而显著高于其他组(P＜0.05).结论:VEGF165基因修饰的脂肪干细胞在糖尿病大鼠体内具有良好的成骨及成血管作用,有望成为修复糖尿病特定骨质条件下骨缺损的一种有效手段.
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