淫羊藿苷优化大鼠脂肪干细胞促进超长随意皮瓣存活的实验研究

Experimental study on icariin optimizing rat adipose-derived stem cells to promote the survival of ultra-long random skin flaps

目的 探讨淫羊藿苷(ICA)优化大鼠脂肪干细胞(rASCs)促进超长随意皮瓣存活的作用及机制。方法 选择6周龄雌性健康无特定病原体(SPF)级SD大鼠作为实验动物。提取rASCs并鉴定其多向分化能力及表面标志物。通过蛋白免疫印迹(WB)实验检测不同浓度ICA(0、0.1、1、10μM)对rASCs中血管生成因子[血管内皮生长因子(VEGF)、血小板源性生长因子-BB(PDGF-BB)、成纤维细胞生长因子-2(FGF-2)、胰岛素样生长因子-2(IGF-2)]表达的影响。设置rASCs组(予未处理的rASCs)、ICA+rASCs组(予1μM ICA预处理的rASCs)及空白对照组(予等体积PBS),每组3只大鼠,根据分组在皮瓣蒂部注射相应细胞悬液。于治疗后7、14、28 d评估皮瓣存活情况。治疗后28 d取皮瓣组织行组织学及WB实验,检测组织中微血管密度(MVD)、胶原含量及各血管生成因子的表达情况。结果 rASCs具有成脂、成骨和成软骨分化的能力,CD29及CD44呈高表达,不表达CD45。WB结果提示ICA处理能显著提高rASCs中血管生成因子的表达(P<0.05)。动物实验结果显示,与空白对照组比较,rASCs组及ICA+rASCs组治疗后7 d、14 d的皮瓣存活面积比更高(P<0.05);在治疗后28 d,仅ICA+rASCs组的皮瓣存活面积比显著高于空白对照组(P<0.05)。组织学实验结果显示,与空白对照组相比,rASCs组和ICA+rASCs组的皮瓣组织结构排列更为紧密,MVD、胶原含量显著提高(P<0.05)。WB结果显示,rASCs组和ICA+rASCs组的VEGF、PDGF-BB和FGF-2蛋白表达水平均显著高于空白对照组(P<0.05),且ICA+rASCs组的VEGF、PDGF-BB蛋白表达水平较rASCs组更高,差异有统计学意义(P<0.05)。结论 ICA能够提高rASCs血管生成因子的表达水平,这可能是其优化rASCs促进大鼠超长随意皮瓣存活的机制。

Objective To explore the effect and mechanisms of icariin(ICA)to optimize rat adipose-derived stem cells(rASCs)in promoting the survival of ultra-long random skin flaps.Methods Six-week-old female healthy specific-pathogen-free(SPF)Sprague-Dawley(SD)rats were selected as the experimental animals.The rASCs were extracted and their multilineage differentiation ability and surface markers were identified.Western blot(WB)was used to detect the protein expressions of angiogenesis factors[vascular endothelial growth factor(VEGF),platelet-derived growth factor-BB(PDGF-BB),fibroblast growth factor-2(FGF-2),insulin-like growth factor-2(IGF-2)]in the rASCs stimulated by ICA at different concentrations(0,0.1,1,10μM).The rASCs group(untreated rASCs),the ICA+rASCs group(rASCs pretreated with 1μM ICA),and the blank control group(an equal volume of PBS)were set up,with 3 rats in each group.According to the grouping,the corresponding cell suspensions were injected into the pedicle of the flaps,respectively.The survival rates of the flaps were evaluated 7 days,14 days and 28 days after treatment.The survival tissues of the flaps were dissected to detect the microvessel density(MVD),collagen content(%)and the protein expressions of angiogenesis factors in each group 28 days after treatment by histological and WB experiments.Results The rASCs had the ability of adipogenesis,osteogenesis,and cartilage differentiation.The rASCs had high expressions of CD29 and CD44,and no expression of CD45.The results of WB experiment suggested that 1-10μM ICA could significantly improve the expressions of angiogenesis factors in rASCs(P<0.05).The results of the animal experiments showed that the flap survival area ratios in the rASCs group and the ICA+rASCs group were elevated 7 days and 14 days after treatment compared with those in the blank control group(P<0.05).However,only the flap survival area rate in the ICA+rASCs group was significantly higher than that in the blank control group 28 days after treatment(P<0.05).The results of the histological experiments showed that the rASCs group and ICA+rASCs group had a more tightly arranged flap structure,higher MVD and collagen content than the blank control group(P<0.05).The results of the WB experiments suggested that the protein expression levels of VEGF,PDGF-BB,and FGF-2 in the rASCs group and the ICA+rASCs group were significantly higher than those in the blank control group(P<0.05),and the protein expression levels of VEGF and PDGF-BB in the ICA+rASCs group were higher than those in the rASCs group,and the differences were statistically significant(P<0.05).Conclusion ICA can elevate the expression levels of angiogenesis factors in rASCs,which may be the mechanism of its optimization of rASCs to promote the survival of ultra-long random skin flaps in rat models.